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Role of Satiety Signaling in the Beneficial Effects of Calorie Restriction in Mice

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Title:
Role of Satiety Signaling in the Beneficial Effects of Calorie Restriction in Mice
Creator:
Minor, Robin K
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[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (128 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Percival, Susan S.
Committee Members:
Langkamp-Henken, Bobbi
Knutson, Mitchell D.
Leeuwenburgh, Christiaan
Graduation Date:
12/14/2007

Subjects

Subjects / Keywords:
Body weight ( jstor )
Food intake ( jstor )
Insulin ( jstor )
Liver ( jstor )
Mice ( jstor )
Neurons ( jstor )
Neuropeptides ( jstor )
Obesity ( jstor )
Rats ( jstor )
Tumors ( jstor )
Food Science and Human Nutrition -- Dissertations, Academic -- UF
appetite, cancer, cognition, cr, npy
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Food Science and Human Nutrition thesis, Ph.D.

Notes

Abstract:
Laboratory studies consistently demonstrate extended lifespan in animals on calorie restriction (CR), where total caloric intake is reduced by 10-40% but adequate nutrition is otherwise maintained. CR has been further shown to delay the onset and severity of chronic diseases associated with aging such as cancer, and to extend the functional health span of important faculties like cognition. Less understood are the underlying mechanisms through which CR might act to induce such alterations. One theory postulates that CR?s beneficial effects are intimately tied to the neuroendocrine response to low energy availability, of which the arcuate nucleus in the hypothalamus plays a pivotal role. Neuropeptide Y (NPY), a neurotransmitter in the front line of the arcuate response to low energy availability, is the primary hunger signal affected by CR. It was hypothesized that the arcuate nucleus and NPY are critical not only for certain key physiological alterations, but also for increased stress resistance, decreased cancer risk and enhanced cognition noted with CR. These hypotheses were tested using two mouse models (one chemically treated to impair arcuate function and another genetically modified to lack NPY) maintained on CR or unlimited feeding. Physical performance on locomotor tasks was improved by CR in all mice but benefits to cognition were not observed in either of the neuroendocrine-impaired models. Similarly, while improvements to body composition and reduced serum leptin were induced by CR in all mice, these alterations did not manifest in certain trademark alterations in glucose homeostasis in the models. Resistance to oxidative stress as assessed by survivorship following treatment with the liver toxin diquat and tumorigenicity following a skin tumor induction regimen also suggested the models of impaired hunger sensing faired less well than control mice; liver stress was lethal in NPY knockout mice on CR, and both NPY and arcuate-damaged CR mice were most susceptible to induced skin tumor formation. Taken together these results support the hypothesis that the neuroendocrine response to CR is critical for eliciting some of the beneficial effects of CR. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2007.
Local:
Adviser: Percival, Susan S.
Statement of Responsibility:
by Robin K Minor.

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Copyright Minor, Robin K. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ............................................................................................................... 4

L IST O F T A B L E S .................................................................................................. . 8

LIST OF FIGURES ................................. .. ..... ..... ................. .9

L IST O F A B B R E V IA TIO N S ......... .............. .................................................... ..................... 1

A B S T R A C T ............ ................... ............................................................ 14

CHAPTER

1 L IT E R A T U R E R E V IE W .................................................................................... ........... .... 16

A ppetite.... ................ ............. ...... .................................16
The Role of the Hypothalamic Arcuate Nucleus in Hunger............... .... ........... 17
Neuropeptide Y ..................................... ................. .............. .. 20
U biquitou s expression ............ ......... ........ .................................... ............ .... ..20
Mechanism of action: Signaling through Y receptors..............................21
Physiological pluripotency ............................................. ..... ....................... 22
Neuropeptide Y in feeding behavior ......................... ................... .. ............. 23
Neuropeptide Y in neuroendocrine coordination......... .................................... 26
A g in g .............................................................................2 8
C alorie R estriction ........ ........................................................................ .. ....... .. ....... .. 30
M echanism s of action ............... ... ............. ............ .. ...... ..... ......... 31
Neuropeptide Y: How and Y calorie restriction extends lifespan? ........................32
Overall Rationale .............. ................. ... .... ............... .... ...... ......... 33
Hypothesis #1: The behavioral response to calorie restriction is blunted by impaired
neuroendocrinological signaling ..... ....................... .. ... ...... ................ 34
Hypothesis #2: The physiological response to calorie restriction is blunted by
impaired neuroendocrinological signaling................................................................34
Hypothesis #3: Resistance to oxidative stress by calorie restriction is blunted by
impaired neuroendocrinological signaling................................................................34
Hypothesis #4: Resistance to tumor formation by calorie restriction is blunted by
impaired neuroendocrinological signaling .... ........... ....................................... 34

2 CALORIE RESTRICTION ALTERS PHYSICAL PERFORMANCE BUT NOT
COGNITION IN TWO MODELS OF ALTERED NEUROENDOCRINE SIGNALING ...35

M materials and M methods ................. .................................... .......... ........ .. .............36
A n im als an d D iets ...................................................... ................ 3 6
R otaro d .................................................................................3 7
In clin ed S screen ................... ................................................................... 3 7
Open Field Locom otor A activity .............................................. ............... 37









LIST OF FIGURES

Figure page

1-1 Central integration of satiety signals........................................... ........................... 17

1-2 The hypothalamus, its major appetite regulatory centers, and NPY signaling during
fastin g .......................................... ........ .......................................... 19

1-3 N europeptide Y .................. .............. ............................... ......... ..... 21

2-1 Mean group body weight for the six weeks prior to behavioral testing...........................40

2-2 Effects of ARC function and diet on physical performance........ ...........................41

2-3 Open field locomotor activity and exploratory behavior........................................42

2-4 M orris w ater m aze perform ance ............................................... ............................. 44

2-5 Visual assessment ........................ ........ .. .... .. .. ........ .... .... 45

3-1 Body temperature among the groups. ........................................ .......................... 57

3-2 Fasted leptin and adiponectin levels. ............................................................................ 58

3-3 Fasting blood glucose. .......................... ........................... .... ........ ........ 59

3-4 Oral glucose tolerance test response with area under the curve comparisons. ..................60

3-5 Oral glucose tolerance test insulin response with area under the curve comparisons. ......61

3-6 HOM A calculations ..................................... ... .. .......... ....... ...... 62

4-1 Liver histology of diquat-treated 129S1 mice ....................................... ............... 70

4-2 Liver histology of diquat-treated B6 mice................................ ................................. 71

4-3 Fatty change in the liver in response to diquat ...................................... ............... 72

4-4 Liver antioxidant and stress-related protein expression.........................................74

4 -5 N Q O 1 active ity in th e liv er....................................................................... .....................7 5

5-1 Tum or induction procedure............................................. ................... ............... 80

5-2 Body weights of tum or study mice ....................................................... ............... 82

5-3 Tum or onset profiles. .......................... ........................... .... ...... ...........83









Rotarod

A five-station Rota-Rod Treadmill for mice (Med Associates, St. Albans, VT) consisting

of a mechanized rotating bar (3 cm diameter) suspended 16 cm above a platform was used for

this experiment. On the first day the mice were habituated to the rotarod for 120 s at a constant

speed of 4 rpm. The following day testing was completed in 3 trials of increasing speed from 4 to

40 rpm. Mice were tested for a maximum of 300 s in each trial. The latency to fall was recorded

for each trial and averaged for each group. Results shown are group means of the averaged three

trials per mouse.

Inclined Screen

Mice were placed in a tilted, open field (55 cm2 surface area constructed from a 0.6 cm2

wire mesh grid with black sides extending 15 cm above the grid) and movement was recorded

for 300 s using Field 2020 tracking software from HVS Image (Buckingham, UK). Results were

averaged for total distance traveled for each mouse.

Open Field Locomotor Activity

Mice were placed in a level, open field (same as for inclined screen) and movement was

recorded for 300 s using Field 2020 tracking software from HVS Image. Results were averaged

for both total distance traveled and time spent in the center of the field (comprised of the interior

40 cm2) or around the periphery (the area extending 7 cm inside around the sides of the field).

Morris Water Maze

The apparatus consisted of a white circular plastic pool (100 cm diameter and 70 cm high)

which was filled with water (24 + 1 C) rendered opaque by addition of white Dry Temp paint

powder (Palmer Paint Products Inc., Troy, MI, USA). Spatial navigation cues were affixed to a

clear, plastic cylinder concentric with the pool and extending approximately 30 cm above the top

of the pool. A circular (10 cm diameter) escape platform (the target) was submerged just below









profiles and the points on an unbiased point grid using a high magnification objective (oil-

immersion 100x, n.a. 1.4), as shown in Figure 4-1c. Finally, the total vacuolar load was

calculated as the product of vacuole fraction and total section volume.

Western Blotting

Livers homogenates from six mice from each group were prepared in RIPA buffer and

protein concentration was determined by the Bradford method. The samples were subjected to

SDS-PAGE on 8-16% tris-glycine gels and transferred to nitrocellulose membranes (Invitrogen,

Carlsbad, CA). Rabbit anti-SIRT1, mouse anti-HSP70, rabbit anti-SOD2, (Santa Cruz

Biotechnology, Santa Cruz, CA) were the primary antibodies used to detect proteins. Membranes

were incubated with primary antibody solutions at 1:1,000 (SIRT-1, HSP70) or 1:2,000 (SOD2)

dilution. Secondary antibodies (goat anti-mouse and bovine anti-rabbit horseradish peroxidase-

conjugates from Santa Cruz Biotechnology) were applied at 1:3000 dilutions. Immunolabeled

proteins were detected by chemiluminiscence (ECL Plus Western Blotting Detection System,

Amersham, Buckinghamshire, UK).

NAD(P)H:Quinone Reductase 1 (NQO1) Activity

Liver tissues were homogenized in Tris-sucrose buffer (10 mM Tris-HC1, pH 7.4 and 25

mM sucrose) and lysates were centrifuged at 100,000 x g for 40 min at 40C. Reactions were

carried out in a final volume of 1.0 ml containing 25 mM Tris-HCl (pH 7.4), 0.01% Tween-20,

0.1% BSA, 80 iM 2,6-dichloroindophenol (DCIP), 0 or 40 iM dicumarol, 200 iM NADH, and

an appropriate volume of cytosolic sample. Reduction of DCIP was determined

spectrophotometrically at 600 nm for 3 min using a Perkin Elmer Lambda 25 spectrophotometer

(Waltham, MA). Specific NQO1 activity is described as the dicumarol-sensitive decrease in

DCIP absorbance (extinction coefficient 2100 M-cm-1) and is expressed in nmol DCIP reduced

per minute per microgram of protein as determined by the Bradford method.









CHAPTER 3
REDUCTIONS IN ADIPOSITY AND LEPTIN FOLLOWING CALORIE RESTRICTION DO
NOT FULLY EXTEND TO IMPROVED GLUCOSE HOMEOSTASIS IN MODELS OF
IMPAIRED NEUROENDOCRINOLOGICAL SIGNALING

Among the mechanisms through which CR has been postulated to extend lifespan,

modulation of endocrine systems and glucose homeostasis are recurring, interrelated themes.

Proponents of the endocrine and glucose homeostasis theories of aging hypothesize the profiles

of circulating hormones and glucose levels imposed by CR provide a protective environment that

decelerates aging (reviewed in Sinclair, 2005). Indeed, it has been known for many years that CR

results in reduced blood glucose levels and altered hormone levels, and that these alterations are

associated with enhanced longevity (for further reviews see Bartke, 2005, and Tatar et al., 2003).

For example, aging is associated with increasing levels of circulating insulin (Ma et al., 2002)

mirrored by increasing insulin resistance (Ferrannini et al., 1996). By significantly decreasing

pancreatic insulin output, CR minimizes this detrimental cycle (Dean et al., 1998).

Reducing adipose stores is another way CR profoundly alters the hormonal milieu in

circulation. Increasingly recognized for its impact on health through chronic disease, adipose

tissue is an endocrine organ that secretes numerous factors with diverse physiological effects

(Ahima and Flier, 2000). Among these, leptin and adiponectin are expressed by adipocytes

differentially depending on adiposity and aging. Leptin is known to increase with adiposity

(Frederich et al., 1995) and age (Ma et al., 2002); adiponectin conversely decreases with

adiposity (Combs et al., 2002, Stefan et al., 2002) and age (Zhu et al., 2007). Maintenance of

youthful adipokine levels by CR (Zhu et al., 2007) may forestall development of insulin

resistance (Berg et al., 2001) and its consequences with aging.

Poised at the nexus of the peripheral circulation and the hypothalamus, the arcuate nucleus

has been nominated as a critical mediator of CR's physiological effects (Bartke, 2007, Speakman









range of species, and CR remains the most robust intervention to manipulate the rate of aging yet

studied (Ingram, 2006).

Mechanisms of action

The earliest documented effect of CR was its potent antitumor activity (Rous, 1914). Since

then CR has been demonstrated to affect nearly every physiological process (Sinclair, 2005).

From an evolutionary viewpoint the effect of CR seems to be explained by organisms having

evolved mechanisms to maximize survival when faced with food scarcity (Holliday, 1989).

These mechanisms remain to be definitively identified, but may include reduced adiposity,

metabolic rate, body temperature, oxidative stress and insulin/IGF-1 signaling, and increased

antioxidant protection, damage repair, and protein turnover rates (Masoro, 2007).

Given that several of CR's effects pertain to neuroendocrine adaptations, the

neuroendocrine system has been hypothesized to be a critical mediator of the beneficial effects

secondary to negative energy balance (Bishop and Guarente, 2007; Lamberts et al., 1997;

Meites, 1989; Nelson et al., 1995; Rehman and Masson, 2001; Speakman and Hambly, 2007).

Fuel sensing systems are likely to play an important role in the initial response to altered caloric

intake, since the organism must first recognize reduced caloric intake in order to respond to it.

Sensation of food withdrawal occurs primarily within the gastrointestinal tract and the central

nervous system. It is unlikely that the coordinated systemic response is mediated by the

gastrointestinal system alone, and that the gut communicates with the brain both directly and

humorally further suggests systemic control mediated by neural and endocrine factors.

Neuroendocrine-related effects of CR include frequent periods of moderate

hyperadrenocorticism (Ahima et al., 1996; Masoro, 2007), reduced serum thyroid hormones

(Ahima et al., 1996; Herlihy et al., 1990), and inhibition of gonadal axes (Ahima et al., 1996). In










altogether (Larhammar and Salaneck, 2004). NPY acts through Y receptors to inhibit adenylyl

cyclase (Herzog et al., 1992) and increase intracellular calcium levels (Jacques et al., 2000). In

some instances NPY receptor activity has been shown to result in activation of mitogen-activated

protein kinase (Nie and Selbie, 1998) and protein kinase C (Mannon and Raymond, 1998). The

NPY Yi and Y5 receptors expressed in the hypothalamus are considered to be the most active in

the regulation of appetitive behavior and energy balance in mammals (Duhault et al., 2000; Hu et

al., 1996; Lecklin et al., 2002; Parker and Herzog, 1999).

Physiological pluripotency

The relative abundance of NPY and its receptors combined with their widespread

distribution suggests NPY is involved in multiple important physiological roles beyond the

regulation of food intake. This is indeed the case, and the gamut of NPY's systemic and central

effects is depicted in Table 1-1. Of particular interest to this review are NPY's involvement in

food intake and neuroendocrine coordination, to be elaborated below.

Table 1-1. Diverse effects of central and systemic NPY.
Reviewed by*:
Central Effects
Alcohol Intake Carvajal et al. (2006), Thorsell (2007)
Circadian Rhythms Kallingal and Mintz (2007), Yannielli and Harrington (2001)
Emotion Carvajal et al. (2006), Heilig (2004)
Feeding Behavior Arora and Anubhuti (2006), Beck (2006)
Learning Redrobe et al. (1999)
Locomotion Karlsson et al. (2005)
Neuroendocrine Coordination Magni (2003), Plant and Shahab (2002)
Reproductive Function Kalra and k iI1 ., '- -i W6jcik-Gladysz and Polkowska (2006)
Seizures Dube (2007), Redrobe et al. (1999)
Systemic Effects
Adipose Function Kos et al. (2007)
Adrenal Function Spinazzi et al. (2005)
Cardiovascular Function Zukowska et al. (2003)
Gastrointestinal Function Cox (2007)
Pancreatic Function Imai et al. (2007)
*See review by Thorsell and Heilig (2002) for an overview of many of NPY's actions










Tumor Characteristics

Tumor onset began 8 weeks after DMBA tumor induction in the 129S1 mice (Fig. 5-2a),

and 25 weeks after DMBA treatment all 129S1 mice have tumors except one NPYF+ CR mouse.

B6 mice presented with their first tumors 11 weeks after DMBA treatment (Fig. 5-2b) and at 25

weeks only one SAL AL mouse remains without any tumors.


A] 129S1 Tumor Progression B6 Tumor Progression
100 P 0 0 :100

S80 -- NPY+/+ AL 0 O 80 -*- SAL AL
E -- NPY-/- AL E --- MSG AL
NPY+/+ CR SAL CR p -a
60 NPY-/- CR 60 0 MSGCR

S40 40
2. 20S..
20 20 -

0 0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time (weeks) Time (weeks)




















Figure 5-3. Tumor onset profiles for the 129S1 (A) and B6 (B) mice, with representative mice
and papillomas 13 weeks after DMBA tumor initiation shown below. (Mean +
S.E.M., n = 6 except NPY AL n = 5 and NPY/ CR n = 3.)

Tumor onset was most rapid in the NPY CR mice and this was significantly different

from that of the NPY/+ CR mice. When total number of tumors was assessed at sacrifice, NPY/











Table 3-1. NMR body composition data.


129S1


Week 0


Week 4


Week 8 Week 12


Week 0


Week 4


Week 8


Week 12


NPY +* AL 26.30.5 28.91.2 29.01.1a 30.21.2a SAL AL 21.40.3 24.70.5a 26.40.5a 29.31.1 b
Body NPY AL 26.90.8 28.70.9 29.10.9 33.41.0t MSG AL 18.40.6 25.50.6at 27.10.7at 33.0o0.5b'
Weight NPY*'* CR 26.10.7 22.30.3* 22.00.3a'* 22.30.2a'* SAL CR 21.10.3 17.60.2b'* 19.70.1a* 19.7-0.3a

(g) NPY-- CR 27.21.0 23.80.6a*'t 21.70.4b'* 21.90.3b,* MSG CR 190.6 18.70.3* 18.110.2*t 19.70.2
NPY** AL 15.70.3 16.90.6a 16.30.6 17.20.6a SAL AL 13.10.2 15.30.3a 16.10.3b 17.50.6c
Lean NPY- AL 16.00.4 16.90.5 16.50.4 18.90.5at MSG AL 10.90.4 14.70.4at 15.00.4at 18.40.3b
Mass (g) NPY*'* CR 15.60.4 13.30.2'* 12.90.1a'* 13.40.1a'* SAL CR 13.10.2 10.80.1a'* 11.90.1a'* 12.20.2a'*

NPY CR 16.20.5 14.40.3a*'t 12.70.2b'* 13.4+0.2b* MSG CR 11.20.3t 11.1+0.1* 10.7+0.1* 12.00.1a'*
NPY/ AL 6.10.2 7.40.6a 8.30.6a 8.80.6a SAL AL 4.00.1 4.80.2a 6.00.2b 7.30.4c
Fat NPY AL 6.20.4 7.50.4a 8.20.4a 10.00.5b MSG AL 4.710.2 8.10.2at 9.90.2b't 12.210.20t
Mass (g) NPY' CR 5.90.2 4.60.1a'* 4.80.2a'* 4.50.1a'* SAL CR 3.60.1 2.90.1a'* 4.00.1* 3.30.1*

_NPY-- CR 6.20.5 4.90.2a* 4.20.2* 3.80.1 b,*,t MSG CR 5.00.3 4.910.2*' 4.50.2* 4.40.3*'
NPY+ AL 2.10.1 2.00.1 2.10.1 2.20.1 SAL AL 1.60.1 1.80.1a 2.00.1ab 2.20.1b
Fluid NPY-- AL 2.10.1 2.10.1 2.10.1 2.30.1 MSG AL 1.20.1t 1.70.1a 1.90.1ab 2.20.1b
Mass (g) NPY'+ CR 2.10.1 1.60.1a'* 1.60.1a'* 1.60.1a'* SAL CR 1.60.1 1.30.1* 1.50.1* 1.50.1*

____ NPY CR 2.10.1 1.70.1a* 1.60.1a'* 1.60.1a'* MSG CR 1.30.1t 1.30.1* 1.30.1* 1.40.1*
Fat, NPY/ AL 23.12.6 25.40.9a 28.41.0b 29.00.8b SAL AL 18.60.4 19.20.3 22.70.3a 24.70.5b
as Oo of NPY- AL 22.70.8 26.00.5a 27.90.8ab 29.80.6b MSG AL 25.30.7 3.8.4at 36.50.3b't 37.10.5b't
total mass NPY/ CR 22.70.4 20.70.4* 21.80.6* 20.10.5a'* SAL CR 17.10.4 16.50.4* 20.30.3a'* 17.00.2*

___ NPY CR 22.51.0 20.30.6* 19.30.5a* 17.20.3b'*'t MSG CR 26.00.8 25.90.8* 25.011.1*' 22.11.0a*'t
NPY/ AL 2.60.1 2.30.1a 2.00.1b 2.00.1b SAL AL 3.30.1 3.20.1 2.70.1a 2.40.1b
LeanlFat NPY- AL 2.70.1 2.30.1a 2.110.1ab 1.90.1b MSG AL 2.40.1t 1.80.1at 1.5s0.1b't 1.5s0.1b,
Ratio NPY CR 2.60.1 2.90.1* 2.70.1* 3.00.1a'* SAL CR 3.70.1 3.70.1* 3.00.1a'* 3.70.1*

__NPY-- CR 2.70.1 3.00.1* 3.10.1'* 3.60.1b'*'t MSG CR 2.30.1 2.30.1* 2.40.1a''t 2.80.1b'*'t

p < 0.05 CR vs AL within same phenotype and time point.
p < 0.05 NPY- vs NPY+/+ or MSG vs SAL within same diet and time point.
Letters denote p < 0.05 from baseline; different letters are significantly different.









seasonal hyperphagia in preparation for hibernation (Boswell et al., 1993; Lakhdar-Ghazal et al.,

1995).

Hypothalamic NPY is also increased in genetic models of obesity involving hyperphagia

including obese (ob/ob) mice (Jang and Romsos, 1998), diabetic (db/db) mice (de Luca et al.,

2005), Zucker fatty (fa/fa) rats (Dryden et al., 1995), and the Koletsky corpulent (cp/cp) rat

(Williams et al., 1992). Tubby (tub/tub) mice are unique in that they show significantly reduced

ARC NPY, however high levels of NPY are found in the VMH and DMH, indicating increased

neurotransmission of NPY from the ARC to the VMH and DMH could explain their hyperphagia

(Guan et al., 1998). Whether increased NPY is associative or causative to obesity in these models

remains to be definitively proven, however, at least infafa rats NPY overexpression occurs pre-

obesity and is thought to be a driving factor in their weight gain (Bchini-Hooft et al., 1993). The

reverse association has also been observed; anorexic (anx/anx) mice display reduced NPY

signaling concomitant with their reduced food intake (Broberger et al., 1997).

Research investigating the effects of genetic manipulation of NPY expression and feeding

behavior belies the complexity of food intake regulation. For example, mice engineered to

overexpress central NPY by 15% did not display increased feeding (Inui et al., 1998), and NPY

knockout (NPY -) mice on a mixed background have normal food intake and body weight and

are able to respond to fasting with hyperphagia (Erickson et al., 1996b). C57BL/6 (B6) NPY -

mice even develop mild obesity when access to food is unrestricted (Segal-Lieberman et al.,

2003). Concomitant knockout of NPY and AgRP-mutually expressed by ARC neurons in

response to negative energy balance-did not prevent mice from developing to normal body

weight or displaying hyperphagia following fasting (Qian et al., 2002). Given that roughly two

dozen neurotransmitters have been identified to play a role in regulating feeding behavior (Kalra









In conclusion, these results indicate that while CR results in alterations to body

composition and leptin output from adipose tissue, further downstream changes related to

glucose homeostasis and body temperature are unable to be fully realized without proper

hypothalamic function.










AL mice had significantly more papillomas than CR mice. This difference was not continued to

average tumor size. Among the B6 groups, no significant difference was found in average onset

time although the MSG groups predated their SAL counterparts in both diet conditions. MSG CR

mice presented with roughly double the average number of tumors seen per mouse in the other

groups and this was significantly different from the MSG AL mice and SAL CR mice. Tumor

size, like onset, was not significantly different but MSG mice tended to show larger tumors at

sacrifice than the SAL mice.

Table 5-1. Characteristics of tumor incidence by latency, multiplicity, and size of tumors.
129S1 B6
AL CR AL CR
Tumor Onset (weeks) Tumor Onset (weeks)
NPY+/+ 13.4(1.1) 16.9 (2.1) NS SAL 18.3 (2.8) 19.0 (1.9) NS
NPY- 14.0 (1.2) 11.0 (1.0)* NS MSG 16.6 (0.8) 15.3 (2.3) NS

Tumor Multiplicity Tumor Multiplicity
NPY+/+ 5.5 (1.4) 2.0 (0.6) NS SAL 0.7 (0.3) 0.7 (0.5) NS
NPY- 8.3 (2.6) 2.3 (0.9) p = 0.03 MSG 0.8 (0.3) 2.2 (0.6)t p = 0.05

Tumor Size (mm) Tumor Size (mm)
NPY+ 1.9(0.2) 2.1(0.3) NS SAL 1.7(0.2) 1.8(0.2) NS
NPY- 2.0 (0.2) 1.6(0.1) NS MSG 2.3 (0.3) 2.3 (0.3) NS

*p < 0.05 NPY' vs NPY+
p < 0.05 MSGvs SAL
NS, not significant

Protein Conformation Effect

In general proteins from treated skin samples were assessed to show greater ultrastructural

compromise as reflected in greater hydrophobic amino acid presence on the outer surface as

predicted (Fig. 5-3), although unexpectedly high levels were found in the NPY+Y CR mice.









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Results


Body Weight

Treatment-related changes to body weight in the mice are shown in Figure 5-1. Show how

they lose weight and consume more. DMBA treatment did not appear to affect body weight in

any group. Following TPA treatment initiation, however, the 129S1 AL mice began a long

period of gradual weight loss which continued for 10 weeks and ultimately induced weight loss

of approximately 20% compared with weight at treatment onset (Fig. 5-la). Weight loss was also

seen in the 129S1 CR mice and 3 deaths occurred between weeks 10 and 16 likely due to

starvation. TPA-associated weight loss is not apparent in the B6 mice (Fig. 5-1b), although

weight gain is attenuated for the first 6 weeks in the AL groups. A slight, transient reduction in

food intake in the MSG AL mice was observed which necessitated a corresponding reduction in

MSG CR feeding and accounts for their weight loss after week 12.


X 129S1 Body Weights B6 Body Weights
40 40


30 30




O NPY+/+ AL SAL AL
C0 10 NPY-/- AL CO 10 MSGAL
-NPY+/+ CR -* SAL CR
NPY-/- CR -MSG CR

0 5 10 15 20 0 5 10 15 20
Weeks on Diets Weeks on Diets

Figure 5-2. Body weights of tumor study mice with 129S1 mice represented in panel A and B6
mice in panel B. Solid circle represents week in which DMBA was applied, dotted
circle when TPA treatments were initiated. (Mean S.E.M., n = 6 except NPY+/+ AL
andNPY- CR n = 5.)











D


Figure 1-2. The hypothalamus, its major appetite regulatory centers, and NPY signaling during
fasting. The panel shows an enlargement of the central hypothalamus and its
symmetrical architecture. The ARC is situated along the third ventricle (3V) above
the median eminence (ME) and communicates with the DMH, LHA, PVN and VMH.
The right side shows that during fasting ARC-produced NPY is released to the LHA,
PVN and VMH in the primary response. Fasting also decreases ARC POMC/CART
output.

Hypothalamic ARC circuits are directly responsive to an array of circulating hunger and

satiety signals such as hormones (e.g. ghrelin, insulin and leptin), and metabolites (e.g. glucose)

and thus monitor input regarding both short-term fuel status as well as long-term energy stores.

Registered in concert these signals convey the current state of energy availability such that an

array communicating energy sufficiency leads to low ARC NPY/AgRP expression and high









3. A final NMR was performed on these mice during week twelve. In week 13 the mice were

injected with diquat, euthanized, and tissues were harvested for the work presented in Chapter 4.

Mice randomized to the tumorigenesis study were treated with the tumor inducer DMBA

in week eight after the NMR analysis was performed. No further treatment was then performed

for two weeks. Starting in week ten, mice were treated with the tumor promoter TPA until they

presented with papillomas and were subsequently euthanized. These procedures and the results

constitute Chapter 5.

Body Weights and Food Intake

Body weight and food consumption for the first 12 weeks of study are presented in Figure

A-3. The data reflect consumption and body weight for all mice through week 8 and the

oxidative stress study mice from weeks 10-12. Body weights for the mice randomized to the

tumorigenesis study are shown in Chapter 5.

Food restriction was established gradually for all CR groups. The initial drop in body

weight of the 129S1 CR mice reflects their heavier initial body weight (at 3-5 months of age

these mice were full adults) at diet onset. Body weight loss stops at approximately 20g for these

mice at 70% CR, and AL-fed 129S1 mice continue to gain weight throughout the feeding period

(Fig. A-3A). The B6 mice were younger (at 2.5 months of age these mice were young adults) and

lighter at study start, and consequently CR in these mice resulted in weight maintenance rather

than weight loss (Fig. A-3B).









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L129S1 Food Consumption Rate


NPY+/+


- B6 Food Consumption Rate
100 1


NPY-/-


MSG


Figure A-5. Feeding rate among the CR groups. In the 129S1 mice (A) NPY'+ control mice
tended to consume their food more quickly than NPY~ mice. In the B6 mice (B), SAL
mice ate more rapidly than MSG mice.









Maximum lifespan, defined as the average lifespan of the longest-lived decile of a cohort

(Holloszy, 2000), is often used as the gold standard in gerontology research because valid

biomarkers of physiological aging have not yet been identified (Johnson, 2006). The oldest

documented person in recent history, Jean Louise Calment, died in 1997 at the age of 122,

representing what has been considered the near-maximum life span for humans (Cole and LA-

GRG, 2004). Despite concerted effort, the mechanisms underlying the aging process that set the

maximum lifespan of species have not been completely elucidated. It is likely that multiple

mechanisms impact lifespan, and many potential contributors have been nominated based on

commonly observed consequences of aging. These include oxidative damage (Beckman and

Ames, 1998; Muller et al., 2007), aggravation of inflammatory processes (Chung et al., 2001),

increased fat mass (Enzi et al., 1986; Shimokata et al., 1989), decreased muscle mass (Morley,

2001), insulin resistance (Fraze et al., 1987; Ma et al., 2002), and shifting hormonal profiles

(Bartke, 2005). Subsequently, a number of theories have been developed to explain aging in the

context of these phenomena.

Theories of aging can be divided into two major categories-'programmed' theories and

'wear and tear' theories-outlined in Table 1-3. Programmed theories view aging as the result of

an innate genetic program that dictates the rate of aging and maximum lifespan (Butler et al.,

2003). In contrast, wear and tear theories envision aging as the result of spontaneous events

(Muller et al., 2007). They hypothesize an organism is subject to continual stress (especially

oxidative stress) from the environment and metabolism, and that ability to repair the damage

declines with age such that mutated DNA, proteins, and lipids accumulate and lead to impaired

function of cells and tissues.









entered into the reading chamber of the Minispec and the scan was initiated. Scanning takes

approximately 1 min, after which mice were freed from restraint and returned to the home cage.

Body Temperature Measurement

129S1 mice were assayed for temperature by rectal probe. C57 mice were microchipped

subcutaneously with implantable electronic transponders (Bio Medic Data Systems, Inc.,

Seaford, DE).

Hormonal Assessments

During the eleventh week of the study (see Appendix A, Fig. A-2) mice were fasted

overnight (16h) and blood was collected without anesthesia by retro-orbital puncture. After

centrifugation sera were stored at -800 C until assay.

Leptin

Measurements were taken with the Luminex-based bead array method using the

LINCOplex simultaneous multianalyte detection system (Linco Research, Inc., St. Charles, MO)

following the manufacturer's instructions.

Adiponectin

Adiponectin levels were assayed using the mouse ELISA kit from Alpco Diagnostics

(Salem, NH) following the manufacturer's protocol except sera were diluted 1:40,000 (instead of

1:20,000) prior to assay.

Glucose Homeostasis

Plasma samples for glucose and insulin quantification were collected simultaneously by

retro-orbital puncture from animals without the use of anesthesia. The baseline blood draw was

taken from animals that had been fasted overnight (16h).









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF SATIETY SIGNALING IN THE BENEFICIAL EFFECTS OF CALORIE
RESTRICTION IN MICE

By

Robin Kaye Minor

December 2007

Chair: Susan S. Percival
Major: Food Science and Human Nutrition

Laboratory studies consistently demonstrate extended lifespan in animals on calorie

restriction (CR), where total caloric intake is reduced by 10-40% but adequate nutrition is

otherwise maintained. CR has been further shown to delay the onset and severity of chronic

diseases associated with aging such as cancer, and to extend the functional health span of

important faculties like cognition. Less understood are the underlying mechanisms through

which CR might act to induce such alterations. One theory postulates that CR's beneficial effects

are intimately tied to the neuroendocrine response to low energy availability, of which the

arcuate nucleus in the hypothalamus plays a pivotal role. Neuropeptide Y (NPY), a

neurotransmitter in the front line of the arcuate response to low energy availability, is the

primary hunger signal affected by CR.

It was hypothesized that the arcuate nucleus and NPY are critical not only for certain key

physiological alterations, but also for increased stress resistance, decreased cancer risk and

enhanced cognition noted with CR. These hypotheses were tested using two mouse models-one

chemically treated to impair arcuate function and another genetically modified to lack NPY-

maintained on CR or unlimited feeding.









the reductions in thermogenesis and body temperature that accompany CR (Kotz et al., 1998).

Most importantly, transgenic rats that overexpress NPY have been found to have improved stress

resistance as demonstrated through reduced blood pressure in response to novelty stress and

increased mean (but not maximum) lifespan (Michalkiewicz et al., 2003). Lifespan benefits with

increased NPY may be reflected in humans as well, as long-lived female centenarians have high

plasma NPY levels compared with younger women (Baranowska et al., 2006).

Because CR is so successful in multiple species there is increasing interest in the

therapeutic potential for CR to extend maximum lifespan in humans (Ingram, 2006). In fact, the

prospect of CR in humans is already a reality and there are societies, books and internet sites

devoted to CR in humans (see http://www.calorierestriction.org/). Despite the willful adherence

of this CRonie minority, the current hunch of gerontologists is that most humans will prefer not

to regimen their diet in the presence of an abundant food supply so long as alternatives, or

mimetics, to CR may be found. Indeed, candidate CR mimetics are already under investigation

(Ingram, 2006) with the hopes of rendering CR's benefits attainable to the overeating masses.

This movement presupposes one critical notion: that CR's beneficial effects can be separated

from those imposed on appetite. Taking into account the profound effects of CR on the appetite-

regulating machinery of the hypothalamus and NPY, the key central hunger signal that may play

a critical role in transducing CR's benefits, it may be that the very effectiveness of CR stems

from the same hunger-inducing phenomena that CR mimetic research seeks to repress.

Overall Rationale

It is well established that CR promotes beneficial adaptations to laboratory animals that

slow the aging process. Such changes include reduced rates of cancer, improved glucose

homeostasis, enhanced oxidative stress resistance, and promotion of cognitive capabilities. The

involvement of neuroendocrinological mechanisms in mediating these effects-and in particular









In the broadest terms, NPY's central actions include stimulating hunger, fat storage and

weight gain (Beck et al., 1992; Stanley et al., 1986; Zarjevski et al., 1993) while decreasing sex

drive, locomotion, energy expenditure and body temperature (Billington et al., 1991; Hwa et al.,

1999; Kulkosky et al., 1988; Lopez-Valpuesta et al., 1996; Menendez et al., 1990). These effects

contrast the actions of NPY in the periphery where stress is a major factor influencing the release

ofNPY (Bernet et al., 1998; Han et al., 2005; Kuo et al., 2007b) and the consequences are

strongly registered by the adrenal and cardiovascular systems. It has been hypothesized that in

the current Western lifestyle-which antagonizes central NPY through leptin resistance and

peripheral NPY through stress-NPY could contribute to a number of diseases such as

hypertension, diabetes, and obesity where calories are in plentiful supply (Chronwall and

Zukowska, 2004; Kuo et al., 2007b).

Neuropeptide Y in feeding behavior

Within two years following its discovery NPY became well known for inducing a robust

feeding response in rats (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz,

1984), and to this day NPY is the most potent endogenous orexigenic stimulant known

(Chamorro et al., 2002; Edwards et al., 1999). The feeding effect of NPY appears to be well

conserved, as central injection induces food intake in a wide range of species as depicted in

Table 1-2. The only known exception is the baboon, in which intracerebroventricular injection of

NPY did not induce feeding (Sipols et al., 1996).









CHAPTER 1
LITERATURE REVIEW

Appetite

Deprived of food, an organism becomes hungry. As seemingly straightforward as this

relationship appears externally, internally it is mediated by a decidedly complex interaction of

numerous alterations both psychological and physiological. For example, hunger may be

described in behavioral terms as the state in which an organism is motivated to eat. The feeding

response, the most overt outcome subsequent to negative energy balance, belies numerous

physiological and neurophysiological alterations imperceptible to the naked eye: neural signaling

patterns, body temperature, metabolism, and various blood-borne hormones and metabolites are

just a few of the many processes responsive to hunger and satiety in animals. As an organism

shifts from a fed to a fasted state, its empty gut ceases signaling fullness and commences to call

for refeeding. Circulating metabolites shift from energetic macronutrients to byproducts of

metabolism which trigger a compensatory adjustment in pancreatic exocrine output. The sum of

these signals among others is tallied by the brain, where the peripheral call to eat is put into

action.

Central recipients of the peripheral messages relaying the status of satiety are primarily the

hypothalamus and the brainstem (Berthoud, 2004). The brainstem functions in the control of

autonomous feeding behavior via the caudal nucleus of the solitary tract (NTS). Gastrointestinal,

circulatory and central cues all reach the NTS and influence the determination of meal size

(Schwartz, 2006). The NTS alone, however, has been shown to be insufficient for a full response

to long-term food deprivation (Seeley et al., 1994) as this requires the action of another CNS

satiety center: the hypothalamus. This critical difference relates to the primary signal input for

these brain regions; the NTS acts as the main port of entry for gastrointestinal signals while the









5-4 Detection of changes in surface hydrophobicity in proteins................... ................85

A-1 Representative mice of the genotype and treatment groups. .............................................92

A-2 Representative hypothalamic brain sections stained for NPY................ ..................94

A-3 Project design for the three major studies and other periodic assessments ..................96

A-4 Food consumption and body weight ............ .... ........ ....................... 98

A-5 Feeding rate am ong the CR groups...................... ......... .......................... ............... 99









which has been found to substitute in part for NPY activity in MSG-treated rats (Moreno et al.,

2005).

Another limit in the methodology of the studies was the use of young instead of aged mice.

This led to the inability to assess the prevention of cognitive decline seen with CR (Ingram et al.,

1987; Means et al., 1993) and therefore to fully assess the impact of impaired neuroendocrine

function on this effect. This same issue may have contributed to lack of differences seen in

antioxidant protein expression in chapter 4, as the attenuation in age-related pro-oxidant changes

associated with CR (Seo et al., 2006) would not yet be apparent in mice aged 4-6 months.

One question that remains unresolved from the current studies is whether lifespan

extension could be obtained with CR in these models of altered hunger signaling. Extrapolating

from our results, lifespan extension would be expected to be reduced as the mice displayed

diminished improvements to glucose homeostasis, reduced resistance to oxidative stress and

increased tumorigenicity-all conditions associated with reduced longevity (Finkel et al., 2007;

Kenyon, 2005). Lifespan data are still needed to evaluate this hypothesis.

In conclusion, the current project showed measures of physical performance and body

composition are improved by CR even without full neuroendocrine competency whereas

downstream effects of CR on physiological systems are more sensitive to neuroendocrine status.

The models employed in these studies are well-suited for research designed to test the

significance of hunger to diet-induced physiological changes and lifespan. The aim of research to

find agents that mimic CR may be unsuccessful in their effort to sidestep hunger as the current

results indicate hunger is a critical component in the relay of CR's beneficial effects through the

neuroendocrine system.









Of the B6 groups, MSG AL mice showed significantly higher vacuole content after the 5h

treatment than SAL AL mice (119.1 + 19.4 versus 35.7 9.3 i3; p < 0.05) (Fig. 4-3c). MSG CR

mouse livers contained significantly less microvesicle load their MSG AL counterparts (21.4 +

11.0 versus 119.1 + 19.4 23;p < 0.05).

Expression of Stress Proteins and Antioxidants

Protein expression in the 129S1 mice is compared with expression levels in two NPY-

saline-injected control mice (for which the bands are not shown) and the results are depicted in

Figure 4-4. SIRT1 and SOD2 expression showed no difference at 24h post-injection in any

groups (Fig. 4-4a & e, respectively) as with glyceraldehyde 3 phosphate dehydrogenase

(GAPDH) used as a loading control (Fig. 4-4g). HSP70, however, is significantly increased in

the CR groups (111.0 + 9.5 versus 58.0 + 9.3 % in NPYt+; 102.7 8.3 versus 53.0 4.3 % in

NPY/ ; p < 0.05) (Fig. 4-4c).

Protein expression in the B6 mice is compared with expression levels in a SAL saline-

injected control mouse (not shown). No significant differences were observed in SIRT1

expression between the groups (Fig. 4-4b). HSP70 was significantly increased in MSG CR mice

compared with MSG AL mice (90.0 10.2 versus 67.5 3.5 %;p < 0.05) (Fig. 4-4d), and SOD2

was significantly higher in the MSG groups compared with their SAL counterparts (159.9 2.7

versus 90.3 14.3 % in AL; 186.2 11.4 versus 102.6 7.1 % in CR; p < 0.05) (Fig. 4-4f).

Protein loading in the MSG mice was not equivalent by GAPDH expression as the MSG CR

level was lower than in AL mice (98.3 2.6 versus 116.8 4.7 %; p < 0.05) (Fig. 4-4h). This

would not affect the increase in MSG CR HSP70 expression but may mask an increase in SIRT1

or SOD2 in these mice.









ROLE OF SATIETY SIGNALING IN THE BENEFICIAL EFFECTS OF CALORIE
RESTRICTION IN MICE





















By

ROBIN KAYE MINOR


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007









Fasting blood glucose

Glucose was assayed at the time of blood draw by applying 5 pL whole blood to an Elite

XL Glucometer (Bayer, Elkhart, IN).

Oral glucose tolerance test

Following the baseline blood draw, mice were gavaged with a 1.5 g/kg glucose solution.

Blood glucose was then assayed as above at 15, 30, 60 and 120 min post-gavage.

Insulin measurement

Blood samples collected during the OGTT were centrifuged and the sera stored at -800 C

until assay. Insulin levels were determined using a commercially-available ELISA kit (Crystal

Chem Inc., Downers Grove, IL). The manufacturer's instructions were followed with the

exception that the volume of serum used was increased to 15 pl (as opposed to 5 pl) per sample.

HOMA calculations

Insulin resistance was determined by the Homeostasis Model Assessment (HOMA) index,

calculated from the fasting blood glucose and insulin values by the HOMA2 Calculator software

available from the Oxford Center for Diabetes, Endocrinology and Metabolism, Diabetes Trials

Unit (http://www.dtu.ox.ac.uk/homa/).

Statistics

Data are expressed as the mean + S.E.M. Statistical analyses were carried out by ANOVA

with repeated measures (body composition) or one-way ANOVA (fasting blood glucose)

followed by Fisher's LSD tests. Statistical significance was inferred atp < 0.05.

Results

Body Composition

Results from the NMR body composition analyses are presented in Table 3-1. Mice from

the 129S1 background started the study at equivalent body composition independent of









groups. As expected, NQO1 activity is increased by CR in the B6 SAL mice. Unexpectedly,

NQO1 activity appears to be constitutively upregulated in the MSG mice as with SOD2 protein

expression, and presumably the same unknown causative factors are responsible.

This study demonstrated some of the protective effects of CR on oxidative stress response

in the liver. Survivorship to oxidative insult is impaired in models of altered neuroendocrine

signaling, suggesting a role for the hypothalamus in the regulation of systemic stress resistance.

CR in NPY-/ mice was found to be anti-protective to survival without apparent impairment of

antioxidant systems. Hypothalamic lesioning by MSG markedly reduced survivorship in AL-fed

mice which correlated with reduced HSP70 expression and extensive accumulation of

intrahepatic microvesicles and CR was able to reverse these effects. Future studies are needed to

characterize the nature of the weakness observed in the NPY- mice in response to oxidative

stress.









Verification of NPY Expression Levels

Immunohistochemistry was employed to verify reduced NPY expression in the NPY- and

MSG mice. Brains were obtained from the mice undergoing the diquat experiment (see Fig. A-

3). Only brains from AL-fed mice were used, however these mice had been fasted overnight

prior to diquat injection. Brains were excised and fixed by immersion in Streck tissue fixative

(Streck Laboratories, Omaha, NE). Prior to sectioning brains were cryoprotected in a 30%

sucrose solution and frozen in liquid nitrogen. Coronal sections (50pm) were cut on a freezing

microtome (Microm HM 400; Microme, Walldorf, Germany) and washed (washing consisted of

3 changes with 0.1 mM phosphate-buffered saline (PBS) for 10 min each). Endogenous

peroxidase activity was then quenched by incubation with 1% hydrogen peroxide/PBS for 30

min. After washing the sections were blocked in 10% normal goat serum for 30 min, followed by

incubation with the primary anti-NPY antibody (1:1000 dilution; Abcam, Cambridge, MA) at

4C overnight. Following washing the sections were incubated at room temperature with a series

of solutions purchased as part of the Vectastain kit from Vector Laboratories (Burlingame, CA):

a goat anti-rabbit biotinylated antibody (1:400 dilution) for 2 h, an avidin-biotin complex

solution for 2 h, and finally diaminobenzidine (DAB) until a strong color reaction was observed.

The stained sections were washed, mounted on slides, dried thoroughly and coverslipped before

microscopy. The results are shown in Figure A-2. As expected, staining for NPY is robust in

NPY/+ and SAL mice, greatly reduced in MSG mice and absent from NPY- mice.









In the current study, no differences were observed in the NPY- mice in the water maze during

training or the probe trial although there was a nonsignificant trend towards better performance

in the NPY~ mice, which contrasts with previous work in which these mice demonstrated a slight

impairment in water maze acquisition (Palmiter et al., 1998). There was no discernable effect

due to diet treatment. In the B6 mice, however, MSG treatment precluded the mice from learning

the task-while the SAL mice improved continuously over the training period and actively swam

in the correct quadrant during the probe trial, the MSG mice performed nearly equivalently every

day of training and swam indiscriminately through all quadrants during the probe trial.

Deficits in spatial learning in the water maze have been observed in MSG-injected rats

(Olvera-Cortes et al., 2005; Saari et al., 1990) and have been attributed to cognitive and visual

defects. To assess the visual ability of our mice, we performed a visual cue test following the

probe trial in the water maze and also a vision-dependent fear conditioning trial. During the

visual cue test, MSG mice swam significantly longer before locating the escape platform,

suggestive of visual impairment. Thus, the deficient performance of the MSG mice in all phases

of water maze testing suggests cognitive defects cannot solely account for the effect but may also

involve defective visual processing. To further assess the visual ability of the mice, we then

employed a modified version of fear conditioning. One study evaluating NIH Swiss and Black

Swiss mice-which are visually impaired due to genetic retinal degeneration-found these mice

were deficient in both the water maze and contextual fear conditioning (Clapcote et al., 2005). In

the present study, the MSG mice were equivalently competent to SAL mice in the fear

conditioning experiment in discerning the visually-cued environment during testing. These data

suggest MSG-induced water maze deficits may be less related to vision than has been previously

thought, and indeed a study with MSG-treated mice in a non-spatial water maze task showed













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200

150

100

50

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Figure 4-4. Liver antioxidant and stress-related protein expression after diquat treatment. (Mean
+ S.E.M., = significantly different from AL; t = significantly different from SAL; n
= 6 except MSG AL n = 5.)


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258. Park, C.H., Choi, S.H., Piao, Y., Kim, S., Lee, Y.J., Kim, H.S., Jeong, S.J., Rah, J.C., Seo,
J.H., Lee, J.H., Chang, K., Jung, Y.J., Suh, Y.H., 2000. Glutamate and aspartate impair
memory retention and damage hypothalamic neurons in adult mice. Toxicol. Lett. 115,
117-125.

259. Parker, R.M., Herzog, H., 1999. Regional distribution of Y-receptor subtype mRNAs in rat
brain. Eur. J. Neurosci. 11, 1431-1448.

260. Parrott, R.F., Heavens, R.P., Baldwin, B.A., 1986. Stimulation of feeding in the satiated
pig by intracerebroventricular injection of neuropeptide Y. Physiol. Behav. 36, 523-525.

261. Pau, M.Y., Pau, K.Y., Spies, H.G., 1988. Characterization of central actions of
neuropeptide Y on food and water intake in rabbits. Physiol. Behav. 44, 797-802.










NQO1 Activity

No significant differences were found among the 129S1 groups in NQO1 activity (Fig. 4-

5a), but among the B6 groups the MSG AL mice showed greater activity than SAL AL mice

(87.5 9.3 versus 35.5 6.3 nmol DCIP/min/tg; p < 0.05) (Fig. 4-5b). SAL CR mice also had

increased NQO1 activity relative to the SAL AL mice (84.4 + 23.8 versus 35.5 + 6.3 nmol

DCIP/min/lg; p < 0.05).


129S1 [ B6
160 160
E 140 E 140
o 120 o 120
S100 E100 t
S80 n 80
60 60
40 40

0 0





Figure 4-5. NQO1 activity in the liver after diquat treatment. NQO1 activity was not affected by
diet or genotype in the 129S1 mice (A). In the B6 groups, MSG AL NQO1 activity
was significantly elevated compared with SAL AL activity and CR increased SAL
NQO1 activity but not MSG NQO1 activity (B). (Mean S.E.M., = significantly
different from AL; f = significantly different from SAL; n = 6 except MSG AL n =
5.)

Discussion

This study was performed to determine the role of the ARC and NPY in the effects of CR

on oxidative stress resistance. The strongest indicator of hardiness to any stress must be

survivorship, and in this case the models of impaired neuroendocrine signaling fared less well

than their intact counterparts. Of the NPY~ CR mice, 2 of 6 mice were unable to survive 24h

after injection. These deaths occurred between 20 to 24h post-injection. MSG AL mice fared the

worst of all groups with 3 of 5 mice unable to survive 5h. Originally it was planned to extend the









M orris W after M aze ....................................... ........ .... ............ ............... .. 37
V isual Platform Training ......... ................................. ....................... ............... 38
C ontextual F ear C conditioning .............................................................. .....................38
S statistic s ......... .......................................................................................... . 3 9
Results ...............................................................39
B o dy W eig hts ................................................................3 9
P hy sical A gility .............................................................4 0
Open Field Locom otor A activity .............................................. ............... 42
Morris Water Maze Performance ................................................43
V isu al A sse ssm en t..................................................................................................... 4 4
C ontextual F ear C conditioning ................................................................................... 46
D isc u ssio n ........................ .......................................................................................................4 6

3 REDUCTIONS IN ADIPOSITY AND LEPTIN FOLLOWING CALORIE
RESTRICTION DO NOT fULLY EXTEND TO IMPROVED GLUCOSE
HOMEOSTASIS IN MODELS OF IMPAIRED NEUROENDOCRINOLOGICAL
S IG N A L IN G ..................................................................................................................... 5 1

M materials an d M eth o d s ...........................................................................................................52
A n im als an d D iets ..................................................................................................... 5 2
Body Com position A nalysis................................................... 52
Body Temperature Measurement ................................................53
H orm on al A ssessm ents ............................................................................................. 53
L e p tin ...............................................................................5 3
A diponectin .................................................................... 53
G lu co se H o m eo stasis................................................................................................. 5 3
Fasting blood glucose .................... ............ ... ............... 54
O ral glu cose tolerance test ................................................................................. 54
Insulin m easurem ent ............................................................54
H OM A calculations............................................ 54
Statistics ......... ........... ............................... ...............54
Results ............. ..... ..... ................................ 54
B o d y C o m p o sitio n ..................................................................................................... 5 4
Body Temperature ............... .. ......... ..................57
H orm on al A ssessm ents ............................................................................................. 57
G lu co se H o m eo stasis................................................................................................. 5 8
Fasting blood glucose ............... ......... ......... .........58
O ral glu cose tolerance test ................................................................................. 59
Assessment of insulin resistance ............................................. ..... 62
D iscu ssio n ......... ........... .................................... ..................................6 2

4 DIVERGENT EFFECTS OF CALORIE RESTRICTION ON OXIDATIVE STRESS
RESISTANCE AND ANTIOXIDANT PROTECTION IN TWO MOUSE MODELS
OF IMPAIRED NEUROENDOCRINE SIGNALING .............. ......................................66

M materials an d M eth o d s ...........................................................................................................6 7
A n im als an d D iets ..................................................................................................... 6 7


6









treatment of the B6 to 24h as with the 129S1 mice, but the experiment was cut short due to these

deaths. B6 mice are generally competent to survive diquat up to 50 mg/kg (Lei, 2001) and the

extreme susceptibility of the MSG mice to the treatment suggests these mice may be deficient in

antioxidant defense systems.

Histological analysis of the mouse livers revealed a striking presence of periportal

microvesicles particularly in the AL groups. It is known that drugs can cause acute

microvacuolar fatty change in the liver, with consequences including liver failure and a high

mortality rate (Oliveira et al., 2002). The mechanism by which this is thought to occur involves

stress-induced production of cytokines such as TNF-a that would impair mitochondrial P-

oxidation of fatty acids which would lead to a buildup of fatty acids in the liver and exacerbate

reactive oxygen species (ROS) production (Oliveira et al., 2002; Yang et al., 2000). ROS-

induced lipid peroxidation follows which activates an inflammatory response as well (Lee et al.,

1995). Because the MSG-treated mice in this study were obesity-prone, as shown in chapter 3,

and the saline-injected control liver section in Figure 4-2 indicates moderate liver steatosis prior

to diquat injection, additional fat accumulation in the livers of the MSG AL mice may have

hastened the fatalities in this group. Reduced liver steatosis may have been one protective

mechanism in the MSG CR mice that allowed them to survive the treatment more successfully.

Indeed, quantification of the intrahepatic microvesicle response to the diquat treatment revealed

a strikingly high presence in the MSG AL mice which may account for their high mortality rate.

Given that ROS protection is a key benefit associated with CR, levels of antioxidant and

stress-related protein profiles were assessed in the mouse livers. There were no statistical

differences in SIRT1 levels in either mouse strain. While the CR groups showed a trend to

greater expression, as would be expected (Cohen et al., 2004), the sample size may have been too











0.05; Student-Newman-Keuls test) showed that MSG mice, regardless of diet, showed


significantly longer latencies to find the cued platform as compared with SAL mice (Fig. 5b).


129S1 Visual Cue


B


E
0 50
4O

4-
40

0 30

U
20
C
) 10
_1


NPY+/+ AL NPY-/ AL NPY+/+ CR


B6 Visual Cue


SAL AL MSG AL SAL CR MSG CR
SALAL MSGAL SALCR MSGCR


10
100 1


B6 Fear Conditioning


SAL AL
m MSG AL


Baseline


Testing


Figure 2-5. Visual assessment via visual cue testing in the water maze and contextual fear
conditioning. 129S1 mice swam to the platform with equivalent acuity (A); whereas
MSG mice, regardless of diet, showed significantly longer latencies to find the cued
platform as compared with SAL mice (B). Another vision-dependent test, contextual
fear conditioning, conversely revealed MSG AL were able to associate the
conditioning chamber with shocks as they froze significantly more during the 5
minute context exposure than during baseline exposure (C). (Mean + S.E.M., n = 10
for panels A and B, n = 5 for panel C.)


60 1


E
0 50

C 40

0
O
220

C-10

_1 0

































To my parents, Nancy and Grady Minor










(Fig. 3-3a). This was not the case in the B6 mice, where CR was able to lower fasting blood

glucose in the SAL CR compared with SAL AL mice (66.6 2.7 versus 107.3 3.8 mg/dl;p <

0.05) but produced no effect in the MSG CR mice (Fig. 3-3b).


S129S1 Fasting Blood Glucose B6 Fasting Blood Glucose
140 140
120 120
S100 100
E 80 E 80 *
S60 60
0 0
40 1 40
20 20
0 0
x 4 4V le V 1 ^



Figure 3-3. Fasting blood glucose was significantly reduced by CR in both NPY groups
regardless of genotype (A). In the B6 mice, only SAL CR mice saw a significant
reduction in fasting glucose (B) (Mean S.E.M., = significantly different from AL;
n =6.)

Oral glucose tolerance test

Overall blood glucose response to the OGTT, as determined by area under the curve

(AUC) comparison computed from the glucose response for the two hours following glucose

gavage, was improved by CR in the NPY/+ mice relative to NPY/+ AL (31,042 2,062 versus

39,305 2,047 mg/dl; p = 0.03) (Fig. 3-4c). CR significantly improved glucose response in both

B6 CR groups (28,555 2,642 versus 45,643 2,116 mg/dl in SAL; 23,471 + 1,740 versus

49,982 4,001 mg/dl in MSG; p < 0.05) (Fig. 3-4d).









compared with AL controls accounts for some of the enhanced motor capability, as all CR mice

either lost weight or were prevented from weight gain during the feeding period. By NMR

assessment we also know that CR prevented a decrease in lean-to-fat ratio in the mice (see Table

3-1), which could account for some of the increased performance in the CR mice. NPY does not

appear to be necessary for the enhanced performance associated with CR as NPY-- CR mice

improved equivalently to NPY'+ CR mice on the rotarod. There is an apparent effect of ARC

integrity on fitness, however, as SAL mice displayed greater endurance on the rotarod than

lesioned MSG mice regardless of diet. Rather than a direct influence of the ARC on motor

ability, this effect may again be attributed to differences in body composition; NMR analysis

showed MSG mice had greater fat mass than SAL mice in their respective diet treatments (Table

3-1).

By measure of total distance traveled, open field behavior of the mice in this study does

not appear to depend much on ARC function or diet treatment. Prior to testing we had

hypothesized CR may increase locomotor activity in the control mice on an open field as a

manifestation of increased drive to forage (Holloszy and Schechtman, 1991; Ingram et al., 1987;

Weed et al., 1997). CR is known to induce NPY expression in the ARC (Bi et al., 2003; Brady et

al., 1990; de Rijke et al., 2005; Kim et al., 1988; Mercer et al., 2001; Widdowson et al., 1997),

and previous work has shown that centrally-injected NPY promotes open field locomotor activity

in mice (Karlsson et al., 2005; Nakajima et al., 1994). In the present study, however, NPY/- CR

mice traveled farthest. Also contrary to the present findings, previous studies have shown that

both mice and rats exposed to MSG were hyperactive in an open field (Saari et al., 1990; Yu et

al., 2006), although differences in the dosing schedule may account in part for this discrepancy.









Distinct neuroendocrine adaptations are also noted with MSG treatment where

hypothalamic function is concerned (Broberger et al., 1998; Meister et al., 1989). It is recognized

that this neurotoxic compound mainly affects ARC neuron cell bodies (Burde et al., 1971;

Cameron et al., 1978; Hu et al., 1998; Seress, 1982), owing to blood-brain barrier permeability at

the median eminence (Peruzzo et al., 2000). Within the ARC, more than 80-90% of the neurons

are eliminated, resulting in ARC shrinkage, widening of the third ventricle, thinning of the

median eminence and marked reorganization among remaining neurons (Broberger et al., 1998,

Elefteriou et al., 2003). Loss of ARC NPY expression has been the most studied of the

orexigenic peptide alterations with MSG treatment (Abe et al., 1990; Broberger et al., 1998;

Kerkerian and Pelletier, 1986; Legradi and Lechan, 1998; Meister et al., 1989; Perell6 et al.,

2004). Since ARC NPY is coexpressed with another orexigenic peptide, AgRP, (Shutter et al.,

1997), MSG treatment would be expected to lower expression of both of these neurotransmitters.

Indeed, erasure of hypothalamic NPY / AgRP neurons by MSG reflects in reduction of both

neuropeptides (Broberger et al., 1998; Tamura et al., 2002).

Mice

Representative mice from the mouse strains used in these studies are pictured in Figure A-

1. Male 129S1 wild-type (NPY /+) controls (129S1/SvImJ) were purchased from the Jackson

Laboratory (Bar Harbor, ME). The mice were 2 months of age upon arrival at the Holabird

Research Facility (Baltimore, MD), where they were acclimated for 1 month before the study

diets were initiated. Male 129S1 NPY-knockout (NPY -) mice were bred at the National Institute

on Aging Gerontology Research Center (Baltimore, MD) from breeder mice obtained from the

Jackson Laboratory (129S-NpytmlRpa/J). Male mice aged 2-4 months were brought to the

Holabird Research Facility where they were acclimated for 1 month before the study diets were

initiated.









Contextual Fear Conditioning

Percent time freezing for B6 AL mice during testing was significantly higher (F(1,8) =

13.046; p = 0.007) than during baseline exposure (i.e. prior to tone-shock trials) to the

conditioning chamber (Fig. 2-5c). Post hoc analysis showed both SAL (p < 0.05; Student-

Newman-Keuls test) and MSG (p < 0.05; Student-Newman-Keuls test) groups spent

significantly more time freezing during testing than during baseline.

Discussion

The present study was designed to assess whether impairment of neuroendocrinological

function would alter the behavioral response to CR in two mouse models. In measures of

physical agility, the mice performed better after CR than AL feeding. The inclined screen, which

is constructed from a wire mesh the mice must grip and hold to traverse, assesses both locomotor

behavior and grip strength. Thus, the greater distance traveled in the B6 CR mice on the inclined

screen is corroborated by previous data showing CR improved ability to hang from a bar and

resist slipping on a tilted platform (Ishihara et al., 2005). The rotarod results also agree with a

previous study in which CR improved rotarod performance in a preclinical mouse model for

amyotrophic lateral sclerosis (Hamadeh et al., 2005) and support the inclined screen data. As the

inclined screen and rotarod are both tests of general physical ability, it is not surprising that the

results of the tests are similar. However, because the trend to improved performance by CR in

the B6 mice is more pronounced on the rotarod than on the inclined screen and a significant

improvement is seen in the 129S1 mice exclusively on the rotarod, our results suggest the rotarod

is a more sensitive task for evaluating diet-induced physical fitness.

Unlike diet, which had a significant effect on motor performance in this study in both the

NPY and the arcuate mouse models, altered neuroendocrine function asserted less of an effect on

measures of physical ability. This suggests that improved body composition in CR animals











+ 10.8 versus 160.7 5.7 pmol/ml;p < 0.05) (Fig. 3-2d), and MSG AL mice were significantly


increased compared with SAL AL mice (210.5 9.7 versus 160.7 5.7 pmol/ml;p < 0.05).


129S1 Leptin


B6 Leptin


300

250
E
S200
E
150

. 100
.)
-J
50



x





300

250
E
S200
C
= 150
0)
0 100

S50


Vd


300


V


*

01- 0^-
s" s^'v


250

0 200
E
S 150
C
S100
-J
50

0





D

300

250

S200

* 150

O 100

< 50

0
0-


129S1 Adiponectin




*







,t


~r A'" A


V9L *
e1 O-, l (-
q; ,^


t
T


e V
V'


*


Figure 3-2. Fasted leptin and adiponectin levels. Leptin was significantly reduced in the CR
groups of both the 129S1 mice (A) and B6 mice (B) regardless of genotype or
treatment. Adiponectin was increased only in NPY++ CR mice (C) and SAL CR mice
(D), although adiponectin was significantly higher in MSG AL mice. (Mean
S.E.M., = significantly different from AL; i = significantly different from SAL; n =
6.)

Glucose Homeostasis


Fasting blood glucose

CR resulted in significantly lower fasting blood glucose in both 129S1 genotypes (56.8


5.0 versus 84.3 4.0 mg/dl in NPY ; 56.7 3.0 versus 79.3 3.9 mg/dl in NPY ; p < 0.05)


B6 Adiponectin


c1L"


I


I?-









BIOGRAPHICAL SKETCH

Robin Kaye Minor grew up in Naples, Florida where she graduated from Naples High

School in 1996, culminating a relatively carefree childhood playing classical Spanish guitar and

competing at quarter horse shows. The next four years found her intermittently at the University

of Florida between studies abroad in Ireland, Brazil and Kenya. Despite nearly succumbing to

the particularly virulent strain of malaria (Plasmodiumfalciparum) following the Kenyan

sojourn in the summer of 1999, she received a bachelor of science degree in psychology with a

minor in Portuguese with highest honors the following spring. A year sabbatical from studies

was spent in Houston teaching remedial fractions to middle-schoolers with Teach for America

and then tending bar for Polynesian immigrants in Los Angeles. A desire to understand more

about insects and their nefarious ways drove her to pursue a master of science in entomology at

UF with Dr. Phil Koehler, from which she graduated in 2002. Declining acceptance to medical

school at the University of Miami for a future less prescribed, Robin spent a year exploring the

culinary arts in New York City and cooking for such venerable establishments as Judson Grill

and Blue Hill. Hungry to marry her interests in food and academics, she entered into a Ph.D.

program in the Food Science and Human Nutrition Department at UF in the fall of 2003 under

the supervision of Dr. Susan S. Percival. Her research interest in nutrition and aging led her to an

extended collaboration with Dr. Rafael de Cabo's laboratory at the National Institute on Aging in

Baltimore, MD, where she collected the data for this dissertation as an NIA Pre-Doctoral Fellow.

In the future Robin will pursue a post-doctoral fellowship at the NIA, collect more degrees, and

continue cultivating a life worth living.









immediately by a foot shock (1 s, 1 mA) through the floor with a 60 second inter-trial interval.

Sixty seconds after the final trial, each mouse was returned to its home cage. The chambers were

cleaned with 70% ethanol between each mouse. Twenty-four hours later, the mice were returned

to the conditioning chamber for 5 minutes. The Video Fear Conditioning System (MED-VFC,

Med Associates) was used to measure freezing behavior, which was considered to be the amount

of time a mouse spent motionless based on an index of motion. From these data a percent-time

freezing score was then calculated for each mouse.

Statistics

For the inclined screen, rotarod and open field data, a two-way analysis of variance

(ANOVA) was used with Student-Newman-Keuls tests performed post hoc. For the water maze

data, a two-way or a two-way with repeated measures ANOVA followed by a Student-Newman-

Keuls test was employed. An ANOVA with repeated measures and Student-Newman-Keuls tests

were used for the contextual fear conditioning data.

Results

Body Weights

Mean cumulative weight change prior to behavioral testing is depicted in Fig. 1. The

129S1 mice (Fig. 2-la) gained body weight on AL feeding and lost weight on CR feeding in both

genotypes. All B6 AL mice gained weight over the feeding period, and MSG AL mice, though

starting at a lower weight, gained weight rapidly to match SAL AL average weight by week 2

(Fig. 2-lb). SAL CR mice lost weight over time but MSG CR mice were lighter at study start

and maintained their initial weight throughout the feeding period.









fact, removal of the pituitary gland with corticosterone replacement leads to lifespan extension in

rats similar to that seen with CR (Everitt et al., 1980).

Neuropeptide Y: How and Y calorie restriction extends lifespan?

It is well known that CR raises ARC NPY levels (Bi et al., 2003; Brady et al., 1990; de

Rijke et al., 2005; Kim et al., 1988; Mercer et al., 2001; Widdowson et al., 1997). Similarly,

methionine restriction extends lifespan (Miller et al., 2005; Orentreich et al., 1993) with a

concomitant increase in hypothalamic NPY (White et al., 1994). Whether the increased NPY is a

necessary precursor to the functional benefits associated with dietary restriction is not known,

but considering NPY's unique long-term response to CR compared with other neuropeptides (Bi

et al., 2003) and its plethora of physiological actions, a causal relationship is certainly plausible.

One way CR may act to extend lifespan through NPY is by prolonging youthful expression

levels of NPY. Aging is associated with reduced levels of NPY in the brain in general

(Gruenewald et al., 1994; Higuchi et al., 1988a; Sohn et al., 2002; Vela et al., 2003) and in

response to fasting (Gruenewald et al., 1996). Reduced NPY has been associated with

Alzheimer's disease (Alom et al., 1995; Edvinsson et al., 1993) and the development of a

condition termed 'anorexia of aging', thought to be responsible for aging-associated

undernutrition and consequent physical deterioration such as osteoporosis, sarcopenia, impaired

immunity and parenchymatous organ failure (Matsumoto et al., 2000; Morley, 2001). Evidence

from the rat showing NPY loss with age is progressive and independent of testosterone levels has

been interpreted to suggest an active role for NPY in the anorexia of aging (Gruenewald et al.,

1994).

CR-driven changes in NPY expression may have downstream consequences that lead to

lifespan extension. For example, NPY increases corticosterone levels, commonly observed in

rodents on CR (Leibowitz et al., 1988; Wahlestedt et al., 1987). NPY is also thought to mediate










A 129S1 Body Weights B6 Body Weights
40 40




>3 >
W 20 20 -

NPY'/+ AL 0 SAL AL
C 10 --- NPY/ AL C 10 -- MSGAL
NPY+/+ --*- SALCR
NPY CR MSG
-- NPY' CR
0 -I 1, 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Weeks Weeks

Figure 2-1. Mean group body weight + S.E.M. for the six weeks prior to behavioral testing in the
129S1 mice (A) and B6 mice (B). (n = 10)

Physical Agility

Performance on the inclined screen did not differ significantly by path length for the 129S1

mice by either diet or NPY expression (Fig. 2-2a). Within the B6 groups, a two-way ANOVA

revealed significant effects of treatment (F(1,36) = 5.36; p = 0.026) and diet (F(1, 36) = 13.92; p

< 0.001) but no significant interaction between treatment and diet on the path length of the

inclined screen. Post hoc analysis (p < 0.05; Student-Newman-Keuls test) revealed SAL and

MSG CR mice traveled significantly farther than their respective AL groups (Fig. 2-2b).

A two-way ANOVA (F(1,36) = 12.14; p = 0.001) revealed a significant effect of diet

among the 129S1 mice on the latency to fall from the rotarod (Fig. 2-2c). Post hoc analysis (p <

0.05; Student-Newman-Keuls test) showed that only the NPY CR mice took significantly

longer to fall compared with NPY / AL mice. Within the B6 groups, a two-way ANOVA

revealed significant effects of treatment (F(1,36) = 14.43; p < 0.001) and diet (F(1,36) = 50.52; p

< 0.001) but no significant interaction between treatment and diet on the latency to fall from the

rotarod. Post hoc analysis (p < 0.05; Student-Newman-Keuls test) showed that CR also









greater escape latencies in MSG mice than controls (Wong et al., 1997). MSG-induced

neurotoxicity beyond the ARC, e.g. in the hippocampus, has also been nominated as a causative

factor in cognitive deficits associated with the treatment (Olvera-Cortes et al., 2005; Wong et al.,

1997). If hippocampal deficits are causal in the present study, the results from our mice lend

further evidence for the dissociation between fear conditioning and spatial learning in the

hippocampus (Bannerman et al., 2004; Maren et al., 1998).

Regardless of the competency of the ARC-lesioned mice in contextual fear conditioning, in

this study CR for 6 weeks was insufficient to boost water maze performance in these mice. Like

environmental enrichment, which has been successfully employed following MSG exposure to

improve rat performance in the water maze (Saari et al., 1990), CR is thought to promote

neurogenesis and synaptic plasticity and thus restore function subsequent to injury (Mattson et

al., 2003). Our lack of benefits may indicate the mice were not on the diets sufficiently long

enough for significant changes in neurogenesis or neuroplasticity to occur. Alternatively,

because the level of CR (30% of AL intake by weight) used in this study was not enough to

induce weight loss in the MSG CR mice relative to baseline it may also have been insufficient to

influence neuroprotective mechanisms.

In conclusion, our results indicate measures of physical performance are more affected by

diet than ARC or NPY signaling while anxiety and cognition are less influenced by diet than

ARC and NPY signaling. Despite established effects of CR on ARC signaling our results suggest

a mechanistic separation between the two where behavior is concerned.









Analysis of open field behavior in terms of spatial preference revealed a strong preference

for the periphery of the field in the models of impaired ARC function. Persistence in peripheral

tracking is known as thigmotaxis and is an index of anxiety in rodents (Simon et al., 1994). NPY

is a potent regulator of stress and anxiety (Heilig, 2004), such that increased central NPY has

anxiolytic effects (Heilig et al., 1989; Karlsson et al., 2005; Kask et al., 1998; Thorsell et al.,

2000) whereas reduced central NPY is anxiogenic (Bannon et al., 2000; Palmiter et al., 1998).

NPY~ mice have previously been shown to demonstrate anxiety through thigmotaxis on an open

field (Bannon et al., 2000). The behavior of the mice in the present study confirms this

phenotype. One neuroendocrine effect of MSG-induced neurotoxicity in mice is a pronounced

reduction in NPY production (Abe et al., 1990; Broberger et al., 1998; Kerkerian and Pelletier,

1986; Legradi and Lechan, 1998; Meister et al., 1989). The MSG mice in this study displayed

increased avoidance of the center field, but this effect was not significant and may reflect the

observation that arcuate lesions induce deficits in multiple neuropeptides in addition to NPY

(Broberger et al., 1998; Meister et al., 1989).

In contrast to the extensive and consistent evidence for NPY in the regulation of anxiety,

the role of NPY in learning and memory-related behaviors is less clear. For example, central

injection of NPY has been shown to enhance memory or induce amnesia depending on the site of

injection (Flood et al., 1989; Flood et al., 1987). Cognitive capability wanes with age (Ingram et

al., 1987; Rapp et al., 1999), as does hypothalamic expression of NPY (Cadiacio et al., 2003;

Higuchi et al., 1988a; Vela et al., 2003), although one study found no correlation between NPY

interneuron loss and spatial deficits (Cadiacio et al., 2003). Adding to the ambiguity, one study

with NPY-overexpressing transgenic rats showed impaired spatial learning (Thorsell et al., 2000)

while another found no significant differences (Carvajal et al., 2004) on the Morris water maze.









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fasting conditions (Bi, 2007). NPY is also found in the peripheral nervous system in sympathetic

nerves (Pemow et al., 1987).

NH2
^ 35 Alpha-helix
25 15


31 20


COOH
1 5 Beta turn

Polyproline helix

Figure 1-3. Neuropeptide Y.

Widespread distribution of NPY continues outside the nervous system. NPY is released

from sympathetic nerves into endocardial endothelial cells (Jacques et al., 2006), the gut (Cox,

2007) and the spleen (Ericsson et al., 1987). Splenic NPY is incorporated by developing blood

cells and ultimately circulates in immune cells and platelets (Ericsson et al., 1987; Kuo et al.,

2007b). NPY is also released into circulation from sympathetic nerves and the adrenal medulla

under stress (Bernet et al., 1998; Han et al., 2005; Kuo et al., 2007b). Recently, adipocytes have

been demonstrated to express NPY where it may play a role in mediating adiposity (Kos et al.,

2007).

Mechanism of action: Signaling through Y receptors

NPY signals through G protein-coupled receptors, seven of which have been named (Y1-

Y7) and five of which have been cloned and described: Y1, Y2, Y4, Y5, and Y6 (Dumont et al.,

1993; Michel et al., 1998). This is not to say that all receptors are relevant to all mammals; the

existence of Y3 has been postulated but not demonstrated (Herzog et al., 1993; Jazin et al., 1993),

Y6 is inactive in primates (Matsumoto et al., 1996), and mammals have lost the Y7 gene















B6


AL



*: .. ." :,..* -. ." .. ; .
.. ; o a . .
3. .. .. ."



.. .- .. .. ** .. :.
3.'"* ', :. *. '. "' "'

-
-- .. 9 : i -


." -' 4 '"9." .
- i '

-. .- *


* .. .



S .. 3 ,. __. 43.. -- ,
: -



"" -" "' o"

a ., 9 ..

". .. 3 ,
o *. *.4
-? i t .




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CR





. :. -.* -, .. .
. *. 4 ."
* .


,-





4. 4. ." ; -
.
SA L : 1. .

t ;.;. .*
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MSG


'--, .. ',' '* "- .- ... o
' "" .. ,.'- ^ / '*. "-

, 4. Jo


~- '' I" ; "', -''.' *-- "-' "'.I
...,.,. *" ., .,y 0. :
....r

,- 1 ct ,-' .S ,, -
:. ; '"_ --^ :.. -* ..
C' -r-
- *1,
..' r- .

43,_...,- ,. -

- .. -:'', .- ,.."*-.
.- % ,. ;, / .-.
* .,--, ..** :,. .' ..' .-' .o .' ,*
* -- -" .. ? "' ." -'
me ^ *" -." .* "* S.. .. q "-/ *


Saline-Injected SAL AL


.* ." a

.-.






' -

S4 .
3 "- .4


.. 0
.* a ,.


Saline-Injected MSG AL



a -


.r... ,
S S ,' 4






*, *- .
9
.



-.

3 *


Figure 4-2. Liver histology of diquat-treated B6 mice (above the line) and saline-treated controls

(below the line).










Body Temperature

As depicted in Figure 3-la, CR lowered the body temperature relative to AL in NPY'+

mice (34.9 0.3 versus 36.7 0.2 C; p < 0.05) but not NPY- mice. In the B6 mice no

differences in body temperature were detected (Fig. 3-1b).


A 129S1 Body Temperature B6 Body Temperature
40 40
39 39
38 38
37 -" U 37
36 36
35 35
34 34
CL33 C 33

ol I I I I I I
31 31
30 30
0 0




Figure 3-1. Body temperature among the groups. Body temperature in the 129S1 mice did not
differ by genotype in the AL groups, but NPY+ CR mice were significantly cooler
than NPY+ AL mice and this drop in temperature was not recapitulated in NPY- CR
mice (A). No differences in temperature were observed in the B6 groups (B). (Mean +
S.E.M., = significantly different from AL; n = 6.)

Hormonal Assessments

CR resulted in significantly lower fasting leptin levels compared with AL in both 129S1

genotypes (16.3 6.7 versus 141.4 19.4 pmol/ml in NPY ; 8.8 5.8 versus 136.5 20.8

pmol/ml in NPY /; p < 0.05) (Fig. 3-2a) and both B6 groups (5.0 + 0.3 versus 132.8 48.4

pmol/ml in SAL; 16.6 2.7 versus 253.4 60.1 pmol/ml in MSG; p < 0.05) (Fig. 3-2b).

Adiponectin was not consistently altered by CR. Only NPY+/+ CR mice showed significantly

higher fasting adiponectin than AL mice (181.8 12.7 versus 139.8 8.4 pmol/ml;p < 0.05)

(Fig. 3-2c). SAL CR mice were also significantly increased compared with SAL AL mice (221.7









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CHAPTER 6
DISCUSSION

This project was conceived to test the relevance of hunger to the physiological effects of

CR. While the neuroendocrine system is popularly hypothesized to figure prominently in the

mediation of CR's effects (Bishop and Guarente, 2007; Lamberts et al., 1997; Meites, 1989;

Nelson et al., 1995; Rehman and Masson, 2001; Speakman and Hambly, 2007), the studies

contained herein mark the first attempt to test the hypothesis from the angle of the hypothalamic

hunger response to CR.

Certainly the salience of the current studies to human health depends on CR's effectiveness

extending beyond laboratory animals. Few studies have evaluated CR in the context of humans,

with the starvation studies by Ancel Keys representing the first to suggest that long-term CR may

confer health benefits to humans by noting food limitations imposed by World War II in Europe

led to a sharp decrease in the incidence of coronary heart disease in the affected populations

(Keys, 1994). More recently, participants in the Biosphere study who were forced to eat a low-

calorie diet over a period of approximately two years exhibited highly significant decreases in

blood pressure, insulin and cholesterol levels (Walford et al., 2002). Another recent study

investigating the feasibility of intermittent fasting in nonobese humans found a significant

decrease in insulin levels after three weeks of intervention (Heilbronn et al., 2005). The impact

CR will have on human lifespan is still open to interpretation, but these studies indicate dietary

restriction can at least offer tangible benefits to healthy-weight individuals.

Increased appetite is perhaps the single most unifying component in dietary restriction

research in all species. Even in the human intermittent fasting study, subjects reported

significantly increased feelings of hunger on fasting days that did not diminish as the study

progressed (Heilbronn et al., 2005). This may become a key obstacle to translating the success of









CHAPTER 5
CALORIE RESTRICTION IS NOT PROTECTIVE AGAINST TUMORIGENICITY IN MICE
WITH IMPAIRED NEUROENDOCRINE SIGNALING

Chapter 4 demonstrated that resistance to oxidative stress is reduced in models of altered

hunger signaling, and that CR does not completely ameliorate this effect. While this oxidative

stress was investigated using the liver toxin diquat, free radicals and oxidative damage to

macromolecules have also been implicated in the pathology of a major age-related disease:

cancer.

Maintenance of DNA stability is a fundamental and continuous challenge faced by every

cell. Genomic instability is generally recognized as a nearly universal feature of both cancer and

aging, fueling efforts to understand these enigmatic processes through their convergence (Finkel

et al., 2007). Cancer and and aging share another important commonality-both can be delayed

by CR. While the lifespan-extending properties of CR were not well appreciated until the 1930s

(McCay et al., 1935; McCay et al., 1939; McCay and Crowell, 1934), observations that CR is

protective against transplanted and induced tumors were first made nearly two decades earlier

(Rous, 1914). Since then, CR has been extensively studied and has consistently shown beneficial

effects on longevity and carcinogenesis across a variety of species (Kritchevsky, 2002;

Weindruch, 1997). As is the case for lifespan extension, the mechanisms behind the protective

effects of CR against cancer remain unknown although decreased oxidative damage is a highly-

regarded possibility (Hursting et al., 2003; Kritchevsky, 2002).

A model for skin tumorigenesis has been developed using 7,12-dimethylbenz[a]anthracene

(DMBA) to initiate tumor formation followed by repeated treatments with 12-0-

tetradecanoylphorbol-13-acetate (TPA) to promote tumor formation (DiGiovanni, 1992). If

neuroendocrine alterations subsequent to the hypothalamic response to CR are responsible for

some of the protection against tumorigenesis seen in CR models, then impairment of hunger











significantly extended latency (Fig. 2-2d), although MSG mice underperformed compared with


their SAL counterparts in both diet conditions.


FA
- 129S1 Inclined Screen
20 -


NPY+ AL NPYAL N + CR N CR
NPY AL NPY' AL NPY CR NPY' CR


2
20 -1


15-
E
--
10-
J -

a. 5


0 1


B6 Inclined Screen


SAL AL MSG AL SAL CR MSG CR


129S1 Rotarod


300
300 -1


250


NPY+/+ AL NPY-- AL NPY+/+ CR NPY-/- CR


4 200

S150

E 100
5i0
50

0


B6 Rotarod


SAL AL MSG AL SAL CR MSG CR


Figure 2-2. Effects of ARC function and diet on physical performance. Path length on the
inclined screen did not differ in the 129S1 mice by either diet or NPY expression (A).
Within the B6 groups (B), CR mice traveled significantly farther than their respective
AL groups. Latency to fall on the rotarod was improved by CR in the 129S1 mice
regardless of genotype (C) although only the NPY/ CR mice took significantly longer
to fall compared with NPY~ AL mice. CR also significantly extended latency within
the B6 rotarod groups (D), although both MSG groups fell faster than their SAL
counterparts. (Mean + S.E.M., = significantly different from AL; t = significantly
different from SAL; n = 10.)


15
e-

,10
,

0. 5


0


]


E
300

250

m 200
0
150

E loo -
1--
50

0









Physical performance on locomotor tasks was improved by CR in all mice but benefits to

cognition were not observed in either of the neuroendocrine-impaired models. Similarly, while

improvements to body composition and reduced serum leptin were induced by CR in all mice,

these alterations did not manifest in certain trademark alterations in glucose homeostasis in the

models. Resistance to oxidative stress as assessed by survivorship following treatment with the

liver toxin diquat and tumorigenicity following a skin tumor induction regimen also suggested

the models of impaired hunger sensing faired less well than control mice; liver stress was lethal

in NPY knockout mice on CR, and both NPY and arcuate-damaged CR mice were most

susceptible to induced skin tumor formation. Taken together these results support the hypothesis

that the neuroendocrine response to CR is critical for eliciting some of the beneficial effects of

CR.









death specifically in the ARC (Burde et al., 1971; Olney, 1969) owing to blood-brain barrier

permeability at the median eminence (Peruzzo et al., 2000), this technique could be employed to

evaluate the role of the ARC in CR-related adaptations. Neonatal MSG-induced neurotoxicity in

mice results in a pronounced reduction in ARC neuropeptide expression (Broberger et al., 1998;

Meister et al., 1989) including NPY (Abe et al., 1990; Broberger et al., 1998; Kerkerian and

Pelletier, 1986; Legradi and Lechan, 1998; Meister et al., 1989) and is a well-characterized

model for evaluating the functional importance ARC neurons (Cameron et al., 1978; Hu et al.,

1998; Seress, 1982).

Evidence suggests the ARC and NPY have functional roles in cognition. For example,

ARC-lesioned mice display impaired memory (Park et al., 2000). Central administration of NPY

has been shown to enhance memory retention in mice (Flood et al., 1987), although a regulatory

role for NPY in learning and cognition is still putative (Redrobe et al., 1999). Whether the ARC

or NPY have been involved in behavioral adaptations following CR is presently unknown

although neuroendocrine signaling has been proposed to play an important role in mediating the

response to CR (Meites, 1989; Speakman and Hambly, 2007; Walford and Spindler, 1997). We

hypothesize that the response by the ARC (and fluctuations in ARC NPY in particular) act to

help mediate downstream effects of CR. To test this hypothesis we employed two mouse models,

an MSG-induced ARC-lesioned mouse and an NPY knockout mouse (NPY ) (Erickson et al.,

1996b) and compared their performance with control animals on a battery of behavioral tests

after CR or ad libitum (AL) feeding.

Materials and Methods

Animals and Diets

Descriptions of the mice and diets used for the following experiments, as well as the

experimental timeline, can be found in Appendix A.










Results


Survivorship

Within minutes of diquat injection all animals entered into a state of torpor followed by

hypothermia. The hypothermic response is recorded in Table 4-1. For individual mice,

temperatures dropping below 26C were strongly associated with fatality. This is exemplified by

the MSG AL mice with their stronger average hypothermic response and mortality. NPY CR

mice were also susceptible to fatality from the treatment although this is not reflected in their

average hypothermic response.

Table 4-1. Hypothermia and mortality after diquat injection.
129S1 B6
AL CR AL CR
Hypothermic Response (oC) Hypothermic Response (oC)
(20h post-injection) (2h post-injection)
NPY+ 29.7 (1.0) 31.8 (1.0) NS SAL 27.3 (0.2) 27.3 (0.2) NS
NPY- 29.5 (0.2) 29.8 (0.5) NS MSG 25.8 (0.2)t 27.5 (0.3) p < 0.001

Mortality (%) Mortality (%)
NPY+/ 0 0 SAL 0 0
NP Y 0 33 MSG 60 0

p <0.001 MSGvs SAL
NS, not significant

Histology

Gross histological effects in the liver from the diquat treatment are shown in Figures 4-1

(129S1 mice) and 4-1 (B6 mice). Livers from the saline-injected control mice shown at the

bottom of each figure are normal for both strains. Periportal microvesicles characteristic of fatty

liver change in response to toxic insult occur in diquat-injected 129S1 AL mice (Fig. 4-1) and B6

MSG mice (Fig. 4-2). CR ablates this microvesicle appearance in 129S1 mice and reduces it in

B6 MSG mice.












S129S1 Water Maze Training

60 -


E
O 50-

40
'a
4-
0 30


U)
C
) 10

0-
0
0-


-0o


B B6 Water Maze Training

60 1


E
O 50

40



C
O 30

20
U.)
a) 10
_j


NPY+/+ AL
NPY-/ AL
NPY+/+ CR
NPY-/- CR


0 1 2 3 4
Training Day


5 6


* *


p
p


SAL AL
MSG AL
SAL CR
MSG CR


0 1 2 3 4
Training Day


5 6


129S1 Probe Trial


100 ,


S NPY+/+ AL
m NPY/ AL
S NPY+/+ CR
SNPY-/- CR


Correct Opposite Right Adj Left Adj
Correct Opposite Right Adj Left Adj


B6 Probe Trial


- SAL AL
I MSG AL
- SAL CR
m MSG CR


Correct Opposite Right Adj Left Adj


Figure 2-4. Morris water maze performance during training and testing (the probe trial). All
129S1 groups performed equivalently in the water maze without effect of genotype or
diet during training (A) and testing (C). After the first day of training, MSG mice,
regardless of diet, showed significantly longer latencies to find the platform compared
with SAL mice (B) and the same MSG groups failed to swim in the correct quadrant
during probe testing (D). (Mean + S.E.M., n = 10.)


Visual Assessment


There were no significant differences in latency to find the cued platform among the 129S1


mice (Fig. 5a). However, for B6 mice a two-way ANOVA revealed a significant treatment effect


(F(1,36) = 90.19; p < 0.001) on the latency during visual cue training. Post hoc analysis (p <


100


80


60


40


20


0










129S1 Open Field
Locomotor Activity


25

S20
4-
S 15
_1
S10

5

0


TI


NPY++ AL NPY' AL NPY+/+ CR NPY' CR


129S1 Open Field
Thigmotaxis

1 NPY+/+ AL
SNPY- AL
m NPY++ CR
i NPY- CR






InI


Periphery


Center


E
20 -
4-
- 15-
_,
-.
S10
a-


0 L_


SALAL


D1
100 i


B6 Open Field
Locomotor Activity


MSGAL SAL CR MSGCR
MSG AL SAL CR MSG CR


B6 Open Field
Thigmotaxis


Periphery


I SAL AL
SMSG AL
I SAL CR
SMSG CR


Center
Center


Figure 2-3. Open field locomotor activity and exploratory behavior of 129S1 and B6 mice. By
measure of total distance traveled both NPY- groups traversed a greater distance in
the open field than NPY"+ mice, and the NPY CR mice traveled significantly farther
than NPY"+ CR mice (A). MSG mice traveled less on average than the SAL mice,
but this difference was not significant (B). Assessing thigmotaxic behavior showed
the relative time spent in the center of the open field was significantly different
among the 129S1 mice (C) such that the NPY- AL and CR mice spent less time in the
center of the field than NPTY CR mice (p = 0.003 and p = 0.006, respectively). MSG
mice also displayed decreased center-field exploration however this difference was
not significant (D). (Mean + S.E.M., n = 10.)

Open Field Locomotor Activity

By measure of total distance, a two-way ANOVA revealed a significant effect of genotype

(F(1,36) = 8.59; p = 0.006) on open field testing such that the NPY mice traveled farther in the


T


CI









CHAPTER 4
DIVERGENT EFFECTS OF CALORIE RESTRICTION ON OXIDATIVE STRESS
RESISTANCE AND ANTIOXIDANT PROTECTION IN TWO MOUSE MODELS OF
IMPAIRED NEUROENDOCRINE SIGNALING

The results of Chapter 3 demonstrated that interference of the systemic-neuronal interface

by genetic knockout of NPY expression or chemical knockout of ARC function attenuates

improvements in glucose handling associated with CR. While improving glucose handling is one

mechanism CR has been postulated to extend lifespan (Bartke, 2005; Tatar et al., 2003), it is but

one among many (Sinclair, 2005). Another popular mechanism through which CR is theorized to

work is by boosting antioxidant systems and enhancing oxidative stress resistance (Beckman and

Ames, 1998).

Diquat, most commonly used in agricultural industries for its herbicidal properties, is a

member of a family of compounds that react with water to form superoxide anions (02"),

hydrogen peroxide (H202), and hydroxyl radicals (HO') (Farrington et al., 1973). In vivo the

effects of diquat are seen in oxidative modifications to cellular antioxidants (Lauterberg et al.,

1984), lipids (Awad et al., 1994; Burk et al., 1980) and proteins (Lei, 2001). The sensitivity of

the liver to diquat oxidative stress (Ran et al., 2004) makes it a useful model in which to assess

protection by various treatments (e.g., CR, potential CR mimetics, antioxidants). Furthermore,

because CR has been shown to improve hepatic stress resistance to oxidative insult by enhancing

endogenous antioxidant systems (de Cabo et al., 2004; Seo et al., 2006), the liver oxidative stress

via diquat model may be a rapid end-point alternative to lifespan in aging studies.

The relevance of NPY and ARC signaling to increased oxidative stress resistance by CR

was investigated in the current study. Expression profiles of stress-related proteins and

antioxidant activity in mouse liver were assayed, and the stress response to the liver toxin diquat

was also investigated.










Table 1-2. Species in which NPY has been shown to stimulate feeding.
First Described:
Rat Clark et al. (1984),
Levine and Morley (1984),
Stanley and Leibowitz (1984)
Pig Parrott et al. (1986)
Chicken Kuenzel et al. (1987)
Mouse Morley et al. (1987)
Hamster Kulkosky et al. (1988)
Rabbit Pau et al. (1988)
Sheep Miner et al. (1989)
Snake Morris and Crews (1990)
Dog Geoghegan et al. (1993)
Squirrel Boswell et al. (1993)
Sparrow Richardson et al. (1995)
Goldfish Lopez-Patino et al. (1999)
Rhesus Monkey Larsen et al. (1999)
Guinea Pig Lecklin et al. (2002)

The potency of NPY to provoke feeding extends even to sated animals (Levine and

Morley, 1984; Parrott et al., 1986), and chronic administration of NPY results in sustained

hyperphagia and ultimately obesity (Beck et al., 1992; Pierroz et al., 1996; Stanley et al., 1986;

Zarjevski et al., 1993). Conversely, compounds that lower NPY levels or inhibit its activity

reduce feeding and body weight (Akabayashi et al., 1994; Burlet et al., 1995; Hulsey et al., 1995;

Lambert et al., 1998; Shimokawa et al., 2002).

Assessment of NPY expression levels over time and during periods of feeding and fasting

has formed the basis for a close relationship between hypothalamic NPY and food intake that

suggests a critical role for NPY in the long-term control of appetite. For example, rodent NPY

levels peak in the dark phase, which is when the majority of their feeding occurs (Jhanwar-

Uniyal et al., 1990; McKibbin et al., 1991). Furthermore, hypothalamic NPY levels are increased

in several models of hyperphagia, such as after short-term food deprivation (Ahima et al., 1996;

Bi et al., 2003; Brady et al., 1990; Grove et al., 2003; Sahu et al., 1988), long-term CR (Bi et al.,

2003; Boswell et al., 1999; Brady et al., 1990; de Rijke et al., 2005; Lewis et al., 1993; Mercer et

al., 2001), heavy exercise (Chen et al., 2007; Lewis et al., 1993), and in animals displaying









CHAPTER 2
CALORIE RESTRICTION ALTERS PHYSICAL PERFORMANCE BUT NOT COGNITION
IN TWO MODELS OF ALTERED NEUROENDOCRINE SIGNALING

Lifespan extension aside, calorie restriction (CR) consistently confers numerous beneficial

functional adaptations upon laboratory animals (Ingram et al., 2006; Sinclair, 2005). Of these,

the behavioral changes that have been associated with calorie restriction include enhanced

physical and cognitive performance (Ingram et al., 1987; Ishihara et al., 2005; Means et al.,

1993; Stewart et al., 1989). Enhanced physical performance may be attributed in large part to

improved body composition in CR animals compared to fully-fed controls, but the mechanisms

responsible for the cognitive effects are not fully understood. Hypotheses linking CR to altered

cognitive capability include reduction of neural oxidative stress, promotion of synaptic plasticity

and induction of various stress proteins and neurotrophic/neuroprotective factors (Mattson, 2003;

Prolla and Mattson, 2001). How CR might trigger such neuromodulation has not been fully

characterized and is thus the subject of this study.

The arcuate nucleus (ARC) in the hypothalamus of rodents is the first-order processing

unit for nutrient status in the brain (Heijboer et al., 2006; Wynne et al., 2005). The sum of

peripheral signals relating information regarding nutrient status (e.g. leptin from adipose tissue

and ghrelin from the gut) converge on the ARC where they are converted to a neurochemical

report (e.g. Neuropeptide Y (NPY), pro-opiomelanocortin (POMC)) that is transmitted to brain

regions involved in the activation of food-related behavior. As would be expected, ARC output is

altered by CR such that expression of orexigenic NPY is upregulated (Bi et al., 2003; Brady et

al., 1990; de Rijke et al., 2005; Kim et al., 1988; Mercer et al., 2001; Widdowson et al., 1997).

While CR-induced NPY upregulation was established more than 15 years ago (Kim et al.,

1988), causative roles for the ARC and NPY in mediating the effects of CR remain putative.

Since mice and rats treated neonatally with monosodium glutamate (MSG) exhibit neuronal









normal models high leptin produced during obesity would reduce adiponectin, whereas weight

loss would reduce leptin production and thereby permit uninhibited adiponectin production. In

the case of ARC-lesioned MSG mice, leptin is produced in abundance relative to their adiposity

but the signal is not transduced by the ARC and therefore adiponectin production is high. CR

reduces adiposity and leptin output in these mice but has no effect on adiponectin because their

adiponectin production is already set to high as a default due to their defective ARC signaling.

Because adiponectin is strongly associated with improved insulin sensitivity (Yamauchi et al.,

2003; Yamauchi et al., 2002), the increased adiponectin in the MSG-treated mice is likely behind

the relatively normal glucose response of these mice during the OGTT even though increased fat

mass is associated with increased insulin resistance (Ferrannini et al., 1996).

The ultimate predictor of insulin sensitivity for these mice may be their HOMA scores.

HOMA, which mathematically deduces insulin resistance from fasted blood glucose and insulin

values, is becoming the tool of choice for determining insulin resistance (Levy et al., 1998). In

the HOMA model higher values reflect greater resistance while lower values reflect greater

sensitivity to insulin. Unsurprisingly, higher HOMA scores are associated with the AL diets in

this study. Neither NPY status nor MSG-treatment increased HOMA scores relative to control

mice in the AL condition. NPY / mice have been shown to have equivalent fasting insulin and

glucose to wildtypes (Imai et al., 2007), but these are the first data indicating CR is less effective

at improving insulin sensitivity in NPY / mice. MSG treatment has been previously reported to

induce insulin resistance in rats (Balbo et al., 2007; Macho et al., 2000), but our MSG AL mice

are equivalent to the SAL controls. As with the NPY mice, MSG CR mice do not benefit by

HOMA assessment of insulin resistance.










POMC

PP

PVN

PYY

ROS

SAL

SEM

SDS

SDS-PAGE

SIRT

SOD

TBHQ

TPA

UV

V

VMH


Proopiomelanocortin

Pancreatic polypeptide

Paraventricular nucleus

Polypeptide YY

Reactive oxygen species

Saline-injected B6 control mice

Standard error of the mean

Sodium dodecyl sulfate

SDS-polyacrylamide gel electrophoresis

Mammalian sirtuin; Sir2-related protein

Superoxide dismutase

Tertiary butylhydroquinone

12-O-Tetradecanoylphorbol-13-acetate

Ultraviolet

Volts

Ventromedial nucleus









et al., 1999), overlapping mechanisms have likely developed to ensure the feeding instinct is not

easily extinguished and survival jeopardized. Regardless, other data confirm NPY's salience in

appetite control. For example, targeted postembryonic ablation of ARC NPY neurons leads to

reduced food intake and body weight (Bewick et al., 2005). B6 NPY mice show a 25% (Bannon

et al., 2000) to 50% (Segal-Lieberman et al., 2003) reduction in the hyperphagic response to

fasting compared with wild-types. NPY mice also exhibit less hyperphagia in streptozotocin-

induced diabetes (Sindelar et al., 2002).

Neuropeptide Y in neuroendocrine coordination

The neuroendocrine system consists of hormones, hormone -producing and -secreting

glands, and neurons that regulate these glands' activity. In mammals these neurons are found in

the hypothalamus, and they act to ensure coordinated secretion of hormones in response to

environmental cues (e.g., food availability). As a primary messenger at the central-peripheral

crossroads, NPY is produced by the hypothalamus to translate systemic signals about energy

status into the local neurochemical dialect.

Diverse circulating factors influence NPY expression, including leptin (Ahima et al., 1996;

Baskin et al., 1999; Schwartz et al., 1996; Stephens et al., 1995; Tang-Christensen et al., 1999),

insulin (Schwartz et al., 1992) and glucocorticoids (Higuchi et al., 1988b). Various gut hormones

such as ghrelin, pancreatic polypeptide (PP) and peptide YY (PYY) are also known to modulate

NPY expression (Asakwa et al., 2003; Batterham et al., 2002; Challis et al., 2003; Kamegai et

al., 2001; Nakazato et al., 2001; Shintani et al., 2001). NPY expression is also directly responsive

to energy availability via circulating glucose (Mizuno et al., 1999).

The most studied of NPY's relationships is that with leptin. Leptin, a hormone produced in

adipocytes in proportion to fat mass, acts as a feedback signal to the hypothalamus and plays a

fundamental role in maintaining energy homeostasis (Jequier, 2002). Not only is ARC NPY gene









the importance of the sensation of hunger in driving these effects-is unknown. This research

project investigated the effects of CR in two mouse models of handicapped hunger sensing: mice

with a chemical lesion of the ARC and mice with a genetic knockout of NPY. The results of

these studies will have significance in shaping the future directions of life extension research, the

search for CR mimetics, and the applicability of CR to humans. The following hypotheses were

tested:

Hypothesis #1: The behavioral response to calorie restriction is blunted by impaired
neuroendocrinological signaling.

Specific aims

Assess physical agility in CR versus AL mice by inclined screen and rotarod testing.
Assess anxiety-related behavior in CR versus AL mice by open field test.
Assess learning and memory in CR versus AL mice by Morris water maze test.


Hypothesis #2: The physiological response to calorie restriction is blunted by impaired
neuroendocrinological signaling.

Specific aims

Determine the effects of CR on serum factors including leptin, adiponectin, and insulin.
Compare the effects of CR on glucose homeostasis including fasting blood glucose
levels, glucose tolerance and insulin sensitivity.
Hypothesis #3: Resistance to oxidative stress by calorie restriction is blunted by impaired
neuroendocrinological signaling.

Specific aims

Determine the expression levels of stress-related proteins in the liver.
Determine the stress response in the liver to the toxin diquat.


Hypothesis #4: Resistance to tumor formation by calorie restriction is blunted by impaired
neuroendocrinological signaling.

Specific aims

Assess resistance to tumor formation after DMBA/TPA treatment in time to presentation
with tumors, quantity of tumors formed and size of tumors formed.
Measure the levels of oxidatively damaged protein in the skin.









ACKNOWLEDGMENTS

My sincere appreciation goes to my major advisor, Dr. Susan S. Percival, for providing the

backbone of my support group throughout graduate school. Without her committed interest and

confidence in me, this work would not have been possible. I also thank Dr. Rafael de Cabo, who

was instrumental in bringing me to the National Institute on Aging and providing a supportive

environment for my research. My graduate committee members, Drs. Henken, Knutson and

Leeuwenburgh, are also greatly appreciated for their support. I am grateful to my colleagues and

friends in the Percival and de Cabo laboratories, especially Meri Nantz and Cheryl Rowe at UF,

and the students and postdocs of the Laboratory of Experimental Gerontology in Baltimore.

These individuals were critical both for assistance with my project and for enriching my graduate

experience on a personal level. A special thank you goes to David del Pozo, who has been not

only my biggest fan throughout graduate school but also my best role model and friend. Lastly

and most importantly, the thanks of my life go to my parents, Nancy and Grady Minor, for being

my lifelong cheering squad, advisory council, adventure enablers, and all-around support net.









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Table 1-3. General theories of aging.
Programmed Theories
Cell Senescence Theory
Immune Theory
Neuroendocrine Theory
Wear and Tear Theories
DNA Damage Theory
Free Radical Theory
Mitochondrial Theory
Rate of Living Theory

There is great interest in the aging field to identify a unifying theory that can singly

account for the myriad symptoms of senescence. One that continues to garner proponents is the

neuroendocrine theory of aging (Bishop and Guarente, 2007; Speakman and Hambly, 2007).

Because the neuroendocrine system manages the general homeostatic tone of the body and

mediates its response to stress, the neuroendocrine theory of aging hypothesizes these activities

set the pace of aging. For example, menopause and andropause are associated with

neurodegeneration (Atwood et al., 2005), reduced GH/IGF-1 in elderly humans is associated

with decreased muscle strength and frailty (Ceda et al., 2005), and removal of the pituitary gland

with corticosterone replacement has been shown to extend lifespan in rats (Everitt et al., 1980).

Calorie Restriction

Few environmental manipulations have been reported to consistently extend the lifespan of

multiple species. CR, the reduction of macronutrient intake while maintaining sufficient

micronutrient intake, is one notable exception. Early studies by McCay and colleagues at Cornell

University established the effectiveness of CR for extending the lifespan of rats in the 1930s

(McCay et al., 1935; McCay et al., 1939; McCay and Crowell, 1934). Subsequent studies have

demonstrated reductions in caloric intake from 30-60% can increase maximum lifespan in a wide











129S1 B6
10000 10000
< 8000 >, 8000 -
M M


0 6000 o 6000 -

S4000 2 4000
S --- NPY+/+AL I --- SALAL
-- NPY-/- AL --- MSG AL
2000 -- NPY+/+CR u 2000 -- SALCR
S--0- NPY-/-CR --- MSGCR
0 01 0
Untreated Treated Untreated Treated
Skin Samples Skin Samples


Figure 5-4. Detection of changes in surface hydrophobicity in proteins between treated and
untreated skin regions. No significant differences were detected in either the 129S1
(A) groups or the B6 groups (B). (Mean S.E.M., n = 6 except NPY AL and NPY
CRn = 5.)

Discussion

This study was performed to determine the role of the ARC and NPY in the effects of CR

on tumorigenicity in a two-stage skin carcinogenesis model. The mice responding most rapidly

to treatment with tumors were the NPY CR mice. The role of NPY in cancer biology is not yet

well understood, but NPY expression has been observed to occur within tumor cells (Cohen et

al., 1990; deS Senanayake et al., 1995; Korner et al., 2004) and has been shown to be both

repressive (Kitlinska et al., 2005; Ruscica et al., 2006) and stimulatory (Kitlinska et al., 2005;

Ramo et al., 1990) to growth of different in vitro cancer cell lines. The current data may support

the repressive role for NPY in tumorigenesis, but one potential confounding issue exists relating

to the body weight loss in the NPY CR mice. After the initiation of TPA treatments, the 129S1

mice were observed relentlessly grooming their backsides on treatment days. Their increased

physical activity may have contributed to the weight loss observed in this strain, and the NPY/

CR mice began to die from starvation. By the 6th week after DMBA induction 2 of 5 NPY- CR

mice had died, at which point the surviving mice were given unrestricted access to food for 72h









64. Chan, Y.Y., Clifton, D.K., Steiner, R.A., 1996b. Role ofNPY neurones in GH-dependent
feedback signalling to the brain. Horm. Res. 45S, 12-14.

65. Chan-Palay, V., Allen, Y.S., Lang, W., Haesler, U., Polak, J.M. 1985. Cytology and
distribution in normal human cerebral cortex of neurons immunoreactive with antisera
against neuropeptide Y. J. Comp. Neurol. 238, 382-389.

66. Chan-Palay, V., Kohler, C., Haesler, U., Lang, W., Yasargil, G., 1986. Distribution of
neurons and axons immunoreactive with antisera against neuropeptide Y in the normal
human hippocampus. J. Comp. Neurol. 248, 360-375.

67. Chen, J.X., Zhao, X., Yue, G.X., Wang, Z.F., 2007. Influence of acute and chronic
treadmill exercise on rat plasma lactate and brain NPY, L-ENK, DYN Al-13. Cell. Mol.
Neurobiol. 27, 1-10.

68. Chronwall, B.M., DiMaggio, D.A., Massari, V.J., Pickel, V.M., Ruggiero, D.A.,
O'Donohue, T.L., 1985. The anatomy of neuropeptide-Y-containing neurons in rat brain.
Neuroscience. 15, 1159-1181.

69. Chronwall, B.M., Zukowska, Z., 2004. Neuropeptide Y, ubiquitous and elusive. Peptides.
25, 359-363.

70. Chung, H.Y., Kim, H.J., Kim, J.W., Yu, B.P., 2001. The inflammation hypothesis of
aging: molecular modulation by calorie restriction. Ann. NY Acad. Sci. 928, 327-335.

71. Clapcote, S.J., Lazar, N.L., Bechard, A.R., Wood, G.A., Roder, J.C., 2005. NIH Swiss and
Black Swiss mice have retinal degeneration and performance deficits in cognitive tests.
Comp. Med. 55, 310-316.

72. Clark, J.T., Kalra, P.S., Crowley, W.R., Kalra, S.P., 1984. Neuropeptide Y and human
pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology. 115, 427-429.

73. Cohen, H.Y., Miller, C., Bitterman, K.J., Wall, N.R., Hekking, B., Kessler, B., Howitz,
K.T., Gorospe, M., de Cabo, R., Sinclair, D.A. Calorie restriction promotes mammalian
cell survival by inducing the SIRT1 deacetylase. Science. 305, 390-392.

74. Cohen, P.S., Cooper, M.J., Helman, L.J., Thiele, C.J., Seeger, R.C., Israel, M.A., 1990.
Neuropeptide Y expression in the developing adrenal gland and in childhood
neuroblastoma tumors. Cancer Res. 50, 6055-6061.

75. Coles, L.S., Los Angeles Gerontology Research Group (LA-GRG), 2004. Validated
supercentenarian cases aged 114 and above. Rejuvenation Res. 7, 271-273.

76. Combs, T.P., Wagner, J.A., Berger, J., Doebber, T., Wang, W.J., Zhang, B.B., Tanen, M.,
Berg, A.H., O'Rahilly, S., Savage, D.B., Chatterjee, K., Weiss, S., Larson, P.J.,
Gottesdiener, K.M., Gertz, B.J., Charron, M.J., Scherer, P.E., Moller, D.E., 2002.
Induction of adipocytes complement-related protein of 30 kDa by PPARgamma agonists: a
potential mechanism of insulin sensitization. Endocrinology. 143, 998-1007.











A 129S1 Glucose Tolerance Curves
600 1


S500

400

o 300
3
200
0
Oo
100

0


-- NPY+/+AL
NPY-/-AL
-*-- NPY+/+CR
S NPY-/- CR


0 20 40 60 80 100 120
Time (min) post-gavage


60000 n


50000


S40000

,E 30000

20000

10000

0 -


B B6 Glucose Tolerance Curves
600 O


500

400

o 300
3
200
0
S 100


--- SAL AL
MSGAL
SALCR
MSGCR


0 20 40 60 80 100 120
Time (min) post-gavage


60000
60000


50000


E I


AC


A


40000

S30000

S 20000

10000

0


'? '


Figure 3-4. Oral glucose tolerance test response with area under the curve comparisons. The
glucose responses over time for the 129S1 groups are represented in panel A with the
area under the curve plotted in panel C. Only NPY+ CR mice showed a significantly
reduced glucose response (p < 0.05; NPY CR versus NPY- ALp = 0.06). The
glucose response over time for the B6 mice is shown in panel B with the area under
the curve comparisons in panel D. Both SAL CR and MSG CR mice had a
significantly reduced glucose response compared with the AL groups. (Mean +
S.E.M., = significantly different from AL; n = 6.)

Overall insulin response to the OGTT, also determined by total AUC quantification, was


significantly lower in both 129S1 CR groups (93.4 9.2 versus 150.2 16.1 ng/ml in NPY++;


97.8 11.5 versus 133.8 8.0 ng/ml in NPY/; p < 0.05) (Fig. 3-5c). SAL CR mice showed


reduced insulin response compared with SAL AL mice (83.7 + 3.8 versus 111.2 7.2 ng/ml; p <









LIST OF ABBREVIATIONS

ACTH Adrenocorticotropic hormone

AgRP Agouti related peptide

AL Ad libitum

ANOVA Analysis of variance

ARC Arcuate nucleus

AU Arbitrary units

AUC Area under the curve

B6 C57BL/6J mice

BisANS 4,4'-dianilino-1,1'-binaphthyl-5,5'disulfonic acid

BSA Bovine serum albumin

C Celsius

CART Cocaine- and amphetamine-regulated transcript

CCK Cholecystokinin

CNS Central nervous system

CR Calorie restriction or Calorie restricted

DCIP 2,6-Dichloroindophenol

DMBA 7,12-Dimethylbenz[a]anthracene

DMH Dorsomedial nucleus of the hypothalamus

ECL-plus Enhanced chemiluminescence plus

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme-linked immunosorbent assay

FOXO Forkhead box transcription factors

g Gram(s) or Gravity










GAPDH

GH

GLP

h

HOMA

HSP

IGF

Irs

1

LHA

M

min

mol

MSG

NAD(P)H

NMR

NPY

NPY/

NPY'

NQO1

NTS

OGTT

PBS


Glyceraldehyde 3 phosphate dehydrogenase

Growth hormone

Glucagon-like peptide

Hour(s)

Homeostasis Model Assessment of insulin resistance

Heat shock protein

Insulin-like growth factor

Insulin receptor substrate

Liter(s)

Lateral hypothalamic area

Molar

Minutes)

Mole(s)

Monosodium glutamate; MSG-injected C57BL/6J mice

Nicotinamide adenine dinucleotide (phosphate)

Nuclear magnetic resonance

Neuropeptide tyrosine

129S1/SvImJ control mice

NPY-knockout mice (129S-NpytmlRpa/J)

NAD(P)H:quinone reductase 1

Nucleus of the solitary tract, caudal brainstem

Oral glucose tolerance test

0.1 mM Phosphate-buffered saline








The C57BL/6J (B6) mice were bred and treated with MSG at the Jackson Laboratory. On

postnatal day 5, 65 male pups were given subcutaneous injections of MSG (at 4mg/g body

weight), and 65 control pups were given subcutaneous injections of saline solution. At 7 weeks

of age the mice were shipped to the Holabird Research Facility where they were acclimated for 3

weeks before the study diets were initiated.


129S1 B6






,N P,. 9 SAL





NPY-/- MSG

Figure A-1. Representative mice of the genotype and treatment groups.

At the onset of the study, the 129S1 mice were 3-5 months of age and the B6 mice 2.5

months of age when they were randomly divided into diet groups. Ninety six mice in total (12 for

each condition and diet) were used for these studies, all of which were males. The mice were

housed individually in ventilated caging on a 12h light/dark cycle at 220C and 35% humidity

with ad libitum access to water. All testing was performed during the light phase and using

procedures outlined and approved by the Animal Care and Use Committee at the National

Institute on Aging.









energy restriction from controlled settings to free-ranging human populations; despite the variety

of benefits associated with calorie restriction, it is thought that the majority of humans would

decline to embark on a lifelong regimen of conscientious calorie cutting. The advent of appetite-

suppressing pharmaceuticals or other mimetics of calorie restriction may therefore be of great

utility to enable 'painless' calorie restriction. Unfortunately, if these agents act through the

depression of neural signaling pathways that convey hunger to an organism, they may impede

the realization of the full spectrum of CR's benefits to physiology, carcinogenesis, cognition and

perhaps even lifespan.

Future studies are therefore needed to evaluate the use of appetite suppressants for weight

loss compared with CR giving attention to secondary health effects. Drugs that target

hypothalamic neuropeptide signaling pathways that control appetite are thought to hold

considerable promise for the treatment of obesity and work to develop such drugs is well under

way (Halford, 2006). Beyond the treatment of obesity, such pharmaceuticals would be appealing

for those seeking to achieve CR-like effects while minimizing the perception of hunger. The

results from this project, however, imply this may be a self-defeating move as the models of

decreased hunger perception fared less well than normal counterparts in withstanding oxidative

stress and tumor induction.

This research project is not without its limitations. While NPY is recognized to play a

crucial role in mediating hunger, it is but one of a suite of neuropeptides responsive to energy

balance. This is unsurprising as the life-preserving importance of caloric sufficiency has

promoted the evolution of overlap in hypothalamic appetite regulation (Horvath and Diano,

2004). Orexin, for example, is another appetite-stimulating neuropeptide produced in the ARC









Orexigenic (appetite-stimulating) signals activate ARC NPY neurons. Ghrelin, secreted

primarily by the stomach in increasing amounts with fasting (Ariyasu et al., 2001), increases

NPY levels (Cowley et al., 2003; Kamegai et al., 2001; Nakazato et al., 2001; Shintani et al.,

2001). Orexin, which influences the ARC via neuronal innervation from the LHA, stimulates

NPY neurons (Burdakov et al., 2003).

Once activated, NPY has multiple downstream effects. Central administration of NPY, for

example, has been shown to induce the release of glucoregulatory hormones including

adrenocorticotropic hormone (ACTH), corticosterone, and insulin (Akabayashi et al., 1994;

Leibowitz et al., 1988; Moltz and McDonald, 1985; Wahlestedt et al., 1987; Zarjevski et al.,

1994). Central NPY administration also leads to reduced growth hormone (GH) and insulin-like

growth factor 1 (IGF-1) release (Catzeflis et al., 1993). As ARC NPY neurons also express GH

receptor (Chan et al., 1996a), they have been hypothesized to mediate feedback control of this

important pituitary hormone (Chan et al., 1996b).

Aging

Aging, most simply defined as the temporal process of growing older, is not in itself a

deleterious process. Furthermore, while it may be said that the greatest risk factor for all natural

causes of death is old age, aging is not a disease either. Senescence, on the other hand, is the

general term used for the constellation of negative effects associated with aging and deterioration

of the organism. Aging-associated senescence includes the progressive decline of multiple organ

systems linked to dysfunctions in metabolism, reproduction, cognition, and ultimately survival.

Theoretically aging and senescence are separable, but in the human experience advanced age and

senescence are nearly constant companions. Ditto for the majority of the scientific literature in

gerontology, so the two terms are used synonymously in this work as well.











0.05), as did MSG AL mice (46.2 7.4 versus 111.2 7.2 ng/ml; p < 0.05) (Fig. 3-5d). CR had


no effect on insulin response in the MSG mice.


[ 129S1 OGTT Insulin Curves

2000 NPY/+ AL
~ NPY-/- AL
E -* NPY+/+ CR
0 1500 NPY-/-CR
-a.

1000


9 500


0
0 20 40 60 80 100 120
Time (min) post-gavage


200
200 n


150
E
S100


50


0 -
xV
A


x
A>


] B6 OGTT Insulin Curves

2000


MSG AL
-- -- SAL CR
MSG CR


E
S1500


S1000


0 500
Fa


0 20 40 60 80 100 120
Time (min) post-gavage


200
200 1


E
S 100
o

50


0


AX


S/ ^6


Figure 3-5. Oral glucose tolerance test insulin response with area under the curve comparisons.
The insulin responses over time for the 129S1 groups are represented in panel A with
the area under the curve plotted in panel C. Both NPY CR groups showed
significantly reduced insulin response to glucose challenge regardless of genotype.
The insulin response over time for the B6 mice is shown in panel B with the area
under the curve comparisons in panel D. SAL CR mice had a significantly reduced
insulin response compared with the SAL AL group, and the insulin response by the
MSG AL mice was significantly lower than that of the SAL AL mice. (Mean
S.E.M., = significantly different from AL; f = significantly different from SAL; n =
6.)









Assessment of insulin resistance

Only NPY+ mice had significantly lowered insulin resistance as assessed by HOMA

values comparing CR with AL groups (1.67 0.23 versus 3.64 0.81 pmol/mmol;p < 0.05),

although it should be noted that the NPY mice clearly follow the same trend (1.62 0.44 versus

3.64 0.78 pmol/mmol;p = 0.09) (Fig. 3-6a). In the B6 mice only SAL CR mice were

significantly reduced from their AL counterparts (1.19 0.34 versus 2.61 0.24 pmol/mmol; p <

0.05) (Fig. 3-6b).


[A 129S1 HOMA Values B6 HOMA Values
10 10

08 8
E E

06 06
E E







S 0.05; NPY CR versus NPY ALp = 0.09) (A). Of the B6 groups, only SAL CR
c 4 C 4

0 w

0 -0



Figure 3-6. HOMA calculations from the ratio of fasting blood insulin (pmol/l) to glucose
(mmol/l). Only NPYF/+ CR mice have a significantly reduced HOMA values (p <
0.05; NPY- CR versus NPY- ALp = 0.09) (A). Of the B6 groups, only SAL CR
mice had a reduced HOMA score (B). (Mean S.E.M., = significantly different
from AL; n = 6.)

Discussion

These experiments were performed to determine the role of the ARC and NPY in the

effects of CR on physiological measures related to body composition, endocrine status and

glucose homeostasis. With respect to the body composition measurements obtained by NMR, CR

reduced fat mass or inhibited its accumulation in all mice, a well-documented effect of CR

(Fontana and Klein, 2007). Secondary to these effects, percent adiposity was reduced in CR mice









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genotype. Both genotypes also responded similarly to the diet treatments; body weight increased

on AL diets and decreased on CR diets, with corresponding individual changes to lean, fat and

fluid mass and percent body fat. The increase in fat mass was the most robust change over time

in the AL groups, and thus the ratio of lean mass to fat mass was decreased by AL feeding

throughout the study. Conversely, the CR mice increased their ratio of lean to fat mass during the

study and this effect was greatest in the NPY- mice, which lost more fat mass on CR than the

NPY+/+ controls. This contrasts with the data from the NPY~ AL mice which were bigger than

their NPY'+ AL counterparts at week 12 (reflected by the body weight and food intake data in

Fig. A-3 of the appendix) and had significantly higher lean mass at that time.

In the B6 mice, notable differences were observed between the treatment groups starting

from baseline. Specifically, the MSG mice were smaller yet had a higher fat mass than SAL

mice. AL feeding exacerbated the fatty phenotype of these mice and MSG AL mice were

ultimately heavier than their SAL AL counterparts despite starting the diet at lower body weight.

CR in both groups acted primarily to temper fat mass gain such that there was no significant

difference between baseline and week twelve. At all time points the MSG CR mice showed a

greater percentage of body fat than the SAL CR mice.




Full Text

PAGE 1

ROLE OF SATIETY SIGNALING IN THE BENEFICIAL EFFECTS OF CALORIE RESTRICTION IN MICE By ROBIN KAYE MINOR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

PAGE 2

2007 Robin Kaye Minor 2

PAGE 3

To my parents, Nancy and Grady Minor 3

PAGE 4

ACKNOWLEDGMENTS My sincere appreciation goes to my major advisor, Dr. Susan S. Percival, for providing the backbone of my support group throughout graduate school. Without her committed interest and confidence in me, this work would not have been possible. I also thank Dr. Rafael de Cabo, who was instrumental in bringing me to the National Institute on Aging and providing a supportive environment for my research. My graduate committee members, Drs. Henken, Knutson and Leeuwenburgh, are also greatly appreciated for their support. I am grateful to my colleagues and friends in the Percival and de Cabo laboratories, especially Meri Nantz and Cheryl Rowe at UF, and the students and postdocs of the Laboratory of Experimental Gerontology in Baltimore. These individuals were critical both for assistance with my project and for enriching my graduate experience on a personal level. A special thank you goes to David del Pozo, who has been not only my biggest fan throughout graduate school but also my best role model and friend. Lastly and most importantly, the thanks of my life go to my parents, Nancy and Grady Minor, for being my lifelong cheering squad, advisory council, adventure enablers, and all-around support net. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................11 ABSTRACT ...................................................................................................................................14 CHAPTER 1 LITERATURE REVIEW.......................................................................................................16 Appetite...................................................................................................................................16 The Role of the Hypothalamic Arcuate Nucleus in Hunger............................................17 Neuropeptide Y...............................................................................................................20 Ubiquitous expression..............................................................................................20 Mechanism of action: Signaling through Y receptors..............................................21 Physiological pluripotency.......................................................................................22 Neuropeptide Y in feeding behavior........................................................................23 Neuropeptide Y in neuroendocrine coordination.....................................................26 Aging......................................................................................................................................28 Calorie Restriction...........................................................................................................30 Mechanisms of action...............................................................................................31 Neuropeptide Y: How and Y calorie restriction extends lifespan?..........................32 Overall Rationale....................................................................................................................33 Hypothesis #1: The behavioral response to calorie restriction is blunted by impaired neuroendocrinological signaling..................................................................................34 Hypothesis #2: The physiological response to calorie restriction is blunted by impaired neuroendocrinological signaling...................................................................34 Hypothesis #3: Resistance to oxidative stress by calorie restriction is blunted by impaired neuroendocrinological signaling...................................................................34 Hypothesis #4: Resistance to tumor formation by calorie restriction is blunted by impaired neuroendocrinological signaling...................................................................34 2 CALORIE RESTRICTION ALTERS PHYSICAL PERFORMANCE BUT NOT COGNITION IN TWO MODELS OF ALTERED NEUROENDOCRINE SIGNALING...35 Materials and Methods...........................................................................................................36 Animals and Diets...........................................................................................................36 Rotarod............................................................................................................................37 Inclined Screen................................................................................................................37 Open Field Locomotor Activity......................................................................................37 5

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Morris Water Maze..........................................................................................................37 Visual Platform Training.................................................................................................38 Contextual Fear Conditioning.........................................................................................38 Statistics...........................................................................................................................39 Results.....................................................................................................................................39 Body Weights..................................................................................................................39 Physical Agility...............................................................................................................40 Open Field Locomotor Activity......................................................................................42 Morris Water Maze Performance....................................................................................43 Visual Assessment...........................................................................................................44 Contextual Fear Conditioning.........................................................................................46 Discussion...............................................................................................................................46 3 REDUCTIONS IN ADIPOSITY AND LEPTIN FOLLOWING CALORIE RESTRICTION DO NOT fULLY EXTEND TO IMPROVED GLUCOSE HOMEOSTASIS IN MODELS OF IMPAIRED NEUROENDOCRINOLOGICAL SIGNALING...........................................................................................................................51 Materials and Methods...........................................................................................................52 Animals and Diets...........................................................................................................52 Body Composition Analysis............................................................................................52 Body Temperature Measurement....................................................................................53 Hormonal Assessments...................................................................................................53 Leptin.......................................................................................................................53 Adiponectin..............................................................................................................53 Glucose Homeostasis.......................................................................................................53 Fasting blood glucose...............................................................................................54 Oral glucose tolerance test.......................................................................................54 Insulin measurement................................................................................................54 HOMA calculations..................................................................................................54 Statistics...........................................................................................................................54 Results.....................................................................................................................................54 Body Composition...........................................................................................................54 Body Temperature...........................................................................................................57 Hormonal Assessments...................................................................................................57 Glucose Homeostasis.......................................................................................................58 Fasting blood glucose...............................................................................................58 Oral glucose tolerance test.......................................................................................59 Assessment of insulin resistance..............................................................................62 Discussion...............................................................................................................................62 4 DIVERGENT EFFECTS OF CALORIE RESTRICTION ON OXIDATIVE STRESS RESISTANCE AND ANTIOXIDANT PROTECTION IN TWO MOUSE MODELS OF IMPAIRED NEUROENDOCRINE SIGNALING..........................................................66 Materials and Methods...........................................................................................................67 Animals and Diets...........................................................................................................67 6

PAGE 7

Diquat Treatment.............................................................................................................67 Histology and Stereology................................................................................................67 Western Blotting..............................................................................................................68 NAD(P)H:Quinone Reductase 1 (NQO1) Activity.........................................................68 Results.....................................................................................................................................69 Survivorship....................................................................................................................69 Histology.........................................................................................................................69 Expression of Stress Proteins and Antioxidants..............................................................73 NQO1 Activity................................................................................................................75 Discussion...............................................................................................................................75 5 CALORIE RESTRICTION IS NOT PROECTIVE AGAINST TUMORIGENICITY IN MICE WITH IMPAIRED NEUROENDOCRINE SIGNALING..........................................79 Materials and Methods...........................................................................................................80 Animals and Diets...........................................................................................................80 Two-Stage Carcinogenesis..............................................................................................80 Protein Damage Assay....................................................................................................81 Results.....................................................................................................................................82 Body Weight....................................................................................................................82 Tumor Characteristics.....................................................................................................83 Protein Conformation Effect...........................................................................................84 Discussion...............................................................................................................................85 6 DISCUSSION.........................................................................................................................87 APPENDIX GENERAL STUDY DESIGN.......................................................................................................90 Background on the Effects of Monosodium Glutamate on Hypothalamic Function.............90 Mice........................................................................................................................................91 Verification of NPY Expression Levels.................................................................................93 Diets........................................................................................................................................94 Study Design...........................................................................................................................95 Body Weights and Food Intake..............................................................................................97 Hunger Assessment................................................................................................................98 LIST OF REFERENCES.............................................................................................................100 BIOGRAPHICAL SKETCH.......................................................................................................128 7

PAGE 8

LIST OF TABLES Table page 1-1 Diverse effects of central and systemic NPY....................................................................22 1-2 Species in which NPY has been shown to stimulate feeding............................................24 1-3 General theories of aging...................................................................................................30 3-1 NMR body composition data.............................................................................................56 4-1 Hypothermia and mortality after diquat injection..............................................................69 5-1 Characteristics of tumor incidence by latency, multiplicity, and size of tumors...............84 A-1 Diet composition................................................................................................................95 8

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LIST OF FIGURES Figure page 1-1 Central integration of satiety signals..................................................................................17 1-2 The hypothalamus, its major appetite regulatory centers, and NPY signaling during fasting.................................................................................................................................19 1-3 Neuropeptide Y..................................................................................................................21 2-1 Mean group body weight for the six weeks prior to behavioral testing.............................40 2-2 Effects of ARC function and diet on physical performance..............................................41 2-3 Open field locomotor activity and exploratory behavior...................................................42 2-4 Morris water maze performance........................................................................................44 2-5 Visual assessment..............................................................................................................45 3-1 Body temperature among the groups.................................................................................57 3-2 Fasted leptin and adiponectin levels..................................................................................58 3-3 Fasting blood glucose........................................................................................................59 3-4 Oral glucose tolerance test response with area under the curve comparisons...................60 3-5 Oral glucose tolerance test insulin response with area under the curve comparisons.......61 3-6 HOMA calculations...........................................................................................................62 4-1 Liver histology of diquat-treated 129S1 mice...................................................................70 4-2 Liver histology of diquat-treated B6 mice.........................................................................71 4-3 Fatty change in the liver in response to diquat..................................................................72 4-4 Liver antioxidant and stress-related protein expression.....................................................74 4-5 NQO1 activity in the liver..................................................................................................75 5-1 Tumor induction procedure................................................................................................80 5-2 Body weights of tumor study mice....................................................................................82 5-3 Tumor onset profiles..........................................................................................................83 9

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5-4 Detection of changes in surface hydrophobicity in proteins..............................................85 A-1 Representative mice of the genotype and treatment groups..............................................92 A-2 Representative hypothalamic brain sections stained for NPY...........................................94 A-3 Project design for the three major studies and other periodic assessments.......................96 A-4 Food consumption and body weight..................................................................................98 A-5 Feeding rate among the CR groups....................................................................................99 10

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LIST OF ABBREVIATIONS ACTH Adrenocorticotropic hormone AgRP Agouti related peptide AL Ad libitum ANOVA Analysis of variance ARC Arcuate nucleus AU Arbitrary units AUC Area under the curve B6 C57BL/6J mice BisANS 4,4-dianilino-1,1-binaphthyl-5,5disulfonic acid BSA Bovine serum albumin C Celsius CART Cocaineand amphetamine-regulated transcript CCK Cholecystokinin CNS Central nervous system CR Calorie restriction or Calorie restricted DCIP 2,6-Dichloroindophenol DMBA 7,12-Dimethylbenz[a]anthracene DMH Dorsomedial nucleus of the hypothalamus ECL-plus Enhanced chemiluminescence plus EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay FOXO Forkhead box transcription factors g Gram(s) or Gravity 11

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GAPDH Glyceraldehyde 3 phosphate dehydrogenase GH Growth hormone GLP Glucagon-like peptide h Hour(s) HOMA Homeostasis Model Assessment of insulin resistance HSP Heat shock protein IGF Insulin-like growth factor Irs Insulin receptor substrate l Liter(s) LHA Lateral hypothalamic area M Molar min Minute(s) mol Mole(s) MSG Monosodium glutamate; MSG-injected C57BL/6J mice NAD(P)H Nicotinamide adenine dinucleotide (phosphate) NMR Nuclear magnetic resonance NPY Neuropeptide tyrosine NPY +/+ 129S1/SvImJ control mice NPY -/NPY-knockout mice ( 129S-Npy/J) tm1Rpa NQO1 NAD(P)H:quinone reductase 1 NTS Nucleus of the solitary tract, caudal brainstem OGTT Oral glucose tolerance test PBS 0.1 mM Phosphate-buffered saline 12

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POMC Proopiomelanocortin PP Pancreatic polypeptide PVN Paraventricular nucleus PYY Polypeptide YY ROS Reactive oxygen species SAL Saline-injected B6 control mice SEM Standard error of the mean SDS Sodium dodecyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis SIRT Mammalian sirtuin; Sir2-related protein SOD Superoxide dismutase TBHQ Tertiary butylhydroquinone TPA 12-O-Tetradecanoylphorbol-13-acetate UV Ultraviolet V Volts VMH Ventromedial nucleus 13

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF SATIETY SIGNALING IN THE BENEFICIAL EFFECTS OF CALORIE RESTRICTION IN MICE By Robin Kaye Minor December 2007 Chair: Susan S. Percival Major: Food Science and Human Nutrition Laboratory studies consistently demonstrate extended lifespan in animals on calorie restriction (CR), where total caloric intake is reduced by 10-40% but adequate nutrition is otherwise maintained. CR has been further shown to delay the onset and severity of chronic diseases associated with aging such as cancer, and to extend the functional health span of important faculties like cognition. Less understood are the underlying mechanisms through which CR might act to induce such alterations. One theory postulates that CRs beneficial effects are intimately tied to the neuroendocrine response to low energy availability, of which the arcuate nucleus in the hypothalamus plays a pivotal role. Neuropeptide Y (NPY), a neurotransmitter in the front line of the arcuate response to low energy availability, is the primary hunger signal affected by CR. It was hypothesized that the arcuate nucleus and NPY are critical not only for certain key physiological alterations, but also for increased stress resistance, decreased cancer risk and enhanced cognition noted with CR. These hypotheses were tested using two mouse modelsone chemically treated to impair arcuate function and another genetically modified to lack NPYmaintained on CR or unlimited feeding. 14

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Physical performance on locomotor tasks was improved by CR in all mice but benefits to cognition were not observed in either of the neuroendocrine-impaired models. Similarly, while improvements to body composition and reduced serum leptin were induced by CR in all mice, these alterations did not manifest in certain trademark alterations in glucose homeostasis in the models. Resistance to oxidative stress as assessed by survivorship following treatment with the liver toxin diquat and tumorigenicity following a skin tumor induction regimen also suggested the models of impaired hunger sensing faired less well than control mice; liver stress was lethal in NPY knockout mice on CR, and both NPY and arcuate-damaged CR mice were most susceptible to induced skin tumor formation. Taken together these results support the hypothesis that the neuroendocrine response to CR is critical for eliciting some of the beneficial effects of CR. 15

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CHAPTER 1 LITERATURE REVIEW Appetite Deprived of food, an organism becomes hungry. As seemingly straightforward as this relationship appears externally, internally it is mediated by a decidedly complex interaction of numerous alterations both psychological and physiological. For example, hunger may be described in behavioral terms as the state in which an organism is motivated to eat. The feeding response, the most overt outcome subsequent to negative energy balance, belies numerous physiological and neurophysiological alterations imperceptible to the naked eye: neural signaling patterns, body temperature, metabolism, and various blood-borne hormones and metabolites are just a few of the many processes responsive to hunger and satiety in animals. As an organism shifts from a fed to a fasted state, its empty gut ceases signaling fullness and commences to call for refeeding. Circulating metabolites shift from energetic macronutrients to byproducts of metabolism which trigger a compensatory adjustment in pancreatic exocrine output. The sum of these signals among others is tallied by the brain, where the peripheral call to eat is put into action. Central recipients of the peripheral messages relaying the status of satiety are primarily the hypothalamus and the brainstem (Berthoud, 2004). The brainstem functions in the control of autonomous feeding behavior via the caudal nucleus of the solitary tract (NTS). Gastrointestinal, circulatory and central cues all reach the NTS and influence the determination of meal size (Schwartz, 2006). The NTS alone, however, has been shown to be insufficient for a full response to long-term food deprivation (Seeley et al., 1994) as this requires the action of another CNS satiety center: the hypothalamus. This critical difference relates to the primary signal input for these brain regions; the NTS acts as the main port of entry for gastrointestinal signals while the 16

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hypothalamus predominately services an inflow of peripheral signals pertaining to metabolic energy stores (Nslund and Hellstrm, 2007), as depicted in Figure 1-1. Located above the pituitary and below the thalamus, the hypothalamus is found in all mammalian brains and operates as a central regulator of multiple physiological processes and circadian cycles, with the most salient to this review being its role as the major integration center for peripheral satiety signals. BrainstemHypothalamus BrainstemHypothalamus AdiposeCirculationAdiponectin LeptinGlucose GutGhrelinPYY GutGhrelinPYY PancreasGlucagonInsulin PancreasGlucagonInsulin Figure 1-1. Central integration of satiety signals. The two main central targets of peripheral cues regarding energy status are the brainstem and the hypothalamus. Afferent nerves carry sensory information about feeding status directly from the gut to the brainstem while circulating factors derived from metabolism, adipose tissue, the pancreas and the gut signal through the hypothalamus. The appetitive state is determined by the balance of hungerversus satiety-inducing cues, depicted above in green (pro-hunger) and purple (pro-satiety). The Role of the Hypothalamic Arcuate Nucleus in Hunger Appreciation of the hypothalamus as a regulator of appetite became firmly established in the 1950s when it was demonstrated that lesions of the hypothalamic ventromedial nucleus 17

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(VMH) result in hyperphagia and obesity whereas lesions of the lateral hypothalamus (LHA) result in anorexia and weight loss (Anand and Brobeck, 1951a,b). Thus was born the hypothesis for a relay of humoral signals through the brain communicating energy needs (Stellar, 1954) and ensuing research has continued to demonstrate the importance of the hypothalamus to feeding behavior (Hoebel, 1997). Today the hypothalamus is considered to be an essential component in the regulatory system for energy homeostasis (Berthoud, 2006; Elmquist et al., 2005; Meister, 2007). Hypothalamic centers associated with the regulation of energy balance include the arcuate (ARC), dorsomedial (DMH), paraventricular (PVN), and ventromedial (VMH) nuclei and the LHA. Of these, the ARC in particular is a critical locus for food intake regulation as it integrates signals from the brainstem and the periphery (Cone et al., 2001; Cowley et al., 2003), uniquely accessible to the latter because the blood-brain barrier is semi-permeable here (Broadwell and Brightman, 1976; Peruzzo et al., 2000). Nestled at the base of the third ventricle just above the median eminence in an elongated, arc-like bundle, the first-order neurons of the ARC are in direct contact with peripheral satiety factors which they transduce and convey to the second-order neuron centers of the DMH, PVN, VMH and LHA (Heijboer et al., 2006; Schwartz and Porte, 2005). At least two populations of first-order neurons controlling appetite are present in the ARC: (1) neurons coexpressing neuropeptide tyrosine (NPY) and agouti-related protein (AgRP) and (2) neurons coexpressing pro-opiomelanocortin (POMC) and cocaineand amphetamine-regulated transcript (CART). The former (NPY/AgRP) stimulate food intake (Broberger et al., 1998; Hahn et al., 1998; Shutter et al., 1997) while the latter (POMCCART) repress it (Elias et al., 1998; Kristensen et al., 1998). 18

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Brainstem Hypothalamus DMH LHAME3V NPY/AgRPPOMC/CART PVN ARC VMH Figure 1-2. The hypothalamus, its major appetite regulatory centers, and NPY signaling during fasting. The panel shows an enlargement of the central hypothalamus and its symmetrical architecture. The ARC is situated along the third ventricle (3V) above the median eminence (ME) and communicates with the DMH, LHA, PVN and VMH. The right side shows that during fasting ARC-produced NPY is released to the LHA, PVN and VMH in the primary response. Fasting also decreases ARC POMC/CART output. Hypothalamic ARC circuits are directly responsive to an array of circulating hunger and satiety signals such as hormones (e.g. ghrelin, insulin and leptin), and metabolites (e.g. glucose) and thus monitor input regarding both short-term fuel status as well as long-term energy stores. Registered in concert these signals convey the current state of energy availability such that an array communicating energy sufficiency leads to low ARC NPY/AgRP expression and high 19

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POMC/CART expression, which promotes satiety (Ziotopoulou et al., 2000). When the opposite is true and the ARC registers an energy deficit, the resulting neuropeptide balance is shifted to high NPY/AgRP expression and low POMC/CART expression to promote hunger (Pinto et al., 2004). If the energy status of an animal is skewed to the negative end over a long-term period such as during repeated fasting or chronic calorie restriction (CR), the prolonged shift in ARC neuropeptide expression could have profound consequences on the organism. In fact, this response could be essential to driving systemic adaptations noted under conditions of reduced energy availability. If so, ARC neuropeptides highly expressed during a hunger response may carry significant responsibility for instigating downstream physiological effects. Such a peptide would respond to a variety of peripheral satiety signals, actively signal to secondary brain appetite centers, and continue to respond to negative energy balance over the long term. One such hypothalamic peptide has been identified, and that is NPY. Neuropeptide Y Ubiquitous expression NPY, first isolated from the porcine brain in 1982, is a powerfully orexigenic 36-amino-acid protein in the pancreatic polypeptide family (Tatemoto et al., 1982a; Tatemoto, 1982b) that is one of the most abundant peptides in the mammalian central nervous system (Adrian et al., 1983; Chan-Palay et al., 1985; Chan-Palay et al., 1986; Chronwall et al., 1985). Widely distributed throughout the brain, NPY localization is strongest in the hypothalamus wherein immunoreactivity is highest in the neurons of the ARC and the DMH (Allen et al., 1983; Chronwall et al., 1985; Gray and Morley, 1986) because it is these neurons in which NPY is synthesized and stored (Bi and Moran, 2003). Both ARC and DMH NPY neurons project to the LHA, PVN and VMH (Chronwall et al., 1985; Cripps et al., 2005; Jhanwar-Uniyal et al., 1993; Sahu et al., 1988) but ARC NPY is the primary responder to both short-term and long-term 20

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fasting conditions (Bi, 2007). NPY is also found in the peripheral nervous system in sympathetic nerves (Pernow et al., 1987). YR NH2QRTINI COOHLYHRLASYYRLEAPADEGPNDPKSPY D A15101520253135 Beta turn Alpha-helix Polyprolinehelix Figure 1-3. Neuropeptide Y. Widespread distribution of NPY continues outside the nervous system. NPY is released from sympathetic nerves into endocardial endothelial cells (Jacques et al., 2006), the gut (Cox, 2007) and the spleen (Ericsson et al., 1987). Splenic NPY is incorporated by developing blood cells and ultimately circulates in immune cells and platelets (Ericsson et al., 1987; Kuo et al., 2007b). NPY is also released into circulation from sympathetic nerves and the adrenal medulla under stress (Bernet et al., 1998; Han et al., 2005; Kuo et al., 2007b). Recently, adipocytes have been demonstrated to express NPY where it may play a role in mediating adiposity (Kos et al., 2007). Mechanism of action: Signaling through Y receptors NPY signals through G protein-coupled receptors, seven of which have been named (Y 1 -Y 7 ) and five of which have been cloned and described: Y 1 Y 2 Y 4 Y 5 and Y 6 (Dumont et al., 1993; Michel et al., 1998). This is not to say that all receptors are relevant to all mammals; the existence of Y 3 has been postulated but not demonstrated (Herzog et al., 1993; Jazin et al., 1993), Y 6 is inactive in primates (Matsumoto et al., 1996), and mammals have lost the Y 7 gene 21

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altogether (Larhammar and Salaneck, 2004). NPY acts through Y receptors to inhibit adenylyl cyclase (Herzog et al., 1992) and increase intracellular calcium levels (Jacques et al., 2000). In some instances NPY receptor activity has been shown to result in activation of mitogen-activated protein kinase (Nie and Selbie, 1998) and protein kinase C (Mannon and Raymond, 1998). The NPY Y 1 and Y 5 receptors expressed in the hypothalamus are considered to be the most active in the regulation of appetitive behavior and energy balance in mammals (Duhault et al., 2000; Hu et al., 1996; Lecklin et al., 2002; Parker and Herzog, 1999). Physiological pluripotency The relative abundance of NPY and its receptors combined with their widespread distribution suggests NPY is involved in multiple important physiological roles beyond the regulation of food intake. This is indeed the case, and the gamut of NPYs systemic and central effects is depicted in Table 1-1. Of particular interest to this review are NPYs involvement in food intake and neuroendocrine coordination, to be elaborated below. Table 1-1. Diverse effects of central and systemic NPY. Reviewed by*:Central Effects Alcohol Intake Carvajal et al. (2006), Thorsell (2007) Circadian RhythmsKallingal and Mintz (2007), Yannielli and Harrington (2001) Emotion Carvajal et al. (2006), Heilig (2004) Feeding Behavior Arora and Anubhuti (2006), Beck (2006) LearningRedrobe et al. (1999) LocomotionKarlsson et al. (2005) Neuroendocrine CoordinationMagni (2003), Plant and Shahab (2002) Reproductive Function Kalra and Kalra (2004), Wjcik-Gadysz and Polkowska (2006) SeizuresDub (2007), Redrobe et al. (1999)Systemic Effects Adipose FunctionKos et al. (2007) Adrenal FunctionSpinazzi et al. (2005) Cardiovascular Function Zukowska et al. (2003) Gastrointestinal Function Cox (2007) Pancreatic FunctionImai et al. (2007) *See review by Thorsell and Heilig (2002) for an overview of many of NPY's actions 22

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In the broadest terms, NPYs central actions include stimulating hunger, fat storage and weight gain (Beck et al., 1992; Stanley et al., 1986; Zarjevski et al., 1993) while decreasing sex drive, locomotion, energy expenditure and body temperature (Billington et al., 1991; Hwa et al., 1999; Kulkosky et al., 1988; Lopez-Valpuesta et al., 1996; Menendez et al., 1990). These effects contrast the actions of NPY in the periphery where stress is a major factor influencing the release of NPY (Bernet et al., 1998; Han et al., 2005; Kuo et al., 2007b) and the consequences are strongly registered by the adrenal and cardiovascular systems. It has been hypothesized that in the current Western lifestylewhich antagonizes central NPY through leptin resistance and peripheral NPY through stressNPY could contribute to a number of diseases such as hypertension, diabetes, and obesity where calories are in plentiful supply (Chronwall and Zukowska, 2004; Kuo et al., 2007b). Neuropeptide Y in feeding behavior Within two years following its discovery NPY became well known for inducing a robust feeding response in rats (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984), and to this day NPY is the most potent endogenous orexigenic stimulant known (Chamorro et al., 2002; Edwards et al., 1999). The feeding effect of NPY appears to be well conserved, as central injection induces food intake in a wide range of species as depicted in Table 1-2. The only known exception is the baboon, in which intracerebroventricular injection of NPY did not induce feeding (Sipols et al., 1996). 23

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Table 1-2. Species in which NPY has been shown to stimulate feeding. First Described:RatClark et al. (1984), Levine and Morley (1984), Stanley and Leibowitz (1984)PigParrott et al. (1986)ChickenKuenzel et al. (1987)MouseMorley et al. (1987)HamsterKulkosky et al. (1988)RabbitPau et al. (1988)SheepMiner et al. (1989)SnakeMorris and Crews (1990)DogGeoghegan et al. (1993)SquirrelBoswell et al. (1993)SparrowRichardson et al. (1995)GoldfishLopez-Patino et al. (1999)Rhesus MonkeyLarsen et al. (1999)Guinea PigLecklin et al. (2002) The potency of NPY to provoke feeding extends even to sated animals (Levine and Morley, 1984; Parrott et al., 1986), and chronic administration of NPY results in sustained hyperphagia and ultimately obesity (Beck et al., 1992; Pierroz et al., 1996; Stanley et al., 1986; Zarjevski et al., 1993). Conversely, compounds that lower NPY levels or inhibit its activity reduce feeding and body weight (Akabayashi et al., 1994; Burlet et al., 1995; Hulsey et al., 1995; Lambert et al., 1998; Shimokawa et al., 2002). Assessment of NPY expression levels over time and during periods of feeding and fasting has formed the basis for a close relationship between hypothalamic NPY and food intake that suggests a critical role for NPY in the long-term control of appetite. For example, rodent NPY levels peak in the dark phase, which is when the majority of their feeding occurs (Jhanwar-Uniyal et al., 1990; McKibbin et al., 1991). Furthermore, hypothalamic NPY levels are increased in several models of hyperphagia, such as after short-term food deprivation (Ahima et al., 1996; Bi et al., 2003; Brady et al., 1990; Grove et al., 2003; Sahu et al., 1988), long-term CR (Bi et al., 2003; Boswell et al., 1999; Brady et al., 1990; de Rijke et al., 2005; Lewis et al., 1993; Mercer et al., 2001), heavy exercise (Chen et al., 2007; Lewis et al., 1993), and in animals displaying 24

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seasonal hyperphagia in preparation for hibernation (Boswell et al., 1993; Lakhdar-Ghazal et al., 1995). Hypothalamic NPY is also increased in genetic models of obesity involving hyperphagia including obese (ob/ob) mice (Jang and Romsos, 1998), diabetic (db/db) mice (de Luca et al., 2005), Zucker fatty (fa/fa) rats (Dryden et al., 1995), and the Koletsky corpulent (cp/cp) rat (Williams et al., 1992). Tubby (tub/tub) mice are unique in that they show significantly reduced ARC NPY, however high levels of NPY are found in the VMH and DMH, indicating increased neurotransmission of NPY from the ARC to the VMH and DMH could explain their hyperphagia (Guan et al., 1998). Whether increased NPY is associative or causative to obesity in these models remains to be definitively proven, however, at least in fa/fa rats NPY overexpression occurs pre-obesity and is thought to be a driving factor in their weight gain (Bchini-Hooft et al., 1993). The reverse association has also been observed; anorexic (anx/anx) mice display reduced NPY signaling concomitant with their reduced food intake (Broberger et al., 1997). Research investigating the effects of genetic manipulation of NPY expression and feeding behavior belies the complexity of food intake regulation. For example, mice engineered to overexpress central NPY by 15% did not display increased feeding (Inui et al., 1998), and NPY knockout (NPY -/) mice on a mixed background have normal food intake and body weight and are able to respond to fasting with hyperphagia (Erickson et al., 1996b). C57BL/6 (B6) NPY -/mice even develop mild obesity when access to food is unrestricted (Segal-Lieberman et al., 2003). Concomitant knockout of NPY and AgRPmutually expressed by ARC neurons in response to negative energy balancedid not prevent mice from developing to normal body weight or displaying hyperphagia following fasting (Qian et al., 2002). Given that roughly two dozen neurotransmitters have been identified to play a role in regulating feeding behavior (Kalra 25

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et al., 1999), overlapping mechanisms have likely developed to ensure the feeding instinct is not easily extinguished and survival jeopardized. Regardless, other data confirm NPYs salience in appetite control. For example, targeted postembryonic ablation of ARC NPY neurons leads to reduced food intake and body weight (Bewick et al., 2005). B6 NPY -/mice show a 25% (Bannon et al., 2000) to 50% (Segal-Lieberman et al., 2003) reduction in the hyperphagic response to fasting compared with wild-types. NPY -/mice also exhibit less hyperphagia in streptozotocin-induced diabetes (Sindelar et al., 2002). Neuropeptide Y in neuroendocrine coordination The neuroendocrine system consists of hormones, hormone -producing and -secreting glands, and neurons that regulate these glands activity. In mammals these neurons are found in the hypothalamus, and they act to ensure coordinated secretion of hormones in response to environmental cues (e.g., food availability). As a primary messenger at the central-peripheral crossroads, NPY is produced by the hypothalamus to translate systemic signals about energy status into the local neurochemical dialect. Diverse circulating factors influence NPY expression, including leptin (Ahima et al., 1996; Baskin et al., 1999; Schwartz et al., 1996; Stephens et al., 1995; Tang-Christensen et al., 1999), insulin (Schwartz et al., 1992) and glucocorticoids (Higuchi et al., 1988b). Various gut hormones such as ghrelin, pancreatic polypeptide (PP) and peptide YY (PYY) are also known to modulate NPY expression (Asakwa et al., 2003; Batterham et al., 2002; Challis et al., 2003; Kamegai et al., 2001; Nakazato et al., 2001; Shintani et al., 2001). NPY expression is also directly responsive to energy availability via circulating glucose (Mizuno et al., 1999). The most studied of NPYs relationships is that with leptin. Leptin, a hormone produced in adipocytes in proportion to fat mass, acts as a feedback signal to the hypothalamus and plays a fundamental role in maintaining energy homeostasis (Jequier, 2002). Not only is ARC NPY gene 26

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expression downregulated by leptin administration (Ahima et al., 1996; Schwartz et al., 1996; Stephens et al., 1995), leptin also hyperpolarizes NPY neurons and inhibits their signaling activity (Spanswick et al., 1997). In the ideal model, an increase in fat mass would lead to an increase in leptin thereby decreasing NPY levels and signaling which would reduce feeding and restore body mass to the appropriate set point. Evidence from ob/ob mice (which lack leptin and grow to obese adults) supports the importance of the ARC NPY response in this model, as these mice display increased hypothalamic NPY and hyperphagia which can be reduced by either leptin administration (Ahima et al., 1996; Schwartz et al., 1996; Stephens et al., 1995) or NPY ablation (Erickson et al., 1996a). Furthermore, leptin therapy is ineffective in reversing weight gain in ob/ob mice when the ARC is rendered dysfunctional by lesioning (Takeda et al., 2002). Anorexigenic (appetite-suppressing) signals other than leptin have a similar influence on NPY. Insulin hyperpolarizes and inactivates ARC NPY (Spanswick et al., 1997) and the high NPY levels observed in streptozotocin-induced diabetes models (which underproduce insulin) can be normalized by insulin therapy (Jones et al., 1992; Sahu et al., 1997; White et al., 1990; Williams et al., 1989). The gut hormones pancreatic polypeptide (PP) and peptide YY (PYY) are released postprandially and induce a reduction in hypothalamic NPY (Asakawa et al., 2003; Batterham et al., 2002; Challis et al., 2003). Glucagon-like peptide 1 (GLP-1), a cleavage product of preproglucagon released postprandially by the small intestine (Herrmann et al., 1995), has been observed to block the NPY-induced feeding response in chicks (Furuse et al., 1997). Oxyntomodulin, another cleavage product of preproglucagon released postprandially by intestinal endocrine cells, also inhibits food intake (Dakin et al., 2001) and may do so by inhibiting ARC NPY neurons through GLP-1 receptors (Wynne and Bloom, 2006). 27

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Orexigenic (appetite-stimulating) signals activate ARC NPY neurons. Ghrelin, secreted primarily by the stomach in increasing amounts with fasting (Ariyasu et al., 2001), increases NPY levels (Cowley et al., 2003; Kamegai et al., 2001; Nakazato et al., 2001; Shintani et al., 2001). Orexin, which influences the ARC via neuronal innervation from the LHA, stimulates NPY neurons (Burdakov et al., 2003). Once activated, NPY has multiple downstream effects. Central administration of NPY, for example, has been shown to induce the release of glucoregulatory hormones including adrenocorticotropic hormone (ACTH), corticosterone, and insulin (Akabayashi et al., 1994; Leibowitz et al., 1988; Moltz and McDonald, 1985; Wahlestedt et al., 1987; Zarjevski et al., 1994). Central NPY administration also leads to reduced growth hormone (GH) and insulin-like growth factor 1 (IGF-1) release (Catzeflis et al., 1993). As ARC NPY neurons also express GH receptor (Chan et al., 1996a), they have been hypothesized to mediate feedback control of this important pituitary hormone (Chan et al., 1996b). Aging Aging, most simply defined as the temporal process of growing older, is not in itself a deleterious process. Furthermore, while it may be said that the greatest risk factor for all natural causes of death is old age, aging is not a disease either. Senescence, on the other hand, is the general term used for the constellation of negative effects associated with aging and deterioration of the organism. Aging-associated senescence includes the progressive decline of multiple organ systems linked to dysfunctions in metabolism, reproduction, cognition, and ultimately survival. Theoretically aging and senescence are separable, but in the human experience advanced age and senescence are nearly constant companions. Ditto for the majority of the scientific literature in gerontology, so the two terms are used synonymously in this work as well. 28

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Maximum lifespan, defined as the average lifespan of the longest-lived decile of a cohort (Holloszy, 2000), is often used as the gold standard in gerontology research because valid biomarkers of physiological aging have not yet been identified (Johnson, 2006). The oldest documented person in recent history, Jean Louise Calment, died in 1997 at the age of 122, representing what has been considered the near-maximum life span for humans (Cole and LA-GRG, 2004). Despite concerted effort, the mechanisms underlying the aging process that set the maximum lifespan of species have not been completely elucidated. It is likely that multiple mechanisms impact lifespan, and many potential contributors have been nominated based on commonly observed consequences of aging. These include oxidative damage (Beckman and Ames, 1998; Muller et al., 2007), aggravation of inflammatory processes (Chung et al., 2001), increased fat mass (Enzi et al., 1986; Shimokata et al., 1989), decreased muscle mass (Morley, 2001), insulin resistance (Fraze et al., 1987; Ma et al., 2002), and shifting hormonal profiles (Bartke, 2005). Subsequently, a number of theories have been developed to explain aging in the context of these phenomena. Theories of aging can be divided into two major categoriesprogrammed theories and wear and tear theoriesoutlined in Table 1-3. Programmed theories view aging as the result of an innate genetic program that dictates the rate of aging and maximum lifespan (Butler et al., 2003). In contrast, wear and tear theories envision aging as the result of spontaneous events (Muller et al., 2007). They hypothesize an organism is subject to continual stress (especially oxidative stress) from the environment and metabolism, and that ability to repair the damage declines with age such that mutated DNA, proteins, and lipids accumulate and lead to impaired function of cells and tissues. 29

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Table 1-3. General theories of aging. Programmed Theories Cell Senescence Theory Immune Theory Neuroendocrine TheoryWear and Tear Theories DNA Damage Theory Free Radical Theory Mitochondrial Theory Rate of Living Theory There is great interest in the aging field to identify a unifying theory that can singly account for the myriad symptoms of senescence. One that continues to garner proponents is the neuroendocrine theory of aging (Bishop and Guarente, 2007; Speakman and Hambly, 2007). Because the neuroendocrine system manages the general homeostatic tone of the body and mediates its response to stress, the neuroendocrine theory of aging hypothesizes these activities set the pace of aging. For example, menopause and andropause are associated with neurodegeneration (Atwood et al., 2005), reduced GH/IGF-1 in elderly humans is associated with decreased muscle strength and frailty (Ceda et al., 2005), and removal of the pituitary gland with corticosterone replacement has been shown to extend lifespan in rats (Everitt et al., 1980). Calorie Restriction Few environmental manipulations have been reported to consistently extend the lifespan of multiple species. CR, the reduction of macronutrient intake while maintaining sufficient micronutrient intake, is one notable exception. Early studies by McCay and colleagues at Cornell University established the effectiveness of CR for extending the lifespan of rats in the 1930s (McCay et al., 1935; McCay et al., 1939; McCay and Crowell, 1934). Subsequent studies have demonstrated reductions in caloric intake from 30-60% can increase maximum lifespan in a wide 30

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range of species, and CR remains the most robust intervention to manipulate the rate of aging yet studied (Ingram, 2006). Mechanisms of action The earliest documented effect of CR was its potent antitumor activity (Rous, 1914). Since then CR has been demonstrated to affect nearly every physiological process (Sinclair, 2005). From an evolutionary viewpoint the effect of CR seems to be explained by organisms having evolved mechanisms to maximize survival when faced with food scarcity (Holliday, 1989). These mechanisms remain to be definitively identified, but may include reduced adiposity, metabolic rate, body temperature, oxidative stress and insulin/IGF-1 signaling, and increased antioxidant protection, damage repair, and protein turnover rates (Masoro, 2007). Given that several of CRs effects pertain to neuroendocrine adaptations, the neuroendocrine system has been hypothesized to be a critical mediator of the beneficial effects secondary to negative energy balance (Bishop and Guarente, 2007; Lamberts et al., 1997; Meites, 1989; Nelson et al., 1995; Rehman and Masson, 2001; Speakman and Hambly, 2007). Fuel sensing systems are likely to play an important role in the initial response to altered caloric intake, since the organism must first recognize reduced caloric intake in order to respond to it. Sensation of food withdrawal occurs primarily within the gastrointestinal tract and the central nervous system. It is unlikely that the coordinated systemic response is mediated by the gastrointestinal system alone, and that the gut communicates with the brain both directly and humorally further suggests systemic control mediated by neural and endocrine factors. Neuroendocrine-related effects of CR include frequent periods of moderate hyperadrenocorticism (Ahima et al., 1996; Masoro, 2007), reduced serum thyroid hormones (Ahima et al., 1996; Herlihy et al., 1990), and inhibition of gonadal axes (Ahima et al., 1996). In 31

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fact, removal of the pituitary gland with corticosterone replacement leads to lifespan extension in rats similar to that seen with CR (Everitt et al., 1980). Neuropeptide Y: How and Y calorie restriction extends lifespan? It is well known that CR raises ARC NPY levels (Bi et al., 2003; Brady et al., 1990; de Rijke et al., 2005; Kim et al., 1988; Mercer et al., 2001; Widdowson et al., 1997). Similarly, methionine restriction extends lifespan (Miller et al., 2005; Orentreich et al., 1993) with a concomitant increase in hypothalamic NPY (White et al., 1994). Whether the increased NPY is a necessary precursor to the functional benefits associated with dietary restriction is not known, but considering NPYs unique long-term response to CR compared with other neuropeptides (Bi et al., 2003) and its plethora of physiological actions, a causal relationship is certainly plausible. One way CR may act to extend lifespan through NPY is by prolonging youthful expression levels of NPY. Aging is associated with reduced levels of NPY in the brain in general (Gruenewald et al., 1994; Higuchi et al., 1988a; Sohn et al., 2002; Vela et al., 2003) and in response to fasting (Gruenewald et al., 1996). Reduced NPY has been associated with Alzheimers disease (Alom et al., 1995; Edvinsson et al., 1993) and the development of a condition termed anorexia of aging, thought to be responsible for aging-associated undernutrition and consequent physical deterioration such as osteoporosis, sarcopenia, impaired immunity and parenchymatous organ failure (Matsumoto et al., 2000; Morley, 2001). Evidence from the rat showing NPY loss with age is progressive and independent of testosterone levels has been interpreted to suggest an active role for NPY in the anorexia of aging (Gruenewald et al., 1994). CR-driven changes in NPY expression may have downstream consequences that lead to lifespan extension. For example, NPY increases corticosterone levels, commonly observed in rodents on CR (Leibowitz et al., 1988; Wahlestedt et al., 1987). NPY is also thought to mediate 32

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the reductions in thermogenesis and body temperature that accompany CR (Kotz et al., 1998). Most importantly, transgenic rats that overexpress NPY have been found to have improved stress resistance as demonstrated through reduced blood pressure in response to novelty stress and increased mean (but not maximum) lifespan (Michalkiewicz et al., 2003). Lifespan benefits with increased NPY may be reflected in humans as well, as long-lived female centenarians have high plasma NPY levels compared with younger women (Baranowska et al., 2006). Because CR is so successful in multiple species there is increasing interest in the therapeutic potential for CR to extend maximum lifespan in humans (Ingram, 2006). In fact, the prospect of CR in humans is already a reality and there are societies, books and internet sites devoted to CR in humans (see http://www.calorierestriction.org/ ). Despite the willful adherence of this CRonie minority, the current hunch of gerontologists is that most humans will prefer not to regimen their diet in the presence of an abundant food supply so long as alternatives, or mimetics, to CR may be found. Indeed, candidate CR mimetics are already under investigation (Ingram, 2006) with the hopes of rendering CRs benefits attainable to the overeating masses. This movement presupposes one critical notion: that CRs beneficial effects can be separated from those imposed on appetite. Taking into account the profound effects of CR on the appetite-regulating machinery of the hypothalamus and NPY, the key central hunger signal that may play a critical role in transducing CRs benefits, it may be that the very effectiveness of CR stems from the same hunger-inducing phenomena that CR mimetic research seeks to repress. Overall Rationale It is well established that CR promotes beneficial adaptations to laboratory animals that slow the aging process. Such changes include reduced rates of cancer, improved glucose homeostasis, enhanced oxidative stress resistance, and promotion of cognitive capabilities. The involvement of neuroendocrinological mechanisms in mediating these effectsand in particular 33

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the importance of the sensation of hunger in driving these effectsis unknown. This research project investigated the effects of CR in two mouse models of handicapped hunger sensing: mice with a chemical lesion of the ARC and mice with a genetic knockout of NPY. The results of these studies will have significance in shaping the future directions of life extension research, the search for CR mimetics, and the applicability of CR to humans. The following hypotheses were tested: Hypothesis #1: The behavioral response to calorie restriction is blunted by impaired neuroendocrinological signaling. Specific aims Assess physical agility in CR versus AL mice by inclined screen and rotarod testing. Assess anxiety-related behavior in CR versus AL mice by open field test. Assess learning and memory in CR versus AL mice by Morris water maze test. Hypothesis #2: The physiological response to calorie restriction is blunted by impaired neuroendocrinological signaling. Specific aims Determine the effects of CR on serum factors including leptin, adiponectin, and insulin. Compare the effects of CR on glucose homeostasis including fasting blood glucose levels, glucose tolerance and insulin sensitivity. Hypothesis #3: Resistance to oxidative stress by calorie restriction is blunted by impaired neuroendocrinological signaling. Specific aims Determine the expression levels of stress-related proteins in the liver. Determine the stress response in the liver to the toxin diquat. Hypothesis #4: Resistance to tumor formation by calorie restriction is blunted by impaired neuroendocrinological signaling. Specific aims Assess resistance to tumor formation after DMBA/TPA treatment in time to presentation with tumors, quantity of tumors formed and size of tumors formed. Measure the levels of oxidatively damaged protein in the skin. 34

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CHAPTER 2 CALORIE RESTRICTION ALTERS PHYSICAL PERFORMANCE BUT NOT COGNITION IN TWO MODELS OF ALTERED NEUROENDOCRINE SIGNALING Lifespan extension aside, calorie restriction (CR) consistently confers numerous beneficial functional adaptations upon laboratory animals (Ingram et al., 2006; Sinclair, 2005). Of these, the behavioral changes that have been associated with calorie restriction include enhanced physical and cognitive performance (Ingram et al., 1987; Ishihara et al., 2005; Means et al., 1993; Stewart et al., 1989). Enhanced physical performance may be attributed in large part to improved body composition in CR animals compared to fully-fed controls, but the mechanisms responsible for the cognitive effects are not fully understood. Hypotheses linking CR to altered cognitive capability include reduction of neural oxidative stress, promotion of synaptic plasticity and induction of various stress proteins and neurotrophic/neuroprotective factors (Mattson, 2003; Prolla and Mattson, 2001). How CR might trigger such neuromodulation has not been fully characterized and is thus the subject of this study. The arcuate nucleus (ARC) in the hypothalamus of rodents is the first-order processing unit for nutrient status in the brain (Heijboer et al., 2006; Wynne et al., 2005). The sum of peripheral signals relating information regarding nutrient status (e.g. leptin from adipose tissue and ghrelin from the gut) converge on the ARC where they are converted to a neurochemical report (e.g. Neuropeptide Y (NPY), pro-opiomelanocortin (POMC)) that is transmitted to brain regions involved in the activation of food-related behavior. As would be expected, ARC output is altered by CR such that expression of orexigenic NPY is upregulated (Bi et al., 2003; Brady et al., 1990; de Rijke et al., 2005; Kim et al., 1988; Mercer et al., 2001; Widdowson et al., 1997). While CR-induced NPY upregulation was established more than 15 years ago (Kim et al., 1988), causative roles for the ARC and NPY in mediating the effects of CR remain putative. Since mice and rats treated neonatally with monosodium glutamate (MSG) exhibit neuronal 35

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death specifically in the ARC (Burde et al., 1971; Olney, 1969) owing to blood-brain barrier permeability at the median eminence (Peruzzo et al., 2000), this technique could be employed to evaluate the role of the ARC in CR-related adaptations. Neonatal MSG-induced neurotoxicity in mice results in a pronounced reduction in ARC neuropeptide expression (Broberger et al., 1998; Meister et al., 1989) including NPY (Abe et al., 1990; Broberger et al., 1998; Kerkerian and Pelletier, 1986; Legradi and Lechan, 1998; Meister et al., 1989) and is a well-characterized model for evaluating the functional importance ARC neurons (Cameron et al., 1978; Hu et al., 1998; Seress, 1982). Evidence suggests the ARC and NPY have functional roles in cognition. For example, ARC-lesioned mice display impaired memory (Park et al., 2000). Central administration of NPY has been shown to enhance memory retention in mice (Flood et al., 1987), although a regulatory role for NPY in learning and cognition is still putative (Redrobe et al., 1999). Whether the ARC or NPY have been involved in behavioral adaptations following CR is presently unknown although neuroendocrine signaling has been proposed to play an important role in mediating the response to CR (Meites, 1989; Speakman and Hambly, 2007; Walford and Spindler, 1997). We hypothesize that the response by the ARC (and fluctuations in ARC NPY in particular) act to help mediate downstream effects of CR. To test this hypothesis we employed two mouse models, an MSG-induced ARC-lesioned mouse and an NPY knockout mouse (NPY -/) (Erickson et al., 1996b) and compared their performance with control animals on a battery of behavioral tests after CR or ad libitum (AL) feeding. Materials and Methods Animals and Diets Descriptions of the mice and diets used for the following experiments, as well as the experimental timeline, can be found in Appendix A. 36

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Rotarod A five-station Rota-Rod Treadmill for mice (Med Associates, St. Albans, VT) consisting of a mechanized rotating bar (3 cm diameter) suspended 16 cm above a platform was used for this experiment. On the first day the mice were habituated to the rotarod for 120 s at a constant speed of 4 rpm. The following day testing was completed in 3 trials of increasing speed from 4 to 40 rpm. Mice were tested for a maximum of 300 s in each trial. The latency to fall was recorded for each trial and averaged for each group. Results shown are group means of the averaged three trials per mouse. Inclined Screen Mice were placed in a tilted, open field (55 cm 2 surface area constructed from a 0.6 cm 2 wire mesh grid with black sides extending 15 cm above the grid) and movement was recorded for 300 s using Field 2020 tracking software from HVS Image (Buckingham, UK). Results were averaged for total distance traveled for each mouse. Open Field Locomotor Activity Mice were placed in a level, open field (same as for inclined screen) and movement was recorded for 300 s using Field 2020 tracking software from HVS Image. Results were averaged for both total distance traveled and time spent in the center of the field (comprised of the interior 40 cm 2 ) or around the periphery (the area extending 7 cm inside around the sides of the field). Morris Water Maze The apparatus consisted of a white circular plastic pool (100 cm diameter and 70 cm high) which was filled with water (24 1 C) rendered opaque by addition of white Dry Temp paint powder (Palmer Paint Products Inc., Troy, MI, USA). Spatial navigation cues were affixed to a clear, plastic cylinder concentric with the pool and extending approximately 30 cm above the top of the pool. A circular (10 cm diameter) escape platform (the target) was submerged just below 37

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the water surface. Each acquisition trial (4 trials per day for 6 days) was started by placing a mouse in the water facing the wall of the tank. The location of entry of the mouse changed every trial such that mice entered the maze from each direction once each day, and the order of start position was set randomly for each mouse. A trial lasted until the mouse found the platform or until 60 s had elapsed. If a mouse did not find the platform within 60 s, it was placed on the platform for 30 s in order to familiarize it with the location of the platform. After completion of the fourth trial on each day, the mouse was returned to its home cage. A camera was mounted on the ceiling in the center of the pool to track the swim route of the mouse. Data were collected using HVS Image with Water 2020 Software (Buckingham, UK). On day 7 each mouse performed a probe trial where the platform was removed and each mouse was allowed 60 s to swim freely. Time (%) spent in each quadrant was recorded and averaged for each group. Visual Platform Training On the 8 th day of the Morris water maze experiment 4 trials of a visual cue test were administered. For each trial the platform was located in a different quadrant of the pool. The visual cue was a metal camera mount attached to the escape platform. Each mouse had 60 s to find the platform. If the mouse failed to find the platform in the allotted time, it was placed on the platform for 15 s. The latency to find the platform was recorded for each mouse. Contextual Fear Conditioning The apparatus consisted of a mouse modular test chamber (30x24x21 cm) enclosed in a sound-attenuating cubicle (56x38x36 cm), both from Med Associates. The sides of the test chamber were aluminum, the ceiling and front door were Plexiglas and the floor was constructed from stainless steel rods wired to a shock generator. A light, ventilation fan, and speaker were mounted to the walls of the chamber. Testing was performed in a quiet, isolated room. Each mouse received 5 trials of a tone (15 s, 80 decibels, 3000 Hz, 10 ms rise/fall time) followed 38

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immediately by a foot shock (1 s, 1 mA) through the floor with a 60 second inter-trial interval. Sixty seconds after the final trial, each mouse was returned to its home cage. The chambers were cleaned with 70% ethanol between each mouse. Twenty-four hours later, the mice were returned to the conditioning chamber for 5 minutes. The Video Fear Conditioning System (MED-VFC, Med Associates) was used to measure freezing behavior, which was considered to be the amount of time a mouse spent motionless based on an index of motion. From these data a percent-time freezing score was then calculated for each mouse. Statistics For the inclined screen, rotarod and open field data, a two-way analysis of variance (ANOVA) was used with Student-Newman-Keuls tests performed post hoc. For the water maze data, a two-way or a two-way with repeated measures ANOVA followed by a Student-Newman-Keuls test was employed. An ANOVA with repeated measures and Student-Newman-Keuls tests were used for the contextual fear conditioning data. Results Body Weights Mean cumulative weight change prior to behavioral testing is depicted in Fig. 1. The 129S1 mice (Fig. 2-1a) gained body weight on AL feeding and lost weight on CR feeding in both genotypes. All B6 AL mice gained weight over the feeding period, and MSG AL mice, though starting at a lower weight, gained weight rapidly to match SAL AL average weight by week 2 (Fig. 2-1b). SAL CR mice lost weight over time but MSG CR mice were lighter at study start and maintained their initial weight throughout the feeding period. 39

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129S1 Body WeightsWeeks 0123456Body Weight (g) 010203040 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR A B C57 Bo dy WeightsWeeks 0123456 Body Weight (g) 010203040 SAL AL MSG AL SAL CR MSG CR B6 Figure 2-1. Mean group body weight S.E.M. for the six weeks prior to behavioral testing in the 129S1 mice (A) and B6 mice (B). (n = 10) Physical Agility Performance on the inclined screen did not differ significantly by path length for the 129S1 mice by either diet or NPY expression (Fig. 2-2a). Within the B6 groups, a two-way ANOVA revealed significant effects of treatment (F(1,36) = 5.36; p = 0.026) and diet (F(1, 36) = 13.92; p < 0.001) but no significant interaction between treatment and diet on the path length of the inclined screen. Post hoc analysis (p < 0.05; Student-Newman-Keuls test) revealed SAL and MSG CR mice traveled significantly farther than their respective AL groups (Fig. 2-2b). A two-way ANOVA (F(1,36) = 12.14; p = 0.001) revealed a significant effect of diet among the 129S1 mice on the latency to fall from the rotarod (Fig. 2-2c). Post hoc analysis (p < 0.05; Student-Newman-Keuls test) showed that only the NPY -/CR mice took significantly longer to fall compared with NPY -/AL mice. Within the B6 groups, a two-way ANOVA revealed significant effects of treatment (F(1,36) = 14.43; p < 0.001) and diet (F(1,36) = 50.52; p < 0.001) but no significant interaction between treatment and diet on the latency to fall from the rotarod. Post hoc analysis (p < 0.05; Student-Newman-Keuls test) showed that CR also 40

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significantly extended latency (Fig. 2-2d), although MSG mice underperformed compared with their SAL counterparts in both diet conditions. B6 Rotarod SAL ALMSG ALSAL CRMSG CRTime (s) to fall 050100150200250300 A C* BB6 Inclined Screen SAL ALMSG ALSAL CRMSG CRPath Length (m) 05101520 ** BB6 Inclined Screen SAL ALMSG ALSAL CRMSG CRPath Length (m) 05101520 **B6 Inclined Screen SAL ALMSG ALSAL CRMSG CRPath Length (m) 05101520 ** D**129S1 Inclined Screen NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRPath Length (m) 05101520 129S1 Inclined Screen NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRPath Length (m) 05101520 129S1 Rotarod NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRTime (s) to fall 050100150200250300 129S1 Rotarod NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRTime (s) to fall 050100150200250300 Figure 2-2. Effects of ARC function and diet on physical performance. Path length on the inclined screen did not differ in the 129S1 mice by either diet or NPY expression (A). Within the B6 groups (B), CR mice traveled significantly farther than their respective AL groups. Latency to fall on the rotarod was improved by CR in the 129S1 mice regardless of genotype (C) although only the NPY -/CR mice took significantly longer to fall compared with NPY -/AL mice. CR also significantly extended latency within the B6 rotarod groups (D), although both MSG groups fell faster than their SAL counterparts. (Mean S.E.M., = significantly different from AL; = significantly different from SAL; n = 10.) 41

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D129S1 Open FieldLocomotor Activity NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRPath Length (m) 051015202530 A*129S1 Open FieldLocomotor Activity NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRPath Length (m) 051015202530 129S1 Open FieldLocomotor Activity NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRPath Length (m) 051015202530 A*129S1 Open FieldThigmotaxis PeripheryCenter% Time 020406080100 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR ** C129S1 Open FieldThigmotaxis PeripheryCenter% Time 020406080100 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR ** C BB6 Open FieldLocomotor Activity SAL ALMSG ALSAL CRMSG CRPath Length (m) 051015202530 BB6 Open FieldLocomotor Activity SAL ALMSG ALSAL CRMSG CRPath Length (m) 051015202530 B6 Open FieldThigmotaxis PeripheryCenter% Time 020406080100 SAL AL MSG AL SAL CR MSG CR Figure 2-3. Open field locomotor activity and exploratory behavior of 129S1 and B6 mice. By measure of total distance traveled both NPY -/groups traversed a greater distance in the open field than NPY +/+ mice, and the NPY -/CR mice traveled significantly farther than NPY +/+ CR mice (A). MSG mice traveled less on average than the SAL mice, but this difference was not significant (B). Assessing thigmotaxic behavior showed the relative time spent in the center of the open field was significantly different among the 129S1 mice (C) such that the NPY -/AL and CR mice spent less time in the center of the field than NPY +/+ CR mice (p = 0.003 and p = 0.006, respectively). MSG mice also displayed decreased center-field exploration however this difference was not significant (D). (Mean S.E.M., n = 10.) Open Field Locomotor Activity By measure of total distance, a two-way ANOVA revealed a significant effect of genotype (F(1,36) = 8.59; p = 0.006) on open field testing such that the NPY -/mice traveled farther in the 42

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open field than NPY +/+ mice (Fig. 2-3a), though post hoc analyses (p<0.05; Student-Newman Keuls) showed that only the NPY -/CR mice covered significantly more ground than NPY +/+ CR mice. There were no significant differences among the B6 groups in total distance traveled (Fig. 2-3b). Percent time spent in the center of the open field was significantly different among the 129S1 mice (F(7,72)=41.44; p < 0.0001) (Fig. 2-3c), such that the NPY -/AL and CR mice spent less time in the center of the field than NPY +/+ CR mice (p = 0.003 and p = 0.006, respectively). As with total distance traveled there were no significant differences among the B6 groups in time spent in the center or periphery of the field although the MSG mice spent less time on average in the center of the field (Fig. 2-3d). Morris Water Maze Performance There were no significant differences in latency to find the hidden platform during training or time spent in the correct quadrant during the probe trial among the 129S1 mice (Fig. 2-4a and 2-4c). In the B6 mice, however, a two-way repeated ANOVA showed significant effects of group (F(3, 36) = 19.92; p < 0.001), day (F(5, 180) = 16.94; p < 0.001), and interaction of group by days (F(15, 180) = 3.07; p < 0.001) on the latency during the hidden platform training (Fig. 2-4b). The effect of group was significant on days 2-6 such that the MSG mice, regardless of diet, showed significantly longer latencies (p < 0.05; Student-Newman-Keuls test) to find the platform as compared to the SAL groups. A two-way ANOVA revealed a significant effect of treatment (F(1,36) = 41.43; p < 0.001) on the percent time spent in the correct quadrant during the probe trial. Post hoc analysis (p < 0.05; Student-Newman-Keuls test) showed also that during the probe trial the MSG mice, regardless of diet, spent significantly less time in the correct quadrant compared with the respective SAL groups (Fig. 2-4d). 43

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B6 Probe Trial CorrectOppositeRight AdjLeft AdjTime (%) swimming in quadrant 020406080100 SAL AL MSG AL SAL CR MSG CR 129S1 Water Maze TrainingTraining Day 0123456Latency (s) to find platform 0102030405060 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR AB6 Water Maze TrainingTraining Day 0123456Latency (s) to find platform 0102030405060 SAL AL MSG AL SAL CR MSG CR B*****B6 Water Maze TrainingTraining Day 0123456Latency (s) to find platform 0102030405060 SAL AL MSG AL SAL CR MSG CR B***** D** C129S1 Probe Trial CorrectOppositeRight AdjLeft AdjTime (%) swimming in quadrant 020406080100 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR C129S1 Probe Trial CorrectOppositeRight AdjLeft AdjTime (%) swimming in quadrant 020406080100 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR Figure 2-4. Morris water maze performance during training and testing (the probe trial). All 129S1 groups performed equivalently in the water maze without effect of genotype or diet during training (A) and testing (C). After the first day of training, MSG mice, regardless of diet, showed significantly longer latencies to find the platform compared with SAL mice (B) and the same MSG groups failed to swim in the correct quadrant during probe testing (D). (Mean S.E.M., n = 10.) Visual Assessment There were no significant differences in latency to find the cued platform among the 129S1 mice (Fig. 5a). However, for B6 mice a two-way ANOVA revealed a significant treatment effect (F(1,36) = 90.19; p < 0.001) on the latency during visual cue training. Post hoc analysis (p < 44

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0.05; Student-Newman-Keuls test) showed that MSG mice, regardless of diet, showed significantly longer latencies to find the cued platform as compared with SAL mice (Fig. 5b). B6 Visual Cue SAL ALMSG ALSAL CRMSG CRLatency (s) to find platform 0102030405060 A129S1 Visual Cue NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRLatency (s) to find platform 0102030405060 A129S1 Visual Cue NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRLatency (s) to find platform 0102030405060 129S1 Visual Cue NPY+/+ALNPY-/-ALNPY+/+CRNPY-/-CRLatency (s) to find platform 0102030405060 B**B6 Fear Conditioning BaselineTesting% Time Freezing 020406080100 SAL AL MSG AL C**B6 Fear Conditioning BaselineTesting% Time Freezing 020406080100 SAL AL MSG AL C** Figure 2-5. Visual assessment via visual cue testing in the water maze and contextual fear conditioning. 129S1 mice swam to the platform with equivalent acuity (A); whereas MSG mice, regardless of diet, showed significantly longer latencies to find the cued platform as compared with SAL mice (B). Another vision-dependent test, contextual fear conditioning, conversely revealed MSG AL were able to associate the conditioning chamber with shocks as they froze significantly more during the 5 minute context exposure than during baseline exposure (C). (Mean S.E.M., n = 10 for panels A and B, n = 5 for panel C.) 45

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Contextual Fear Conditioning Percent time freezing for B6 AL mice during testing was significantly higher (F(1,8) = 13.046; p = 0.007) than during baseline exposure (i.e. prior to tone-shock trials) to the conditioning chamber (Fig. 2-5c). Post hoc analysis showed both SAL (p < 0.05; Student-Newman-Keuls test) and MSG (p < 0.05; Student-Newman-Keuls test) groups spent significantly more time freezing during testing than during baseline. Discussion The present study was designed to assess whether impairment of neuroendocrinological function would alter the behavioral response to CR in two mouse models. In measures of physical agility, the mice performed better after CR than AL feeding. The inclined screen, which is constructed from a wire mesh the mice must grip and hold to traverse, assesses both locomotor behavior and grip strength. Thus, the greater distance traveled in the B6 CR mice on the inclined screen is corroborated by previous data showing CR improved ability to hang from a bar and resist slipping on a tilted platform (Ishihara et al., 2005). The rotarod results also agree with a previous study in which CR improved rotarod performance in a preclinical mouse model for amyotrophic lateral sclerosis (Hamadeh et al., 2005) and support the inclined screen data. As the inclined screen and rotarod are both tests of general physical ability, it is not surprising that the results of the tests are similar. However, because the trend to improved performance by CR in the B6 mice is more pronounced on the rotarod than on the inclined screen and a significant improvement is seen in the 129S1 mice exclusively on the rotarod, our results suggest the rotarod is a more sensitive task for evaluating diet-induced physical fitness. Unlike diet, which had a significant effect on motor performance in this study in both the NPY and the arcuate mouse models, altered neuroendocrine function asserted less of an effect on measures of physical ability. This suggests that improved body composition in CR animals 46

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compared with AL controls accounts for some of the enhanced motor capability, as all CR mice either lost weight or were prevented from weight gain during the feeding period. By NMR assessment we also know that CR prevented a decrease in lean-to-fat ratio in the mice (see Table 3-1), which could account for some of the increased performance in the CR mice. NPY does not appear to be necessary for the enhanced performance associated with CR as NPY -/CR mice improved equivalently to NPY +/+ CR mice on the rotarod. There is an apparent effect of ARC integrity on fitness, however, as SAL mice displayed greater endurance on the rotarod than lesioned MSG mice regardless of diet. Rather than a direct influence of the ARC on motor ability, this effect may again be attributed to differences in body composition; NMR analysis showed MSG mice had greater fat mass than SAL mice in their respective diet treatments (Table 3-1). By measure of total distance traveled, open field behavior of the mice in this study does not appear to depend much on ARC function or diet treatment. Prior to testing we had hypothesized CR may increase locomotor activity in the control mice on an open field as a manifestation of increased drive to forage (Holloszy and Schechtman, 1991; Ingram et al., 1987; Weed et al., 1997). CR is known to induce NPY expression in the ARC (Bi et al., 2003; Brady et al., 1990; de Rijke et al., 2005; Kim et al., 1988; Mercer et al., 2001; Widdowson et al., 1997), and previous work has shown that centrally-injected NPY promotes open field locomotor activity in mice (Karlsson et al., 2005; Nakajima et al., 1994). In the present study, however, NPY -/CR mice traveled farthest. Also contrary to the present findings, previous studies have shown that both mice and rats exposed to MSG were hyperactive in an open field (Saari et al., 1990; Yu et al., 2006), although differences in the dosing schedule may account in part for this discrepancy. 47

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Analysis of open field behavior in terms of spatial preference revealed a strong preference for the periphery of the field in the models of impaired ARC function. Persistence in peripheral tracking is known as thigmotaxis and is an index of anxiety in rodents (Simon et al., 1994). NPY is a potent regulator of stress and anxiety (Heilig, 2004), such that increased central NPY has anxiolytic effects (Heilig et al., 1989; Karlsson et al., 2005; Kask et al., 1998; Thorsell et al., 2000) whereas reduced central NPY is anxiogenic (Bannon et al., 2000; Palmiter et al., 1998). NPY -/mice have previously been shown to demonstrate anxiety through thigmotaxis on an open field (Bannon et al., 2000). The behavior of the mice in the present study confirms this phenotype. One neuroendocrine effect of MSG-induced neurotoxicity in mice is a pronounced reduction in NPY production (Abe et al., 1990; Broberger et al., 1998; Kerkerian and Pelletier, 1986; Legradi and Lechan, 1998; Meister et al., 1989). The MSG mice in this study displayed increased avoidance of the center field, but this effect was not significant and may reflect the observation that arcuate lesions induce deficits in multiple neuropeptides in addition to NPY (Broberger et al., 1998; Meister et al., 1989). In contrast to the extensive and consistent evidence for NPY in the regulation of anxiety, the role of NPY in learning and memory-related behaviors is less clear. For example, central injection of NPY has been shown to enhance memory or induce amnesia depending on the site of injection (Flood et al., 1989; Flood et al., 1987). Cognitive capability wanes with age (Ingram et al., 1987; Rapp et al., 1999), as does hypothalamic expression of NPY (Cadiacio et al., 2003; Higuchi et al., 1988a; Vela et al., 2003), although one study found no correlation between NPY interneuron loss and spatial deficits (Cadiacio et al., 2003). Adding to the ambiguity, one study with NPY-overexpressing transgenic rats showed impaired spatial learning (Thorsell et al., 2000) while another found no significant differences (Carvajal et al., 2004) on the Morris water maze. 48

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In the current study, no differences were observed in the NPY -/mice in the water maze during training or the probe trial although there was a nonsignificant trend towards better performance in the NPY -/mice, which contrasts with previous work in which these mice demonstrated a slight impairment in water maze acquisition (Palmiter et al., 1998). There was no discernable effect due to diet treatment. In the B6 mice, however, MSG treatment precluded the mice from learning the taskwhile the SAL mice improved continuously over the training period and actively swam in the correct quadrant during the probe trial, the MSG mice performed nearly equivalently every day of training and swam indiscriminately through all quadrants during the probe trial. Deficits in spatial learning in the water maze have been observed in MSG-injected rats (Olvera-Cortes et al., 2005; Saari et al., 1990) and have been attributed to cognitive and visual defects. To assess the visual ability of our mice, we performed a visual cue test following the probe trial in the water maze and also a vision-dependent fear conditioning trial. During the visual cue test, MSG mice swam significantly longer before locating the escape platform, suggestive of visual impairment. Thus, the deficient performance of the MSG mice in all phases of water maze testing suggests cognitive defects cannot solely account for the effect but may also involve defective visual processing. To further assess the visual ability of the mice, we then employed a modified version of fear conditioning. One study evaluating NIH Swiss and Black Swiss micewhich are visually impaired due to genetic retinal degenerationfound these mice were deficient in both the water maze and contextual fear conditioning (Clapcote et al., 2005). In the present study, the MSG mice were equivalently competent to SAL mice in the fear conditioning experiment in discerning the visually-cued environment during testing. These data suggest MSG-induced water maze deficits may be less related to vision than has been previously thought, and indeed a study with MSG-treated mice in a non-spatial water maze task showed 49

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greater escape latencies in MSG mice than controls (Wong et al., 1997). MSG-induced neurotoxicity beyond the ARC, e.g. in the hippocampus, has also been nominated as a causative factor in cognitive deficits associated with the treatment (Olvera-Cortes et al., 2005; Wong et al., 1997). If hippocampal deficits are causal in the present study, the results from our mice lend further evidence for the dissociation between fear conditioning and spatial learning in the hippocampus (Bannerman et al., 2004; Maren et al., 1998). Regardless of the competency of the ARC-lesioned mice in contextual fear conditioning, in this study CR for 6 weeks was insufficient to boost water maze performance in these mice. Like environmental enrichment, which has been successfully employed following MSG exposure to improve rat performance in the water maze (Saari et al., 1990), CR is thought to promote neurogenesis and synaptic plasticity and thus restore function subsequent to injury (Mattson et al., 2003). Our lack of benefits may indicate the mice were not on the diets sufficiently long enough for significant changes in neurogenesis or neuroplasticity to occur. Alternatively, because the level of CR (30% of AL intake by weight) used in this study was not enough to induce weight loss in the MSG CR mice relative to baseline it may also have been insufficient to influence neuroprotective mechanisms. In conclusion, our results indicate measures of physical performance are more affected by diet than ARC or NPY signaling while anxiety and cognition are less influenced by diet than ARC and NPY signaling. Despite established effects of CR on ARC signaling our results suggest a mechanistic separation between the two where behavior is concerned. 50

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CHAPTER 3 REDUCTIONS IN ADIPOSITY AND LEPTIN FOLLOWING CALORIE RESTRICTION DO NOT FULLY EXTEND TO IMPROVED GLUCOSE HOMEOSTASIS IN MODELS OF IMPAIRED NEUROENDOCRINOLOGICAL SIGNALING Among the mechanisms through which CR has been postulated to extend lifespan, modulation of endocrine systems and glucose homeostasis are recurring, interrelated themes. Proponents of the endocrine and glucose homeostasis theories of aging hypothesize the profiles of circulating hormones and glucose levels imposed by CR provide a protective environment that decelerates aging (reviewed in Sinclair, 2005). Indeed, it has been known for many years that CR results in reduced blood glucose levels and altered hormone levels, and that these alterations are associated with enhanced longevity (for further reviews see Bartke, 2005, and Tatar et al., 2003). For example, aging is associated with increasing levels of circulating insulin (Ma et al., 2002) mirrored by increasing insulin resistance (Ferrannini et al., 1996). By significantly decreasing pancreatic insulin output, CR minimizes this detrimental cycle (Dean et al., 1998). Reducing adipose stores is another way CR profoundly alters the hormonal milieu in circulation. Increasingly recognized for its impact on health through chronic disease, adipose tissue is an endocrine organ that secretes numerous factors with diverse physiological effects (Ahima and Flier, 2000). Among these, leptin and adiponectin are expressed by adipocytes differentially depending on adiposity and aging. Leptin is known to increase with adiposity (Frederich et al., 1995) and age (Ma et al., 2002); adiponectin conversely decreases with adiposity (Combs et al., 2002, Stefan et al., 2002) and age (Zhu et al., 2007). Maintenance of youthful adipokine levels by CR (Zhu et al., 2007) may forestall development of insulin resistance (Berg et al., 2001) and its consequences with aging. Poised at the nexus of the peripheral circulation and the hypothalamus, the arcuate nucleus has been nominated as a critical mediator of CRs physiological effects (Bartke, 2007, Speakman 51

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and Hambly, 2007, Walford and Spindler, 1997). Reduced food intake and energy stores signal negative energy balance to the ARC which adjusts its neurochemical output accordingly, regulating the expression and signaling of several neuropeptides including NPY (Schwartz et al., 2000). A driving force behind the central response to negative energy balance, NPY expression is responsive to insulin (Schwartz et al., 1992), leptin (Stephens et al., 1995) and probably adiponectin (Steinberg and Kemp, 2007). NPY is also directly responsive to circulating glucose levels (Mizuno et al., 1999). Since downstream effects of NPY include reducing thermogenesis (Kotz et al., 1998) and inducing the release of glucoregulatory hormones like ACTH, corticosterone and insulin (Akabayashi et al., 1994; Leibowitz et al., 1988; Moltz and McDonald, 1985; Wahlestedt et al., 1987; Zarjevski et al., 1994), NPY may be critical for certain changes in body temperature and glucose homeostasis to occur under CR. In the current study we hypothesized impairment of ARC function through either MSG-lesioning or NPY knockout would blunt the physiological effects associated with CR, focusing on changes associated with glucose handling. Materials and Methods Animals and Diets Descriptions of the mice and diets used for the following experiments, as well as the experimental timeline, can be found in Appendix A. Body Composition Analysis Measurements of lean, fat and fluid mass were acquired using a Minispec LF90 (Bruker Optics, The Woodlands, TX), an NMR analyzer for assaying whole body composition of live mice without the use of anesthesia. Briefly, mice were placed into a plastic insert device and secured snugly to minimize movement during the scan. The plastic insert with the mouse was 52

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entered into the reading chamber of the Minispec and the scan was initiated. Scanning takes approximately 1 min, after which mice were freed from restraint and returned to the home cage. Body Temperature Measurement 129S1 mice were assayed for temperature by rectal probe. C57 mice were microchipped subcutaneously with implantable electronic transponders (Bio Medic Data Systems, Inc., Seaford, DE). Hormonal Assessments During the eleventh week of the study (see Appendix A, Fig. A-2) mice were fasted overnight (16h) and blood was collected without anesthesia by retro-orbital puncture. After centrifugation sera were stored at 80 C until assay. Leptin Measurements were taken with the Luminex-based bead array method using the LINCOplex simultaneous multianalyte detection system (Linco Research, Inc., St. Charles, MO) following the manufacturers instructions. Adiponectin Adiponectin levels were assayed using the mouse ELISA kit from Alpco Diagnostics (Salem, NH) following the manufacturers protocol except sera were diluted 1:40,000 (instead of 1:20,000) prior to assay. Glucose Homeostasis Plasma samples for glucose and insulin quantification were collected simultaneously by retro-orbital puncture from animals without the use of anesthesia. The baseline blood draw was taken from animals that had been fasted overnight (16h). 53

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Fasting blood glucose Glucose was assayed at the time of blood draw by applying 5 L whole blood to an Elite XL Glucometer (Bayer, Elkhart, IN). Oral glucose tolerance test Following the baseline blood draw, mice were gavaged with a 1.5 g/kg glucose solution. Blood glucose was then assayed as above at 15, 30, 60 and 120 min post-gavage. Insulin measurement Blood samples collected during the OGTT were centrifuged and the sera stored at 80 C until assay. Insulin levels were determined using a commercially-available ELISA kit (Crystal Chem Inc., Downers Grove, IL). The manufacturers instructions were followed with the exception that the volume of serum used was increased to 15 l (as opposed to 5 l) per sample. HOMA calculations Insulin resistance was determined by the Homeostasis Model Assessment (HOMA) index, calculated from the fasting blood glucose and insulin values by the HOMA2 Calculator software available from the Oxford Center for Diabetes, Endocrinology and Metabolism, Diabetes Trials Unit ( http://www.dtu.ox.ac.uk/homa/ ). Statistics Data are expressed as the mean S.E.M. Statistical analyses were carried out by ANOVA with repeated measures (body composition) or one-way ANOVA (fasting blood glucose) followed by Fishers LSD tests. Statistical significance was inferred at p 0.05. Results Body Composition Results from the NMR body composition analyses are presented in Table 3-1. Mice from the 129S1 background started the study at equivalent body composition independent of 54

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55 genotype. Both genotypes also responded similarl y to the diet treatments; body weight increased on AL diets and decreased on CR diets, with corresponding indivi dual changes to lean, fat and fluid mass and percent body fat. The increase in fat mass was the most robust change over time in the AL groups, and thus the ratio of lean mass to fat mass was decreased by AL feeding throughout the study. Conversely, the CR mice increased their ratio of lean to fat mass during the study and this effect was greatest in the NPY -/mice, which lost more fat mass on CR than the NPY +/+ controls. This contrasts with the data from the NPY -/AL mice which were bigger than their NPY +/+ AL counterparts at week 12 (reflected by the body weight and food intake data in Fig. A-3 of the appendix) and had signifi cantly higher lean mass at that time. In the B6 mice, notable differences were observed between the treatment groups starting from baseline. Specifically, the MSG mice were smaller yet had a higher fat mass than SAL mice. AL feeding exacerbated the fatty phe notype of these mice and MSG AL mice were ultimately heavier than their SAL AL counterparts despite starting the diet at lower body weight. CR in both groups acted primarily to temper fat mass gain such that there was no significant difference between baseline and week twelve. At all time points the MSG CR mice showed a greater percentage of body fat than the SAL CR mice.

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Week 0 Week 4 Week 8 Week 12 Week 0 Week 4 Week 8 Week 12 NPY+/+ AL26.30.528.91.229.01.1a30.21.2aSAL AL21.40.324.70.5a26.40.5a29.31.1bNPY-/AL26.90.828.70.929.10.933.41.0MSG AL18.40.625.50.6a,27.10.7a,33.00.5b,WeightNPY+/+ CR26.10.722.30.3*22.00.3a,*22.30.2a,*SAL CR21.10.317.60.2b,*19.70.1a,*19.70.3a(g)NPY-/CR27.21.023.80.6a,*,21.70.4b,*21.90.3b,*MSG CR190.618.70.3*,18.10.2*,19.70.2NPY+/+ AL15.70.316.90.6a16.30.617.20.6aSAL AL13.10.215.30.3a16.10.3b17.50.6cNPY-/AL16.00.416.90.516.50.418.90.5a,MSG AL10.90.414.70.4a,15.00.4a,18.40.3bNPY+/+ CR15.60.413.30.2a,*12.90.1a,*13.40.1a,*SAL CR13.10.210.80.1a,*11.90.1a,*12.20.2a,*NPY-/CR16.20.514.40.3a,*,12.70.2b,*13.40.2b,*MSG CR11.20.311.10.1*10.70.1*,12.00.1a,*NPY+/+ AL6.10.27.40.6a8.30.6a8.80.6aSAL AL4.00.14.80.2a6.00.2b7.30.4cNPY-/AL6.20.47.50.4a8.20.4a10.00.5bMSG AL4.70.28.10.2a,9.90.2b,12.20.2c,NPY+/+ CR5.90.24.60.1a,*4.80.2a,*4.50.1a,*SAL CR3.60.12.90.1a,*4.00.1*3.30.1*NPY-/CR6.20.54.90.2a,*4.20.2b,*3.80.1b,*,MSG CR5.00.34.90.2*,4.50.2*4.40.3*,NPY+/+ AL2.10.12.00.12.10.12.20.1SAL AL1.60.11.80.1a2.00.1ab2.20.1bNPY-/AL2.10.12.10.12.10.12.30.1MSG AL1.20.11.70.1a1.90.1ab2.20.1bNPY+/+ CR2.10.11.60.1a,*1.60.1a,*1.60.1a,*SAL CR1.60.11.30.1*1.50.1*1.50.1*NPY-/CR2.10.11.70.1a,*1.60.1a,*1.60.1a,*MSG CR1.30.11.30.1*1.30.1*1.40.1*Fat,NPY+/+ AL23.12.625.40.9a28.41.0b29.00.8bSAL AL18.60.419.20.322.70.3a24.70.5bas % ofNPY-/AL22.70.826.00.5a27.90.8ab29.80.6bMSG AL25.30.731.80.4a,36.50.3b,37.10.5b,NPY+/+ CR22.70.420.70.4*21.80.6*20.10.5a,*SAL CR17.10.416.50.4*20.30.3a,*17.00.2*NPY-/CR22.51.020.30.6*19.30.5a,*17.20.3b,*,MSG CR26.00.825.90.8*,25.01.1*,22.11.0a,*,NPY+/+ AL2.60.12.30.1a2.00.1b2.00.1bSAL AL3.30.13.20.12.70.1a2.40.1bNPY-/AL2.70.12.30.1a2.10.1ab1.90.1bMSG AL2.40.11.80.1a,1.50.1b,1.50.1b,NPY+/+ CR2.60.12.90.1*2.70.1*3.00.1a,*SAL CR3.70.13.70.1*3.00.1a,*3.70.1*NPY-/CR2.70.13.00.1*3.10.1a,*3.60.1b,*,MSG CR2.30.12.30.1*,2.40.1a,*,2.80.1b,*,* p < 0.05 CR vs AL within same phenotype and time point. p < 0.05 NPY-/vs NPY+/+ or MSG vs SAL within same diet and time point. Letters denote p < 0.05 from baseline; different letters are significantly different.Ratio129S1BodyFluidMass (g)total massLeanMass (g)FatMass (g)Lean/FatB6 56 Table 3-1. NMR body composition data. 56

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Body Temperature As depicted in Figure 3-1a, CR lowered the body temperature relative to AL in NPY +/+ mice (34.9 0.3 versus 36.7 0.2 C; p < 0.05) but not NPY -/mice. In the B6 mice no differences in body temperature were detected (Fig. 3-1b). 129S1 Body Temperature NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRTemperature (oC) 03031323334353637383940 B A*B6 Body Temperature SAL ALMSG ALSAL CRMSG CRTemperature (oC) 03031323334353637383940 Figure 3-1. Body temperature among the groups. Body temperature in the 129S1 mice did not differ by genotype in the AL groups, but NPY +/+ CR mice were significantly cooler than NPY +/+ AL mice and this drop in temperature was not recapitulated in NPY -/CR mice (A). No differences in temperature were observed in the B6 groups (B). (Mean S.E.M., = significantly different from AL; n = 6.) Hormonal Assessments CR resulted in significantly lower fasting leptin levels compared with AL in both 129S1 genotypes (16.3 6.7 versus 141.4 19.4 pmol/ml in NPY +/+ ; 8.8 5.8 versus 136.5 20.8 pmol/ml in NPY -/; p < 0.05) (Fig. 3-2a) and both B6 groups (5.0 0.3 versus 132.8 48.4 pmol/ml in SAL; 16.6 2.7 versus 253.4 60.1 pmol/ml in MSG; p < 0.05) (Fig. 3-2b). Adiponectin was not consistently altered by CR. Only NPY +/+ CR mice showed significantly higher fasting adiponectin than AL mice (181.8 12.7 versus 139.8 8.4 pmol/ml; p < 0.05) (Fig. 3-2c). SAL CR mice were also significantly increased compared with SAL AL mice (221.7 57

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10.8 versus 160.7 5.7 pmol/ml; p < 0.05) (Fig. 3-2d), and MSG AL mice were significantly increased compared with SAL AL mice (210.5 9.7 versus 160.7 5.7 pmol/ml; p < 0.05). B6 Leptin SAL ALMSG ALSAL CRMSG CRLeptin (pmol/ml) 050100150200250300 129S1 Leptin NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRLeptin (pmol/ml) 050100150200250300 129S1 Adiponectin NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRAdiponectin (g/ml) 050100150200250300 B6 Adiponectin SAL ALMSG ALSAL CRMSG CRAdiponectin (g/ml) 050100150200250300 ****** D C B A Figure 3-2. Fasted leptin and adiponectin levels. Leptin was significantly reduced in the CR groups of both the 129S1 mice (A) and B6 mice (B) regardless of genotype or treatment. Adiponectin was increased only in NPY +/+ CR mice (C) and SAL CR mice (D), although adiponectin was significantly higher in MSG AL mice. (Mean S.E.M., = significantly different from AL; = significantly different from SAL; n = 6.) Glucose Homeostasis Fasting blood glucose CR resulted in significantly lower fasting blood glucose in both 129S1 genotypes (56.8 5.0 versus 84.3 4.0 mg/dl in NPY +/+ ; 56.7 3.0 versus 79.3 3.9 mg/dl in NPY -/; p < 0.05) 58

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(Fig. 3-3a). This was not the case in the B6 mice, where CR was able to lower fasting blood glucose in the SAL CR compared with SAL AL mice (66.6 2.7 versus 107.3 3.8 mg/dl; p < 0.05) but produced no effect in the MSG CR mice (Fig. 3-3b). B A***129S1 Fasting Blood Glucose NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRGlucose (mg/dl) 020406080100120140 B6 Fasting Blood Glucose SAL ALMSG ALSAL CRMSG CRGlucose (mg/dl) 020406080100120140 Figure 3-3. Fasting blood glucose was significantly reduced by CR in both NPY groups regardless of genotype (A). In the B6 mice, only SAL CR mice saw a significant reduction in fasting glucose (B) (Mean S.E.M., = significantly different from AL; n = 6.) Oral glucose tolerance test Overall blood glucose response to the OGTT, as determined by area under the curve (AUC) comparison computed from the glucose response for the two hours following glucose gavage, was improved by CR in the NPY +/+ mice relative to NPY +/+ AL (31,042 2,062 versus 39,305 2,047 mg/dl; p = 0.03) (Fig. 3-4c). CR significantly improved glucose response in both B6 CR groups (28,555 2,642 versus 45,643 2,116 mg/dl in SAL; 23,471 1,740 versus 49,982 4,001 mg/dl in MSG; p < 0.05) (Fig. 3-4d). 59

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SAL ALMSG ALSAL CRMSG CRAUC (mg/dl) 0100002000030000400005000060000 NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRAUC (mg/dl) 0100002000030000400005000060000 129S1 Glucose Tolerance Curves B A D C**` *Time (min) post-gavage 020406080100120Blood Glucose (mg/dl) 0100200300400500600 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR B6 Glucose Tolerance CurvesTime (min) post-gavage 020406080100120Blood Glucose (mg/dl) 0100200300400500600 SAL AL MSG AL SAL CR MSG CR Figure 3-4. Oral glucose tolerance test response with area under the curve comparisons. The glucose responses over time for the 129S1 groups are represented in panel A with the area under the curve plotted in panel C. Only NPY +/+ CR mice showed a significantly reduced glucose response (p < 0.05; NPY -/CR versus NPY -/AL p = 0.06). The glucose response over time for the B6 mice is shown in panel B with the area under the curve comparisons in panel D. Both SAL CR and MSG CR mice had a significantly reduced glucose response compared with the AL groups. (Mean S.E.M., = significantly different from AL; n = 6.) Overall insulin response to the OGTT, also determined by total AUC quantification, was significantly lower in both 129S1 CR groups (93.4 9.2 versus 150.2 16.1 ng/ml in NPY +/+ ; 97.8 11.5 versus 133.8 8.0 ng/ml in NPY -/; p < 0.05) (Fig. 3-5c). SAL CR mice showed reduced insulin response compared with SAL AL mice (83.7 3.8 versus 111.2 7.2 ng/ml; p < 60

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0.05), as did MSG AL mice (46.2 7.4 versus 111.2 7.2 ng/ml; p < 0.05) (Fig. 3-5d). CR had no effect on insulin response in the MSG mice. SAL ALMSG ALSAL CRMSG CRAUC (ng/ml) 050100150200 B A NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRAUC (ng/ml) 050100150200 C D***129S1 OGTT Insulin CurvesTime (min) post-gavage 020406080100120Blood Insulin (pg/ml) 0500100015002000 NPY+/+ AL NPY-/-AL NPY+/+ CR NPY-/-CR B6 OGTT Insulin CurvesTime (min) post-gavage 020406080100120Blood Insulin (pg/ml) 0500100015002000 SAL AL MSG AL SAL CR MSG CR Figure 3-5. Oral glucose tolerance test insulin response with area under the curve comparisons. The insulin responses over time for the 129S1 groups are represented in panel A with the area under the curve plotted in panel C. Both NPY CR groups showed significantly reduced insulin response to glucose challenge regardless of genotype. The insulin response over time for the B6 mice is shown in panel B with the area under the curve comparisons in panel D. SAL CR mice had a significantly reduced insulin response compared with the SAL AL group, and the insulin response by the MSG AL mice was significantly lower than that of the SAL AL mice. (Mean S.E.M., = significantly different from AL; = significantly different from SAL; n = 6.) 61

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Assessment of insulin resistance Only NPY +/+ mice had significantly lowered insulin resistance as assessed by HOMA values comparing CR with AL groups (1.67 0.23 versus 3.64 0.81 pmol/mmol; p < 0.05), although it should be noted that the NPY -/mice clearly follow the same trend (1.62 0.44 versus 3.64 0.78 pmol/mmol; p = 0.09) (Fig. 3-6a). In the B6 mice only SAL CR mice were significantly reduced from their AL counterparts (1.19 0.34 versus 2.61 0.24 pmol/mmol; p < 0.05) (Fig. 3-6b). 129S1 HOMA Values NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRHOMA-IR (pmol/mmol) 0246810 B6 HOMA Values SAL ALMSG ALSAL CRMSG CRHOMA-IR (pmol/mmol) 0246810 ** B A Figure 3-6. HOMA calculations from the ratio of fasting blood insulin (pmol/l) to glucose (mmol/l). Only NPY +/+ CR mice have a significantly reduced HOMA values (p < 0.05; NPY -/CR versus NPY -/AL p = 0.09) (A). Of the B6 groups, only SAL CR mice had a reduced HOMA score (B). (Mean S.E.M., = significantly different from AL; n = 6.) Discussion These experiments were performed to determine the role of the ARC and NPY in the effects of CR on physiological measures related to body composition, endocrine status and glucose homeostasis. With respect to the body composition measurements obtained by NMR, CR reduced fat mass or inhibited its accumulation in all mice, a well-documented effect of CR (Fontana and Klein, 2007). Secondary to these effects, percent adiposity was reduced in CR mice 62

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and their lean to fat ratio was increased. Recent studies have implicated systemic NPY in the promotion of adiposity, as its expression in adipocytes lowers lipolysis and stimulates adipogenesis (Kos et al., 2007; Kuo et al., 2007a). These results show NPY is not required for fat accumulation under normal, free-feeding conditions as NPY -/mice were equivalent to controls in measures of absolute fat mass and % body fat. One key adaptation commonly associated with CR that is nullified by NPY deficiency is a reduction in body temperature. NPY is known to decrease thermogenesis by reducing uncoupling (Billington et al., 2001; Kotz et al. 1998) and our data suggest increased NPY during CR in normal models is critical to their decrease in temperature. We do not show significant differences among the B6 mice, likely because the measurements were obtained during light hours, when the thermogenic gap is narrowest (Tokuyama and Himms-Hagen, 1986). Secondary to changes in adiposity with CR are adjustments in adipose endocrine output. In the current study CR led to reduced serum leptin, as expected (Zhu et al., 2007). With adiponectin, however, the expected increases in serum levels with CR (Zhu et al., 2007) were only seen in the control (NPY +/+ and SAL) mice. Serum adiponectin was not increased by CR in NPY -/mice. While adiponectin knockout mice are known to have reduced NPY and appetite (Kubota et al., 2007), the current finding where the NPY knockout ablates the effect of CR on adiponectin is novel and suggests these proteins may be mutually regulatory. The surprising increase in adiponectin in MSG mice relative to SAL mice on AL feeding further supports the hypothesis that ARC signaling impacts adipose function because models of non-hypothalamic-dependent obesity are characterized by reduced adiponectin output (Kadowaki and Yamauchi, 2005). It has been hypothesized that leptin controls adiponectin production through its action on hypothalamic activity (Huypens, 2007), which is consistent with the results of this study. In 63

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normal models high leptin produced during obesity would reduce adiponectin, whereas weight loss would reduce leptin production and thereby permit uninhibited adiponectin production. In the case of ARC-lesioned MSG mice, leptin is produced in abundance relative to their adiposity but the signal is not transduced by the ARC and therefore adiponectin production is high. CR reduces adiposity and leptin output in these mice but has no effect on adiponectin because their adiponectin production is already set to high as a default due to their defective ARC signaling. Because adiponectin is strongly associated with improved insulin sensitivity (Yamauchi et al., 2003; Yamauchi et al., 2002), the increased adiponectin in the MSG-treated mice is likely behind the relatively normal glucose response of these mice during the OGTT even though increased fat mass is associated with increased insulin resistance (Ferrannini et al., 1996). The ultimate predictor of insulin sensitivity for these mice may be their HOMA scores. HOMA, which mathematically deduces insulin resistance from fasted blood glucose and insulin values, is becoming the tool of choice for determining insulin resistance (Levy et al., 1998). In the HOMA model higher values reflect greater resistance while lower values reflect greater sensitivity to insulin. Unsurprisingly, higher HOMA scores are associated with the AL diets in this study. Neither NPY status nor MSG-treatment increased HOMA scores relative to control mice in the AL condition. NPY -/mice have been shown to have equivalent fasting insulin and glucose to wildtypes (Imai et al., 2007), but these are the first data indicating CR is less effective at improving insulin sensitivity in NPY -/mice. MSG treatment has been previously reported to induce insulin resistance in rats (Balbo et al., 2007; Macho et al., 2000), but our MSG AL mice are equivalent to the SAL controls. As with the NPY -/mice, MSG CR mice do not benefit by HOMA assessment of insulin resistance. 64

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In conclusion, these results indicate that while CR results in alterations to body composition and leptin output from adipose tissue, further downstream changes related to glucose homeostasis and body temperature are unable to be fully realized without proper hypothalamic function. 65

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CHAPTER 4 DIVERGENT EFFECTS OF CALORIE RESTRICTION ON OXIDATIVE STRESS RESISTANCE AND ANTIOXIDANT PROTECTION IN TWO MOUSE MODELS OF IMPAIRED NEUROENDOCRINE SIGNALING The results of Chapter 3 demonstrated that interference of the systemic-neuronal interface by genetic knockout of NPY expression or chemical knockout of ARC function attenuates improvements in glucose handling associated with CR. While improving glucose handling is one mechanism CR has been postulated to extend lifespan (Bartke, 2005; Tatar et al., 2003), it is but one among many (Sinclair, 2005). Another popular mechanism through which CR is theorized to work is by boosting antioxidant systems and enhancing oxidative stress resistance (Beckman and Ames, 1998). Diquat, most commonly used in agricultural industries for its herbicidal properties, is a member of a family of compounds that react with water to form superoxide anions (O 2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (HO ) (Farrington et al., 1973). In vivo the effects of diquat are seen in oxidative modifications to cellular antioxidants (Lauterberg et al., 1984), lipids (Awad et al., 1994; Burk et al., 1980) and proteins (Lei, 2001). The sensitivity of the liver to diquat oxidative stress (Ran et al., 2004) makes it a useful model in which to assess protection by various treatments (e.g., CR, potential CR mimetics, antioxidants). Furthermore, because CR has been shown to improve hepatic stress resistance to oxidative insult by enhancing endogenous antioxidant systems (de Cabo et al., 2004; Seo et al., 2006), the liver oxidative stress via diquat model may be a rapid end-point alternative to lifespan in aging studies. The relevance of NPY and ARC signaling to increased oxidative stress resistance by CR was investigated in the current study. Expression profiles of stress-related proteins and antioxidant activity in mouse liver were assayed, and the stress response to the liver toxin diquat was also investigated. 66

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Materials and Methods Animals and Diets Descriptions of the mice and diets used for the following experiments, as well as the experimental timeline, can be found in Appendix A. Diquat Treatment Following an overnight fast (16h) mice were administered an intraperitoneal injection at 8:00 am of diquat dibromide monohydrate (ChemService, West Chester, PA) dissolved in saline. 129S1 mice were injected at 75 mg/kg and euthanized 24 h post-injection; B6 mice were injected at 50 mg/kg and were euthanized 5h post-injection. For all groups n = 6 except for MSG AL mice for which n = 5. Histology and Stereology Upon euthanization a cross section of liver from each mouse was immersed in Streck tissue fixative (Streck Laboratories, Omaha, NE). The livers were paraffin-embedded, sliced, and stained with hematoxylin by Mass Histology Service (Worcester, MA). Computerized stereology was conducted by a trained operator without knowledge of animal identification. For each liver a single 50 section at approximately the same level was cut for stereological analysis. Sections from three separate mice per group were analyzed for vacuolar load using the volume fraction application of the Delesse principle (Delesse, 1857; for detailed review of mathematics and methodology see Mouton, 2002). Total volume of each analyzed liver section (Vliver) was quantified with the Cavalieri principle using point counting (Gunderson et al., 1999). These studies were performed with assistance from Stereologer, a computerized hardwaresoftware system (Stereology Resource Center, Chester, MD). First, liver sections were outlined using a low-power objective (4), and the technician counted intersections between vacuole 67

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profiles and the points on an unbiased point grid using a high magnification objective (oil-immersion 100, n.a. 1.4), as shown in Figure 4-1c. Finally, the total vacuolar load was calculated as the product of vacuole fraction and total section volume. Western Blotting Livers homogenates from six mice from each group were prepared in RIPA buffer and protein concentration was determined by the Bradford method. The samples were subjected to SDS-PAGE on 8-16% tris-glycine gels and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Rabbit anti-SIRT1, mouse anti-HSP70, rabbit anti-SOD2, (Santa Cruz Biotechnology, Santa Cruz, CA) were the primary antibodies used to detect proteins. Membranes were incubated with primary antibody solutions at 1:1,000 (SIRT-1, HSP70) or 1:2,000 (SOD2) dilution. Secondary antibodies (goat anti-mouse and bovine anti-rabbit horseradish peroxidase-conjugates from Santa Cruz Biotechnology) were applied at 1:3000 dilutions. Immunolabeled proteins were detected by chemiluminiscence (ECL Plus Western Blotting Detection System, Amersham, Buckinghamshire, UK). NAD(P)H:Quinone Reductase 1 (NQO1) Activity Liver tissues were homogenized in Tris-sucrose buffer (10 mM Tris-HCl, pH 7.4 and 25 mM sucrose) and lysates were centrifuged at 100,000 x g for 40 min at 4C. Reactions were carried out in a final volume of 1.0 ml containing 25 mM Tris-HCl (pH 7.4), 0.01% Tween-20, 0.1% BSA, 80 M 2,6-dichloroindophenol (DCIP), 0 or 40 M dicumarol, 200 M NADH, and an appropriate volume of cytosolic sample. Reduction of DCIP was determined spectrophotometrically at 600 nm for 3 min using a Perkin Elmer Lambda 25 spectrophotometer (Waltham, MA). Specific NQO1 activity is described as the dicumarol-sensitive decrease in DCIP absorbance (extinction coefficient 2100 M -1 cm -1 ) and is expressed in nmol DCIP reduced per minute per microgram of protein as determined by the Bradford method. 68

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Results Survivorship Within minutes of diquat injection all animals entered into a state of torpor followed by hypothermia. The hypothermic response is recorded in Table 4-1. For individual mice, temperatures dropping below 26C were strongly associated with fatality. This is exemplified by the MSG AL mice with their stronger average hypothermic response and mortality. NPY CR mice were also susceptible to fatality from the treatment although this is not reflected in their average hypothermic response. Table 4-1. Hypothermia and mortality after diquat injection. ALCRALCRH y pothermic Response (C)H y pothermic Response (C) (20h post-injection) (2h post-injection)NPY+/+29.7 (1.0)31.8 (1.0)NSSAL27.3 (0.2)27.3 (0.2)NSNPY-/-29.5 (0.2)29.8 (0.5)NSMSG 25.8 (0.2)27.5 (0.3)p < 0.001Mortality (%)Mortality (%)NPY+/+00SAL00NPY-/-033MSG600 p < 0.001 MSG vs SAL NS, not significant129S1B6 Histology Gross histological effects in the liver from the diquat treatment are shown in Figures 4-1 (129S1 mice) and 4-1 (B6 mice). Livers from the saline-injected control mice shown at the bottom of each figure are normal for both strains. Periportal microvesicles characteristic of fatty liver change in response to toxic insult occur in diquat-injected 129S1 AL mice (Fig. 4-1) and B6 MSG mice (Fig. 4-2). CR ablates this microvesicle appearance in 129S1 mice and reduces it in B6 MSG mice. 69

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NPY+/+NPY-/-Saline-Injected NPY-/-ALALCR129S1 Figure 4-1. Liver histology of diquat-treated 129S1 mice (above the line) and a saline-treated control (below the line). 70

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SALMSGSaline-Injected MSG ALALCRB6 Saline-Injected SAL AL Figure 4-2. Liver histology of diquat-treated B6 mice (above the line) and saline-treated controls (below the line). 71

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Quantification of the microvesicles observed in the liver after diquat treatment was performed using stereological analysis and the results are graphed in Figure 4-3. After 24h diquat exposure, NPY -/AL mice showed a significantly greater vacuole load than NPY +/+ AL mice (144.0 29.4 versus 44.5 17.5 3 ; p < 0.05) (Fig. 4-3b). In both genotypes, the CR diet significantly reduced the appearance of vesicles (44.5 17.5 versus 2.6 1.0 3 in NPY +/+ ; 144.0 29.4 versus 2.7 1.5 3 in NPY -/; p < 0.05). B6 SAL ALMSG ALSAL CRMSG CRVacuole Load (3) 050100150200 129S1 NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRVacuole Load (3) 050100150200 C B A*** Figure 4-3. Fatty change in the liver in response to diquat (panels B and C). For quantification, vesicle area was identified on a grid at high magnification with panel A showing a representative field with intravesicular points highlighted bright green. Vacuole load was significantly reduced in the CR groups of both the 129S1 mice (B). Only MSG CR mice showed significantly less vacuole accumulation in the B6 mice (C). (Mean S.E.M., = significantly different from AL; = significantly different from SAL; n = 6 except MSG AL n = 5.) 72

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Of the B6 groups, MSG AL mice showed significantly higher vacuole content after the 5h treatment than SAL AL mice (119.1 19.4 versus 35.7 9.3 3 ; p < 0.05) (Fig. 4-3c). MSG CR mouse livers contained significantly less microvesicle load their MSG AL counterparts (21.4 11.0 versus 119.1 19.4 3 ; p < 0.05). Expression of Stress Proteins and Antioxidants Protein expression in the 129S1 mice is compared with expression levels in two NPY -/saline-injected control mice (for which the bands are not shown) and the results are depicted in Figure 4-4. SIRT1 and SOD2 expression showed no difference at 24h post-injection in any groups (Fig. 4-4a & e, respectively) as with glyceraldehyde 3 phosphate dehydrogenase (GAPDH) used as a loading control (Fig. 4-4g). HSP70, however, is significantly increased in the CR groups (111.0 9.5 versus 58.0 9.3 % in NPY +/+ ; 102.7 8.3 versus 53.0 4.3 % in NPY -/; p < 0.05) (Fig. 4-4c). Protein expression in the B6 mice is compared with expression levels in a SAL saline-injected control mouse (not shown). No significant differences were observed in SIRT1 expression between the groups (Fig. 4-4b). HSP70 was significantly increased in MSG CR mice compared with MSG AL mice (90.0 10.2 versus 67.5 3.5 %; p < 0.05) (Fig. 4-4d), and SOD2 was significantly higher in the MSG groups compared with their SAL counterparts (159.9 2.7 versus 90.3 14.3 % in AL; 186.2 11.4 versus 102.6 7.1 % in CR; p < 0.05) (Fig. 4-4f). Protein loading in the MSG mice was not equivalent by GAPDH expression as the MSG CR level was lower than in AL mice (98.3 2.6 versus 116.8 4.7 %; p < 0.05) (Fig. 4-4h). This would not affect the increase in MSG CR HSP70 expression but may mask an increase in SIRT1 or SOD2 in these mice. 73

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B6 SOD2 SAL ALMSG ALSAL CRMSG CR% Control Expression 050100150200250 B6 GAPDH SAL ALMSG ALSAL CRMSG CR% Control Expression 050100150200250 129S1 SOD2 NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CR% Control Expression 050100150200250 129S1 HSP70 NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CR% Control Expression 050100150200250 129S1 SIRT1 NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CR% Control Expression 050100150200250 129S1 GAPDH NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CR% Control Expression 050100150200250 A B C D E F G HB6 SIRT1 SAL ALMSG ALSAL CRMSG CR% Control Expression 050100150200250 B6 HSP70 SAL ALMSG ALSAL CRMSG CR% Control Expression 050100150200250 **** Figure 4-4. Liver antioxidant and stress-related protein expression after diquat treatment. (Mean S.E.M., = significantly different from AL; = significantly different from SAL; n = 6 except MSG AL n = 5.) 74

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NQO1 Activity No significant differences were found among the 129S1 groups in NQO1 activity (Fig. 4-5a), but among the B6 groups the MSG AL mice showed greater activity than SAL AL mice (87.5 9.3 versus 35.5 6.3 nmol DCIP/min/g; p < 0.05) (Fig. 4-5b). SAL CR mice also had increased NQO1 activity relative to the SAL AL mice (84.4 23.8 versus 35.5 6.3 nmol DCIP/min/g; p < 0.05). B6 SAL ALMSG ALSAL CRMSG CRNQO1 Activity (nmol DCIP/min/g) 020406080100120140160 B A129S1 NPY+/+ ALNPY-/ALNPY+/+ CRNPY-/CRNQO1 Activity (nmol DCIP/min/g) 020406080100120140160 Figure 4-5. NQO1 activity in the liver after diquat treatment. NQO1 activity was not affected by diet or genotype in the 129S1 mice (A). In the B6 groups, MSG AL NQO1 activity was significantly elevated compared with SAL AL activity and CR increased SAL NQO1 activity but not MSG NQO1 activity (B). (Mean S.E.M., = significantly different from AL; = significantly different from SAL; n = 6 except MSG AL n = 5.) Discussion This study was performed to determine the role of the ARC and NPY in the effects of CR on oxidative stress resistance. The strongest indicator of hardiness to any stress must be survivorship, and in this case the models of impaired neuroendocrine signaling fared less well than their intact counterparts. Of the NPY -/CR mice, 2 of 6 mice were unable to survive 24h after injection. These deaths occurred between 20 to 24h post-injection. MSG AL mice fared the worst of all groups with 3 of 5 mice unable to survive 5h. Originally it was planned to extend the 75

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treatment of the B6 to 24h as with the 129S1 mice, but the experiment was cut short due to these deaths. B6 mice are generally competent to survive diquat up to 50 mg/kg (Lei, 2001) and the extreme susceptibility of the MSG mice to the treatment suggests these mice may be deficient in antioxidant defense systems. Histological analysis of the mouse livers revealed a striking presence of periportal microvesicles particularly in the AL groups. It is known that drugs can cause acute microvacuolar fatty change in the liver, with consequences including liver failure and a high mortality rate (Oliveira et al., 2002). The mechanism by which this is thought to occur involves stress-induced production of cytokines such as TNFthat would impair mitochondrial -oxidation of fatty acids which would lead to a buildup of fatty acids in the liver and exacerbate reactive oxygen species (ROS) production (Oliveira et al., 2002; Yang et al., 2000). ROS-induced lipid peroxidation follows which activates an inflammatory response as well (Lee et al., 1995). Because the MSG-treated mice in this study were obesity-prone, as shown in chapter 3, and the saline-injected control liver section in Figure 4-2 indicates moderate liver steatosis prior to diquat injection, additional fat accumulation in the livers of the MSG AL mice may have hastened the fatalities in this group. Reduced liver steatosis may have been one protective mechanism in the MSG CR mice that allowed them to survive the treatment more successfully. Indeed, quantification of the intrahepatic microvesicle response to the diquat treatment revealed a strikingly high presence in the MSG AL mice which may account for their high mortality rate. Given that ROS protection is a key benefit associated with CR, levels of antioxidant and stress-related protein profiles were assessed in the mouse livers. There were no statistical differences in SIRT1 levels in either mouse strain. While the CR groups showed a trend to greater expression, as would be expected (Cohen et al., 2004), the sample size may have been too 76

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small to detect significance. Heat shock protein 70 (HSP70), a member of a ubiquitously-expressed class of proteins important for their involvement in protein folding and protection, can be upregulated by stress to serve defensively. In the case of these mice, HSP70 was not observed to be upregulated compared with saline-injected controls, but rather decreased over time in some of the groups. Namely, the AL-fed 129S1 mice showed a decrease in HSP70 to about half of normal from the diquat, while CR served to maintain normal levels of this protein. In the B6 groups, the SAL AL mice still show normal HSP70 expression at the end of the experiment, but MSG AL mice have already dropped below normal. This may further account for their poor survivorship to the diquat treatment, and the maintenance of normal levels in the MSG CR mice likely played a role in their relative protection. Superoxide dismutase (SOD2), associated with both ROS defense and increased longevity (Kenyon, 2005), was not changed in the 129S1 groups. Surprisingly, SOD2 was found to be overexpressed in both MSG groups. Upregulation of antioxidant systems has been noted to occur in the liver as a response to obesity-imposed oxidative stress (Yang et al., 2000) which may account for the relative increase in the MSG AL mice. Assessment of GAPDH as a loading control indicated MSG CR samples were underloaded compared to MSG AL samples, which may mean the CR SOD2 expression is underrepresented in this study even though it is still significantly higher than SAL CR mice. In order to estimate functional antioxidant activity in these mice, NQO1 activity was assayed. NQO1 functions in the plasma membrane redox system, which is important for maintaining a functional barrier in the plasma membrane against the extracellular environment (Hunt et al., 2006; Navas et al., 2007) and its activity in the liver has been shown to be enhanced by CR (de Cabo et al., 2004). While there were no significant differences in the NQO1 activity levels in the 129S1 mice, there were tendencies toward increased activity in the NPY +/+ and CR 77

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groups. As expected, NQO1 activity is increased by CR in the B6 SAL mice. Unexpectedly, NQO1 activity appears to be constitutively upregulated in the MSG mice as with SOD2 protein expression, and presumably the same unknown causative factors are responsible. This study demonstrated some of the protective effects of CR on oxidative stress response in the liver. Survivorship to oxidative insult is impaired in models of altered neuroendocrine signaling, suggesting a role for the hypothalamus in the regulation of systemic stress resistance. CR in NPY -/mice was found to be anti-protective to survival without apparent impairment of antioxidant systems. Hypothalamic lesioning by MSG markedly reduced survivorship in AL-fed mice which correlated with reduced HSP70 expression and extensive accumulation of intrahepatic microvesicles and CR was able to reverse these effects. Future studies are needed to characterize the nature of the weakness observed in the NPY -/mice in response to oxidative stress. 78

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CHAPTER 5 CALORIE RESTRICTION IS NOT PROECTIVE AGAINST TUMORIGENICITY IN MICE WITH IMPAIRED NEUROENDOCRINE SIGNALING Chapter 4 demonstrated that resistance to oxidative stress is reduced in models of altered hunger signaling, and that CR does not completely ameliorate this effect. While this oxidative stress was investigated using the liver toxin diquat, free radicals and oxidative damage to macromolecules have also been implicated in the pathology of a major age-related disease: cancer. Maintenance of DNA stability is a fundamental and continuous challenge faced by every cell. Genomic instability is generally recognized as a nearly universal feature of both cancer and aging, fueling efforts to understand these enigmatic processes through their convergence (Finkel et al., 2007). Cancer and and aging share another important commonalityboth can be delayed by CR. While the lifespan-extending properties of CR were not well appreciated until the 1930s (McCay et al., 1935; McCay et al., 1939; McCay and Crowell, 1934), observations that CR is protective against transplanted and induced tumors were first made nearly two decades earlier (Rous, 1914). Since then, CR has been extensively studied and has consistently shown beneficial effects on longevity and carcinogenesis across a variety of species (Kritchevsky, 2002; Weindruch, 1997). As is the case for lifespan extension, the mechanisms behind the protective effects of CR against cancer remain unknown although decreased oxidative damage is a highly-regarded possibility (Hursting et al., 2003; Kritchevsky, 2002). A model for skin tumorigenesis has been developed using 7,12-dimethylbenz[a]anthracene (DMBA) to initiate tumor formation followed by repeated treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA) to promote tumor formation (DiGiovanni, 1992). If neuroendocrine alterations subsequent to the hypothalamic response to CR are responsible for some of the protection against tumorigenesis seen in CR models, then impairment of hunger 79

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sensing should negate this effect. We tested tumorigenicity following CR in mice with impaired hunger signaling due to MSG-induced ARC lesioning or NPY knockout. Tumor expression profiles were monitored, and skin protein damage as measured by structural mutation was also assayed. Materials and Methods Animals and Diets Descriptions of the mice and diets used for the following experiments, as well as the experimental timeline, can be found in Appendix A. Two-Stage Carcinogenesis All groups began treatment with an n = 6 except for the NPY +/+ AL and NPY -/CR groups where n = 5 due to prior spontaneous deaths. Several mice were euthanized or found dead during the study and their data was included until the time of death. Figure 5-1 outlines the treatment procedure and its carcinogenic effects. DMBA is administered once TPA treatment begins twice weekly Tumor growth occurs TPA Induction of ROS in keratinocytes Epithelial hyperplasia Skin tumor growth DMBABegin caloric restriction Covalent binding to DNA Point mutations in the Ha-Rasgene -802 ????weeks Figure 5-1. Tumor induction procedure. 80

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Immediately before DMBA tumor initiation a 2 cm 2 treatment area was shaved into the back just above the base of the tail. All mice were treated with a single dose of 25 g DMBA dissolved in 100 L of acetone. Cautionary to the carcinogenicity of DMBA, the mice were not handled for the following 72h after which the cages and bedding were changed. Tumor promotion with TPA (4 g dissolved in 100 L acetone) began two weeks after DMBA initiation and continued twice weekly until at least 1 papilloma with a radius > 1 mm was recorded for tumor incidence data. Papilloma-positive mice were euthanized when >50% of all groups presented with tumors (15 weeks after DMBA for 129S1 mice; 18 weeks after DMBA for B6 mice). Periodic euthanization of the remaining mice was performed in the morning on non-fasted animals until all mice presented with tumors. Final papilloma count and size were measured upon euthanization and sections of treated and untreated skin were collected separately for storage. Brains and livers were also collected and all tissues were flash-frozen in liquid nitrogen and stored at -80C. Protein Damage Assay The surface hydrophobicity of skin proteins was assayed via the UV-induced photoincorporation of 4,4-dianilino-1,1-binaphthyl-5,5disulfonic acid (BisANS) to proteins. Skin samples were homogenized in RIPA and protein content was assayed by the Bradford method. Samples were diluted to 1 mg/ml with buffer (50 mM Tris-HCl, 10 mM MgSO 4 at pH 7.4) and 200 l was loaded into a 96-well plate to which 100 M BisANS was added. The plate was incubated on ice for 1 h under direct UV (115 V) exposure (UVL-21 Compact UV Lamp, UVP LLC, Upland, CA). Afterwards, Laemmli buffer was added to the samples and they were subjected to SDS-PAGE, which allows unincorporated BisANS to run off the gel. BisANS fluorescence was exposed to UV light and quatified using the Gene Genius Bio Imaging System (Syngene, Frederick, MD). 81

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Results Body Weight Treatment-related changes to body weight in the mice are shown in Figure 5-1. Show how they lose weight and consume more. DMBA treatment did not appear to affect body weight in any group. Following TPA treatment initiation, however, the 129S1 AL mice began a long period of gradual weight loss which continued for 10 weeks and ultimately induced weight loss of approximately 20% compared with weight at treatment onset (Fig. 5-1a). Weight loss was also seen in the 129S1 CR mice and 3 deaths occurred between weeks 10 and 16 likely due to starvation. TPA-associated weight loss is not apparent in the B6 mice (Fig. 5-1b), although weight gain is attenuated for the first 6 weeks in the AL groups. A slight, transient reduction in food intake in the MSG AL mice was observed which necessitated a corresponding reduction in MSG CR feeding and accounts for their weight loss after week 12. B6 Body WeightsWeeks on Diets 05101520Body Weight (g) 010203040 SAL AL MSG AL SAL CR MSG CR 129S1 Body WeightsWeeks on Diets 05101520Body Weight (g) 010203040 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR B A Figure 5-2. Body weights of tumor study mice with 129S1 mice represented in panel A and B6 mice in panel B. Solid circle represents week in which DMBA was applied, dotted circle when TPA treatments were initiated. (Mean S.E.M., n = 6 except NPY +/+ AL and NPY -/CR n = 5.) 82

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Tumor Characteristics Tumor onset began 8 weeks after DMBA tumor induction in the 129S1 mice (Fig. 5-2a), and 25 weeks after DMBA treatment all 129S1 mice have tumors except one NPY +/+ CR mouse. B6 mice presented with their first tumors 11 weeks after DMBA treatment (Fig. 5-2b) and at 25 weeks only one SAL AL mouse remains without any tumors. B6 Tumor ProgressionTime (weeks) 0510152025Mice Presenting Tumors (%) 020406080100 SAL AL MSG AL SAL CR MSG CR 129S1 Tumor ProgressionTime (weeks) 0510152025Mice Presenting Tumors (%) 020406080100 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR B A Figure 5-3. Tumor onset profiles for the 129S1 (A) and B6 (B) mice, with representative mice and papillomas 13 weeks after DMBA tumor initiation shown below. (Mean S.E.M., n = 6 except NPY +/+ AL n = 5 and NPY -/CR n = 3.) Tumor onset was most rapid in the NPY -/CR mice and this was significantly different from that of the NPY +/+ CR mice. When total number of tumors was assessed at sacrifice, NPY -/83

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AL mice had significantly more papillomas than CR mice. This difference was not continued to average tumor size. Among the B6 groups, no significant difference was found in average onset time although the MSG groups predated their SAL counterparts in both diet conditions. MSG CR mice presented with roughly double the average number of tumors seen per mouse in the other groups and this was significantly different from the MSG AL mice and SAL CR mice. Tumor size, like onset, was not significantly different but MSG mice tended to show larger tumors at sacrifice than the SAL mice. Table 5-1. Characteristics of tumor incidence by latency, multiplicity, and size of tumors. ALCRALCRTumor Onset (weeks)Tumor Onset (weeks)NPY+/+13.4 (1.1)16.9 (2.1)NSSAL18.3 (2.8)19.0 (1.9)NSNPY-/-14.0 (1.2)11.0 (1.0)*NSMSG16.6 (0.8)15.3 (2.3)NSTumor MultiplicityTumor MultiplicityNPY+/+5.5 (1.4)2.0 (0.6)NSSAL0.7 (0.3)0.7 (0.5)NSNPY-/-8.3 (2.6)2.3 (0.9)p = 0.03MSG0.8 (0.3)2.2 (0.6)p = 0.05Tumor Size (mm)Tumor Size (mm)NPY+/+1.9 (0.2)2.1 (0.3)NSSAL1.7 (0.2)1.8 (0.2)NSNPY-/-2.0 (0.2)1.6 (0.1)NSMSG2.3 (0.3)2.3 (0.3)NS *p < 0.05 NPY-/vs NPY+/+ p < 0.05 MSG vs SAL NS, not significant129S1B6 Protein Conformation Effect In general proteins from treated skin samples were assessed to show greater ultrastructural compromise as reflected in greater hydrophobic amino acid presence on the outer surface as predicted (Fig. 5-3), although unexpectedly high levels were found in the NPY +/+ CR mice. 84

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B6 Surface Hydrophobicity (A.U.) 0200040006000800010000 SAL AL MSG AL SAL CR MSG CR 129S1 Surface Hydrophobicity (A.U.) 0200040006000800010000 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR B ASkin SamplesUntreatedTreated UntreatedTreatedSkin Samples Figure 5-4. Detection of changes in surface hydrophobicity in proteins between treated and untreated skin regions. No significant differences were detected in either the 129S1 (A) groups or the B6 groups (B). (Mean S.E.M., n = 6 except NPY +/+ AL and NPY -/CR n = 5.) Discussion This study was performed to determine the role of the ARC and NPY in the effects of CR on tumorigenicity in a two-stage skin carcinogenesis model. The mice responding most rapidly to treatment with tumors were the NPY -/CR mice. The role of NPY in cancer biology is not yet well understood, but NPY expression has been observed to occur within tumor cells (Cohen et al., 1990; deS Senanayake et al., 1995; Krner et al., 2004) and has been shown to be both repressive (Kitlinska et al., 2005; Ruscica et al., 2006) and stimulatory (Kitlinska et al., 2005; Rm et al., 1990) to growth of different in vitro cancer cell lines. The current data may support the repressive role for NPY in tumorigenesis, but one potential confounding issue exists relating to the body weight loss in the NPY -/CR mice. After the initiation of TPA treatments, the 129S1 mice were observed relentlessly grooming their backsides on treatment days. Their increased physical activity may have contributed to the weight loss observed in this strain, and the NPY -/CR mice began to die from starvation. By the 6 th week after DMBA induction 2 of 5 NPY -/CR mice had died, at which point the surviving mice were given unrestricted access to food for 72h 85

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in order to prevent further deaths. CR feeding was resumed at the end of this period. Since CR has been shown to be primarily protective during the promotion (i.e. TPA) phase of carcinogenesis (Tannenbaum, 1944), the rapid tumor onset in the refed mice may simply reflect their temporary dietary unrestriction. This would not explain the relatively high number of tumors found on the NPY -/AL mice, however, so the implication for a protective role for NPY in cancer still applies. Furthermore, the tumor characteristics from the B6 indicate CR is antiprotective in the MSG mice with respect to tumor multiplicity, another indication that the ARC hunger response is a necessary component of CRs cancer protection. Two major mechanisms through which CR is postulated to inhibit tumorigenicity are through antioxidant and glucose homeostasis-related pathways (Kritchevsky, 2002). Based on the results shown in chapter 4, the altered neuroendocrine models were found to be equal or greater to control models in protective protein expression and antioxidant activity. In the current study the changes in surface hydrophobicity in the treated skin samples, a sensitive indicator for oxidatively-induced structural alterations to proteins associated with loss of function (Pierce et al., 2006), do not show NPY -/or MSG mice incur more damage than control mice. Taken together these results indicate loss of antioxidant function is not causal in the increased susceptibility to tumorigenesis in these models of neuroendocrine impairment. Glucose homeostasis, however, was not found to be as responsive to CR in NPY -/and MSG mice than in normal controls in chapter 3 and may account for some of the differences in tumor incidence observed in the current study. 86

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CHAPTER 6 DISCUSSION This project was conceived to test the relevance of hunger to the physiological effects of CR. While the neuroendocrine system is popularly hypothesized to figure prominently in the mediation of CRs effects (Bishop and Guarente, 2007; Lamberts et al., 1997; Meites, 1989; Nelson et al., 1995; Rehman and Masson, 2001; Speakman and Hambly, 2007), the studies contained herein mark the first attempt to test the hypothesis from the angle of the hypothalamic hunger response to CR. Certainly the salience of the current studies to human health depends on CRs effectiveness extending beyond laboratory animals. Few studies have evaluated CR in the context of humans, with the starvation studies by Ancel Keys representing the first to suggest that long-term CR may confer health benefits to humans by noting food limitations imposed by World War II in Europe led to a sharp decrease in the incidence of coronary heart disease in the affected populations (Keys, 1994). More recently, participants in the Biosphere study who were forced to eat a low-calorie diet over a period of approximately two years exhibited highly significant decreases in blood pressure, insulin and cholesterol levels (Walford et al., 2002). Another recent study investigating the feasibility of intermittent fasting in nonobese humans found a significant decrease in insulin levels after three weeks of intervention (Heilbronn et al., 2005). The impact CR will have on human lifespan is still open to interpretation, but these studies indicate dietary restriction can at least offer tangible benefits to healthy-weight individuals. Increased appetite is perhaps the single most unifying component in dietary restriction research in all species. Even in the human intermittent fasting study, subjects reported significantly increased feelings of hunger on fasting days that did not diminish as the study progressed (Heilbronn et al., 2005). This may become a key obstacle to translating the success of 87

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energy restriction from controlled settings to free-ranging human populations; despite the variety of benefits associated with calorie restriction, it is thought that the majority of humans would decline to embark on a lifelong regimen of conscientious calorie cutting. The advent of appetite-suppressing pharmaceuticals or other mimetics of calorie restriction may therefore be of great utility to enable painless calorie restriction. Unfortunately, if these agents act through the depression of neural signaling pathways that convey hunger to an organism, they may impede the realization of the full spectrum of CRs benefits to physiology, carcinogenesis, cognition and perhaps even lifespan. Future studies are therefore needed to evaluate the use of appetite suppressants for weight loss compared with CR giving attention to secondary health effects. Drugs that target hypothalamic neuropeptide signaling pathways that control appetite are thought to hold considerable promise for the treatment of obesity and work to develop such drugs is well under way (Halford, 2006). Beyond the treatment of obesity, such pharmaceuticals would be appealing for those seeking to achieve CR-like effects while minimizing the perception of hunger. The results from this project, however, imply this may be a self-defeating move as the models of decreased hunger perception fared less well than normal counterparts in withstanding oxidative stress and tumor induction. This research project is not without its limitations. While NPY is recognized to play a crucial role in mediating hunger, it is but one of a suite of neuropeptides responsive to energy balance. This is unsurprising as the life-preserving importance of caloric sufficiency has promoted the evolution of overlap in hypothalamic appetite regulation (Horvath and Diano, 2004). Orexin, for example, is another appetite-stimulating neuropeptide produced in the ARC 88

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which has been found to substitute in part for NPY activity in MSG-treated rats (Moreno et al., 2005). Another limit in the methodology of the studies was the use of young instead of aged mice. This led to the inability to assess the prevention of cognitive decline seen with CR (Ingram et al., 1987; Means et al., 1993) and therefore to fully assess the impact of impaired neuroendocrine function on this effect. This same issue may have contributed to lack of differences seen in antioxidant protein expression in chapter 4, as the attenuation in age-related pro-oxidant changes associated with CR (Seo et al., 2006) would not yet be apparent in mice aged 4-6 months. One question that remains unresolved from the current studies is whether lifespan extension could be obtained with CR in these models of altered hunger signaling. Extrapolating from our results, lifespan extension would be expected to be reduced as the mice displayed diminished improvements to glucose homeostasis, reduced resistance to oxidative stress and increased tumorigenicityall conditions associated with reduced longevity (Finkel et al., 2007; Kenyon, 2005). Lifespan data are still needed to evaluate this hypothesis. In conclusion, the current project showed measures of physical performance and body composition are improved by CR even without full neuroendocrine competency whereas downstream effects of CR on physiological systems are more sensitive to neuroendocrine status. The models employed in these studies are well-suited for research designed to test the significance of hunger to diet-induced physiological changes and lifespan. The aim of research to find agents that mimic CR may be unsuccessful in their effort to sidestep hunger as the current results indicate hunger is a critical component in the relay of CRs beneficial effects through the neuroendocrine system. 89

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APPENDIX GENERAL STUDY DESIGN Background on the Effects of Monosodium Glutamate on Hypothalamic Function The function of specific neuroanatomical structures has classically been studied using either chemical or electrical ablation techniques (Shah and Jay, 1993). To assess the impact of the hypothalamic arcuate nucleus on homeostasis, selective chemical lesioning of the ARC may be accomplished using monosodium glutamate. Excitatory amino acids such as glutamate are utilized by nearly every central neuronal circuit, and during development these amino acids play a pivotal role in learning, memory, and brain plasticity (Grumbach, 2002). Excitotoxicity was proposed by Olney and colleagues in 1971 (Olney et al., 1971) to explain the pathophysiology of brain ischemia. The ability of glutamate to kill neurons seems to be mediated principally by its interaction with N-methyl-D-aspartate (NMDA) receptors leading to a rise in intracellular calcium (Limbrick et al., 2003). Beyond the laboratory, excitotoxicity has been implicated in such diverse pathologic processes as epilepsy, ischemic brain damage, anxiety, addiction, and neuropsychiastic disorders (Pellicciari and Constantino, 1999; Whetsell, 1996). Neonatal treatment of rodents with MSG induces a phenotype that has been well characterized since its first use (Olney, 1969) to include several metabolic alterations such as hyperadiposity despite hypophagia, HPA axis hyper-responsiveness, and insulin resistance (Balbo et al., 2007; Dawson and Lorden, 1981; Macho et al., 2000; Morris et al., 1998; Olney, 1969; Perell et al., 2004; Tokuyama and Himms-Hagen, 1986). Explanations for the obesity in these hypophagic animals include decreased thermogenesis (Morris et al., 1998; Tokuyama and Himms-Hagen, 1986) and increased metabolic efficiency (Djazayery et al., 1979; Morris et al., 1998). The stunted growth is hypothesized to owe to their impaired growth hormone releasing hormone production (Tamura et al., 2002). 90

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Distinct neuroendocrine adaptations are also noted with MSG treatment where hypothalamic function is concerned (Broberger et al., 1998; Meister et al., 1989). It is recognized that this neurotoxic compound mainly affects ARC neuron cell bodies (Burde et al., 1971; Cameron et al., 1978; Hu et al., 1998; Seress, 1982), owing to blood-brain barrier permeability at the median eminence (Peruzzo et al., 2000). Within the ARC, more than 80% of the neurons are eliminated, resulting in ARC shrinkage, widening of the third ventricle, thinning of the median eminence and marked reorganization among remaining neurons (Broberger et al., 1998, Elefteriou et al., 2003). Loss of ARC NPY expression has been the most studied of the orexigenic peptide alterations with MSG treatment (Abe et al., 1990; Broberger et al., 1998; Kerkerian and Pelletier, 1986; Legradi and Lechan, 1998; Meister et al., 1989; Perell et al., 2004). Since ARC NPY is coexpressed with another orexigenic peptide, AgRP, (Shutter et al., 1997), MSG treatment would be expected to lower expression of both of these neurotransmitters. Indeed, erasure of hypothalamic NPY / AgRP neurons by MSG reflects in reduction of both neuropeptides (Broberger et al., 1998; Tamura et al., 2002). Mice Representative mice from the mouse strains used in these studies are pictured in Figure A-1. Male 129S1 wild-type (NPY +/+ ) controls (129S1/SvImJ) were purchased from the Jackson Laboratory (Bar Harbor, ME). The mice were 2 months of age upon arrival at the Holabird Research Facility (Baltimore, MD), where they were acclimated for 1 month before the study diets were initiated. Male 129S1 NPY-knockout (NPY -/) mice were bred at the National Institute on Aging Gerontology Research Center (Baltimore, MD) from breeder mice obtained from the Jackson Laboratory (129S-Npy tm1Rpa /J). Male mice aged 2-4 months were brought to the Holabird Research Facility where they were acclimated for 1 month before the study diets were initiated. 91

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The C57BL/6J (B6) mice were bred and treated with MSG at the Jackson Laboratory. On postnatal day 5, 65 male pups were given subcutaneous injections of MSG (at 4mg/g body weight), and 65 control pups were given subcutaneous injections of saline solution. At 7 weeks of age the mice were shipped to the Holabird Research Facility where they were acclimated for 3 weeks before the study diets were initiated. NPY+/+NPY-/-MSGSAL129S1 B6 Figure A-1. Representative mice of the genotype and treatment groups. At the onset of the study, the 129S1 mice were 3-5 months of age and the B6 mice 2.5 months of age when they were randomly divided into diet groups. Ninety six mice in total (12 for each condition and diet) were used for these studies, all of which were males. The mice were housed individually in ventilated caging on a 12h light/dark cycle at 22C and 35% humidity with ad libitum access to water. All testing was performed during the light phase and using procedures outlined and approved by the Animal Care and Use Committee at the National Institute on Aging. 92

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Verification of NPY Expression Levels Immunohistochemistry was employed to verify reduced NPY expression in the NPY -/and MSG mice. Brains were obtained from the mice undergoing the diquat experiment (see Fig. A-3). Only brains from AL-fed mice were used, however these mice had been fasted overnight prior to diquat injection. Brains were excised and fixed by immersion in Streck tissue fixative (Streck Laboratories, Omaha, NE). Prior to sectioning brains were cryoprotected in a 30% sucrose solution and frozen in liquid nitrogen. Coronal sections (50m) were cut on a freezing microtome (Microm HM 400; Microme, Walldorf, Germany) and washed (washing consisted of 3 changes with 0.1 mM phosphate-buffered saline (PBS) for 10 min each). Endogenous peroxidase activity was then quenched by incubation with 1% hydrogen peroxide/PBS for 30 min. After washing the sections were blocked in 10% normal goat serum for 30 min, followed by incubation with the primary anti-NPY antibody (1:1000 dilution; Abcam, Cambridge, MA) at 4C overnight. Following washing the sections were incubated at room temperature with a series of solutions purchased as part of the Vectastain kit from Vector Laboratories (Burlingame, CA): a goat anti-rabbit biotinylated antibody (1:400 dilution) for 2 h, an avidin-biotin complex solution for 2 h, and finally diaminobenzidine (DAB) until a strong color reaction was observed. The stained sections were washed, mounted on slides, dried thoroughly and coverslipped before microscopy. The results are shown in Figure A-2. As expected, staining for NPY is robust in NPY +/+ and SAL mice, greatly reduced in MSG mice and absent from NPY -/mice. 93

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NPY+/+NPY-/-MSGSAL ARC3V Figure A-2. Representative hypothalamic brain sections stained for NPY. 3V denotes area pertaining to the third ventricle and the arrow indicates the region of the arcuate nucleus, which are located in the corresponding regions of each frame. Diets The diet used in this research, AIN-93G, was obtained from Bio-Serv (Frenchtown, NJ). The ingredients and macronutrient profile of the diet are defined in Table A-1. AL mice were fed ad libitum on the AIN-93G and CR animals were fed daily the amount equivalent to 70% by weight of AL intake with the same chow. The diet consisted of 59% carbohydrate, 18% protein and 7% fat by weight. 94

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Table A-1. Diet composition. (g)Starch397.50Carbohydrate2.368Casein200.00Protein0.720Cystine3.00Fa t 0.630Vitamin mix10.00Total:3.718Choline2.50Salt mix35.00Sucrose100.00*Lo Dex132.00Fiber50.00**TBHQ0.01Soybean oil70.00Total:1000.00 approximately 5% monoand disaccharides, and about 95% oligoand polysaccharides. **Tertiary butylhydroquinone (TBHQ) is a phenolic compound added as a preservative for its antioxidant properties.AIN-93G IngredientsCalorie Distribution(kcal/g) *Lo Dex is a processed corn starch containing, by weight, less than Study Design Figure A-2 outlines the overarching protocol used for the mice in this research. At the study onset mice were randomized to AL and CR diet groups containing 12 animals from each of the 4 treatment conditions. A baseline body composition analysis was performed by NMR, following which diet treatments were commenced. Body composition was assessed again by NMR during week 4, after which 10 mice were selected randomly from the 8 diet/treatment groups to undergo behavioral assessment. Behavior assessment spanned two weeks; the rotarod, open field and inclined screen tests were performed over the first 2 days and the Morris water maze was performed over the next 8 days. 95

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ALCR ALCR ALCR ALCRNPY+/+NPY-/-SALMSG All Mice (8 groups, n = 12 / group)Week 0 = NMR #1Week 1 = Diets StartWeek 4 = NMR #2Week 6-7 = Behavior StudiesWeek 8 = NMR #3Week 9 = OGTTWeek 11 = Serum CollectionWeek 12 = NMR #4Week 13 = Diquat ExperimentWeek 8 = Tumor Induction Weeks 10+ = Tumor Promotion Mice (8 groups, n = 6 / group) Mice (8 groups, n = 6 / group) Figure A-3. Project design for the three major studies and other periodic assessments. In the eighth week, all mice were assayed by NMR a third time. Later in week eight mice were again divided randomly into groups designated to enter one of two terminal studiesthe oxidative stress study (via exposure to diquat) or the tumorigenesis study. Six animals from each of the diet-treatment groups were allotted to the studies. Mice randomized to the oxidative stress study were given an OGTT in week nine. Their blood was collected in week 11 and serum frozen at 80C for the analyses described in Chapter 96

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3. A final NMR was performed on these mice during week twelve. In week 13 the mice were injected with diquat, euthanized, and tissues were harvested for the work presented in Chapter 4. Mice randomized to the tumorigenesis study were treated with the tumor inducer DMBA in week eight after the NMR analysis was performed. No further treatment was then performed for two weeks. Starting in week ten, mice were treated with the tumor promoter TPA until they presented with papillomas and were subsequently euthanized. These procedures and the results constitute Chapter 5. Body Weights and Food Intake Body weight and food consumption for the first 12 weeks of study are presented in Figure A-3. The data reflect consumption and body weight for all mice through week 8 and the oxidative stress study mice from weeks 10-12. Body weights for the mice randomized to the tumorigenesis study are shown in Chapter 5. Food restriction was established gradually for all CR groups. The initial drop in body weight of the 129S1 CR mice reflects their heavier initial body weight (at 3-5 months of age these mice were full adults) at diet onset. Body weight loss stops at approximately 20g for these mice at 70% CR, and AL-fed 129S1 mice continue to gain weight throughout the feeding period (Fig. A-3A). The B6 mice were younger (at 2.5 months of age these mice were young adults) and lighter at study start, and consequently CR in these mice resulted in weight maintenance rather than weight loss (Fig. A-3B). 97

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Weeks on Diets 024681012Body Weight (g) 010203040 NPY+/+ AL NPY-/AL NPY+/+ CR NPY-/CR A B129S1Weeks on Diets 024681012Body Weight (g) 010203040 SAL AL MSG AL SAL CR MSG CR B6Food Intake (g) 012345 Food Intake (g/d) 012345 Food Intake (g/d) Figure A-4. Food consumption (top) and body weight (bottom) for the 129S1 mice (A) and B6 mice (B). Hunger Assessment During the first month of diet restriction the CR mice were monitored for their food consumption rate on five separate days. Mice were fed their allotted chow for the day in the morning as usual. Hourly checkups were then performed to assess which animals had consumed all of that days food. The average results S.E.M. for the groups (n=12 in each diet group) are graphed in Figure A-4. NPY -/and MSG CR mice consume their food less quickly than the control mice and appear to be less hungry after dietary restriction than the neuroendocrinologically intact mice. This confirms previous phenotypic observations that MSG-treated mice are less hyperphagic in response to fasting (Ma et al., 1988) as are NPY -/mice (Bannon et al., 2000; Segal-Lieberman et al., 2003). 98

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129S1 Food Consumption Rate NPY+/+NPY-/-% Finished at 3h 020406080100 B6 Food Consumption Rate SALMSG% Finished at 2h 020406080100 B A Figure A-5. Feeding rate among the CR groups. In the 129S1 mice (A) NPY +/+ control mice tended to consume their food more quickly than NPY -/mice. In the B6 mice (B), SAL mice ate more rapidly than MSG mice. 99

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BIOGRAPHICAL SKETCH Robin Kaye Minor grew up in Naples, Florida where she graduated from Naples High School in 1996, culminating a relatively carefree childhood playing classical Spanish guitar and competing at quarter horse shows. The next four years found her intermittently at the University of Florida between studies abroad in Ireland, Brazil and Kenya. Despite nearly succumbing to the particularly virulent strain of malaria (Plasmodium falciparum) following the Kenyan sojourn in the summer of 1999, she received a bachelor of science degree in psychology with a minor in Portuguese with highest honors the following spring. A year sabbatical from studies was spent in Houston teaching remedial fractions to middle-schoolers with Teach for America and then tending bar for Polynesian immigrants in Los Angeles. A desire to understand more about insects and their nefarious ways drove her to pursue a master of science in entomology at UF with Dr. Phil Koehler, from which she graduated in 2002. Declining acceptance to medical school at the University of Miami for a future less prescribed, Robin spent a year exploring the culinary arts in New York City and cooking for such venerable establishments as Judson Grill and Blue Hill. Hungry to marry her interests in food and academics, she entered into a Ph.D. program in the Food Science and Human Nutrition Department at UF in the fall of 2003 under the supervision of Dr. Susan S. Percival. Her research interest in nutrition and aging led her to an extended collaboration with Dr. Rafael de Cabos laboratory at the National Institute on Aging in Baltimore, MD, where she collected the data for this dissertation as an NIA Pre-Doctoral Fellow. In the future Robin will pursue a post-doctoral fellowship at the NIA, collect more degrees, and continue cultivating a life worth living. 128