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The Effect of Citrulline Supplementation on Horse Skeletal Muscle Mitochondrial Function and Biogenesis

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The Effect of Citrulline Supplementation on Horse Skeletal Muscle Mitochondrial Function and Biogenesis
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Guzman, Maria
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English

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Biology ( jstor )
Citrates ( jstor )
Cytochromes ( jstor )
Enzyme activity ( jstor )
Horses ( jstor )
Muscles ( jstor )
Oxidases ( jstor )
Placebos ( jstor )
Skeletal muscle ( jstor )
Transponders ( jstor )
Dietary supplements
Horses
Mitochondria
Musculoskeletal system
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Undergraduate Honors Thesis

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Abstract:
In this study, we evaluated the effect of dietary citrulline on mitochondrial function and number in horse skeletal muscle. Untrained horses (n=12; 10.8 ± 2.5 years old) were supplemented daily for a period of 15 days with either citrulline-malate (CIT; 86 mg citrulline/kg body weight; n=6) or placebo (CTR; 25 mg urea/kg body weight; n=6). Skeletal muscle samples were collected prior to supplementation (baseline), and on day 14 of the supplementation. An additional muscle sample was collected on day 15 one hour after a two-hour medium intensity exercise bout. In those skeletal muscle specimens, enzyme activities of cytochrome c oxidase (COX) and citrate synthase (CS) were assessed to evaluate mitochondrial function and number, respectively. Interestingly, we found that horses in the CIT group could be separated in two groups: Horses with lower COX activity on d0 but significant response to CIT treatment (responders), and horses with higher COX activity on d0 but minor response to CIT treatment (non-responders). COX activity in responders and non-responders at d0 were significantly different from each other. COX activity in responders increased by 100% (p=0.027) after 14 days of CIT supplementation whereas there was no significant change in non-responders after supplementation or exercise. Exercise further doubled COX activity in responders (p=0.005). COX activity in control horses increased significantly by d14 (p=0.001), and decreased by 25% after the exercise bout (p=0.069). CS activities were not different between and within treatments at any time point, or between responders and non-responders in the CIT group. Our data suggest that CIT supplementation may support energy supply during exercise in horses that exhibit lower skeletal muscle mitochondrial function under resting conditions. ( en )
General Note:
Awarded Bachelor of Science; Graduated May 7, 2013 summa cum laude. Major: Biology, Emphasis/Concentration: Pre-Professional
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Advisor: Stephanie Wohlgemuth
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College of Agricultural and Life Sciences

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Copyright Maria Guzman. 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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The Effect of C itrulline Supplementation on Horse Skeletal Muscle Mitochondrial Function and Biogenesis March 2 7 2013 ________________________ _____________________ __ __ ____________________ Maria Guzman Stephanie Wohlgemuth Ph.D. Allen Wysocki, Ph.D. CALS Biology Undergrad uate Faculty Advisor CALS Associate Dean

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Maria Guzman Biology Honors Thesis 2 TABLE OF CONTENTS ABSTRACT ................................ ................................ ................................ ................................ ....................... 3 INTRODUCTION ................................ ................................ ................................ ................................ .............. 4 METHODS ................................ ................................ ................................ ................................ ......................... 6 Experimental Design and Tissue Sample Collect ion ................................ ................................ ...................... 6 Tissue Analysis ................................ ................................ ................................ ................................ ............... 6 Sample Preparation ................................ ................................ ................................ ................................ ..... 6 Cytochrome c Oxidase (COX) Activity ................................ ................................ ................................ ..... 7 Citrate Synthase (CS) Activity ................................ ................................ ................................ ................... 7 Statistical Analysis ................................ ................................ ................................ ................................ ..... 7 RESULTS ................................ ................................ ................................ ................................ ........................... 8 Citrate Synthase Activity ................................ ................................ ................................ ................................ 8 Cytochrome c Oxidase Activi ty ................................ ................................ ................................ ...................... 9 COX activity normalized to CS activity ................................ ................................ ................................ ....... 11 DISCUSSION ................................ ................................ ................................ ................................ ................... 13 Citrate Synthase Activity ................................ ................................ ................................ .............................. 13 Cytochrome c Oxidase Activi ty ................................ ................................ ................................ .................... 13 Limitations and further studies ................................ ................................ ................................ ..................... 16 CONCLUSION ................................ ................................ ................................ ................................ ................ 18 REFERENCES ................................ ................................ ................................ ................................ ................. 19

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Maria Guzman Biology Honors Thesis 3 ABSTRACT In this study, w e evaluated the effect of dietary citrulline on mitochondrial function and number in horse skeletal muscle. Untrained horses (n=12; 10.8 2.5 years old) were supplemented daily for a period of 15 days with either citrulline malate (CIT; 86 mg citrulline /kg body weight; n=6) or placebo (CTR; 25 mg u rea/kg body weight; n=6). Skeletal muscle samples were collected prior to supplementation (baseline), and on day 14 of the supplementation. An additional muscle sample was collected on day 15 one hour after a two hour medium intensity exercise bout. In tho se skeletal muscle specimens enzyme activities of cytochrome c oxidase (COX) and citrate synthase (CS) were assessed to evaluate mitochondrial function and number, respectively. Interestingly, we found that horses in the CIT group could be separated in two groups: Horses with lower COX activity on d0 but significant response to CIT treatment (responders), and horses with higher COX activity on d0 but minor response to CIT treatment (non responders). COX activity in responders and non responders at d0 were significantly different from each other. COX activity in responders increased by 100% ( p=0.027 ) after 14 days of CIT supplementation whereas there was no significant change in non responders after supplementation or exercise Exercise furt her doubled COX activity in responders (p= 0.005 ) COX activity in control horses in creased significantly by d14 (p=0.001 ), and decreased by 25 % after the exercise bout (p=0.069 ). CS activities were not different between and within treatments at any time point, or betw een responders and non responders in the CIT group Our data suggest that CIT supplementation may support energy supply during exercise in horses that exhibit lower skeletal muscle mitochondrial function under resting conditions

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Maria Guzman Biology Honors Thesis 4 INTRODUCTION Citrulline (CIT) is a non protein non essential amino acid found la rgely in watermelons Although non essential, c itrulline plays a critical role in ammonium metabolism in the urea cycle ( Fig. 1 ) and is a precursor of the amino acid Arginine (Arg). It ha s been established that CIT plays an important role in both cellular metabolism and organ functionality 1 Recent studies have also shown that citrulline supplementation in rodents enhanced exercise performance by reducing exercise related fatigue and energ etic cost of contraction 2, 3 In fact, citrulline malate supplements are commercially available in the exercise nutrition industry and continue to grow in popularity as enhancers of exercise performance. A possible mechanism through which CIT may benefit exercise performance could be through an increase in Arg availability, shown in a group of professional cyclists supplemented with citrulline 4 T he amino acid Arg is important in protein biosynthesis as well as energy metabolism as it is indirectly required for the primary storage of creatine phosphate, a major energy storing molecule that is immediately available for short term exercise 5 In addition, Arg can be broken down to CIT and Nitric Oxide (NO) by the enzyme Ni tric Oxidase Synthase (NOS). A CIT induced increase in Arg can thereby lead to increased levels of NO, a known vasodilator, subsequently enhanc ing blood flow to the muscles during exercise. Many of the biological effects of NO, such as vasodilation and gro wth of new blood vessels, have already been established, but Nisoli et al. 6 have recently reported that NO triggered the biogenesis of mitochondria, the site of ATP production in the cell in a variety of tissues, including heart muscle. It is possible tha t CIT exerts its exercise supporting effects by increasing circulating Arg l evels and thereby increasing NO, which could result in increased mitochondrial biogenesis. It was further suggested that citrulline may enhance the elimination of the metabolic by products Citrulline as part of plasma lactate and ammonium, a known cause of fatigue, thereby increasing exercise endurance 2,3 Another study provided more evidence that citrulline malate supplementation resulted in a protective effect against a build up of ammonia during exercise Fig. 1 Urea Cycle.

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Maria Guzman Biology Honors Thesis 5 confirming its role in reducing exercise fatigue 7 Bendahan et al. 8 reported that citrulline malate supplementation led to a significant increase in the rate of oxidative ATP pro duction during exercise Their group suggested that increased m alate supply could activate ATP production by increasing activity of the citric acid c ycle 8 However, the exact mechanisms by which CIT leads to increased exercise performance have not yet been clarified. In an attempt to elucidate the biological effects of CIT on equine skeletal muscle, this study evaluated the effect of CIT supplementation on mitochondrial function and biogenesis in horse skeletal muscle Based on the findings described ab ove we hypothesize d that citrulline supplementation would induce mitochondrial biogenesis and improve mitochondrial function ; thereby supporting and potentially enhan cing exercise performance. In the present study, skeletal muscle samples collected from horses enrolled in a citrulline malate supplementation study were analyzed for enzyme activities of cytochrome c oxidase (COX) and citrate synthase (CS) to evaluate mitochondrial function and content, respectively. According to a recent study, the enzyme a ctivity of complex IV (COX) in the electron transport chain is an appropriate marker of muscle oxidative capacity 9 Comparing COX enzyme activity of muscle homogenates before and after CIT supplementation and following an exercise bout could help explain C IT effects on mitochondrial function specifically within the electron transport chain Further, CS activity is a commonly used marker of mitochondrial content as there is a strong association between density of total mitochondria and chang es in citric aci d cycle enzymes 10 To test our hypothesis we utilized muscle samples previously taken from horses that were either feed supplemented with CIT or isonitrogenous placebo daily for a period of 15 days. Muscle samples were collected prior to supplementation (d0) and again on Day 14 (d14) of supplementation. On Day 15, the horses underwent a 2 hour exercise bout and were subsequently biopsied again 1 hour post exercise (d15)

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Maria Guzman Biology Honors Thesis 6 METHODS Experimental Design and Tissue S ample C ollection Twelve untrained mature Thoroughbred and Quarter horses ( mares; average age 10.8 2.5 years of age ) were enrolled in a citrulline malate supplementation study located at the UF/IFAS Equine Sciences Center (ESC) The horses were housed on pasture at the ESC The mares enrolled i n the study were paired by age and breed and randomly assigned to the citrulline supplementation (CIT) or the control group (CTR bermudagrass hay, fortified commercial feed. The feed was supplemented daily with either citrulline malate ( CIT; 86 mg citrulline /kg body weight; n=6) or placebo ( CTR; 25 mg urea/kg body weight ; isonitrogenous ; n=6) for a period of 15 days On d 15, horses underwent a prolonged exercise bout (medium intensity) which consisted of a 2 hour walk trot canter regimen in a free stall exerciser. The total distance achieved was 27.5 km, at a maximum speed of 4.83 m/sec. Average heart rate was 142 2 beats per minute. Muscle biopsies from the gluteus medius were t aken prior to supplementation ( day 0) and again on day 14 (d14) of supplementation. A third muscle biopsy was taken on day 15 (d15) one hour after the exercise bout. Immediately after the muscle biopsy, muscle specimens were freed of visible fat and connec tive tissue and immediately flash frozen in liquid nitrogen. The samples were kept at 80 C upon arrival in the laboratory until further biochemical analyses All procedures were reviewed and approved by the Institute of Food and Agricultural Sciences Animal Research Co mmittee prior to the start of the study (008 10ANS). Tissue Analysis Sample Preparation Muscle samples were pulverized in liquid nitrogen using a Bi o Pulverizer ( Biospec Products, Bartlesville, OK ). Pulverized s amp les were subsequently homogenized in e xtraction buffer (0.1 M KH 2 PO 4 Buffer, 2 mM EDTA, pH 7.2) as described in Adhihetty et al. 11 Briefly, tissue powder was diluted 1:20 (w/v) in extraction buffer, mixed in a thermomixer at room temperature (1 3 00 rpm) for 15 min, and sonicated (3 x 3 s) The homogenates were further diluted to 1:80 with extraction buffer and cen trifuged for 2 min at room temperature ( 14,000 x g ) The pellet was discarded, and the s upernatant kept in ice thereafter, was collected for measurement of both citrate synthase and cytochrome c oxidase activity. Protein concentration of samples was determined using a Bradford assay (BioRad, Hercules, CA), according to th

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Maria Guzman Biology Honors Thesis 7 Cytochrome c Oxidase (COX) Activity COX activity in skeletal muscle homogenates was determined by spectrophotometric analysis modified after Smith 12 Briefly, a solution of reduced horse heart cytochrome c (cyt c; 2 mg/mL; Sigma, St. Louis, MO) serving as substrate for COX was prepared in 100 mM KPO4 buffer (pH 7.0) using 57 mM sodium dithionite (in 10 mM KPO4) as a reducing agent Enzyme activity was determined from the rate of oxidation of reduced cyt c by C OX in the muscle homogenates at 30 C, measured by a reduction in absorbance at 550 nm using a microplate reader (Synergy HT, Biotek Instruments, Winooski, VT; Gen5 operating software). COX activity is reported in mol/min/mg protein. Citrate Synthase (CS) Activity The activity of CS in skeletal muscle homogenates was determined by spectrophotometric analysis as described in Kuznetsov et al 13 In this assay, the production of Coenzyme A by CS from Oxaloacetate and Acetyl CoA is coupled to the irreversibl e reaction of Coenzyme A with DTNB (5,5 dithiobis 2 nitrobezoate), producing TNB (thionitrobenzoic acid). The absorbance of TNB is measured at a wavelength of 412 nm using a microplate reader. The reaction mix contained a final concentration of 0.25% Trito n X 100, 0.31 mM Acetyl CoA (30 mM stock in water), 0.1 mM DTNB (1.01 mM stock in 1.0 M Tris reaction was started by addition of 0.5 mM Oxaloactetate (10 mM stock in 0.1 M triethanolamine pH 8.0). Citrate Synthase activity is reported in mol/min/mg protein. Statistical Analysis Results analyzed by Two Way ANOVA with significance set at p = 0.05, using statistical software (SigmaPlot version 12.0; Systat Software, Inc., San Jose, CA).

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Maria Guzman Biology Honors Thesis 8 RESULTS This study aimed to investigate the effect of a citrulline malate supplementation diet on mitochondrial function and biogenesis in horse skeletal muscle. We used Citrate synthase (CS) and Cytochrome c Oxidase (COX) activit ies to assess mitochondria l content and function respectively in sk letal muscle from horses that were either fed a citrulline malate supplement or an isonitrogenous placebo We compared enzyme activities at baseline (d0), after 14 days of supplementation (d14), and one hour after a medium intensity, two h our exercise bout on Day 15 (d15). Citrate Synthase Activity Statistical analysis of CS activity in CTR samples showed one significant outlier in the data, r educing the sample population to n=5. There were no significant outliers in the CIT samples which kept the sample size of the CIT group at n=6. We found no significant differen ce in average CS activity between CIT and CTR group on d0 (p= 0.115 ), d14 of CI T supplementation (p= 0.329 ), and after exercise (p= 0.325 ). There was also no significant change of CS activity within either treatment group between each of the time points ( CIT group: d0 to d14: p=0.082 d14 to d15 : p= 0.281 ; d0 to d15: p= 0.392 ) ( Fig. 2 ). Although mean CS activity of the control group tended to be lower than that of the treatment group at every time point the standard deviation among the samples is too high to draw significant conclusions CS activities in individual horses in the CTR and CIT groups are depicted in Fig. 3 A and Fig. 3 B respectively Statistical analysis indicat ed a significant time effect of both treatments from d0 to d15 (p=0.036). However, since there was no significant difference between the treatment groups at any time point, we conclude that CIT supplementation and exercise did not have a significant effect on mitochondrial content in horse skeletal muscle. Fig. 2 Enzyme activity of Citrate Synthase ( mol /min/mg protein). No significant difference in Citrate Synthase (CS) activity observed in skeletal muscle from horses fed citrulline (dark green bars; n=6) or placebo (light green bars; control; n=5) after 14 days of supplementation, or after a 2hr exercis e bout on day 15. Furthermore, there were no differences in CS activity between citrulline and control group at any time point. Data are shown as mean SD.

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Maria Guzman Biology Honors Thesis 9 Cytochrome c Oxidase Activity Mean COX activity tended to increase in CIT horses after 14 days of supplementation although not significantly (p=0.09 ; Fig. 4 ). COX activity in the CTR group was significantly higher on d14, but tended to decrease after a 2 hour exercise bout. On the contrary, there was a significant increase in COX activity in the CIT group after the exercise bout compared to baseline activit ies ( p= 0.007; Fi g. 4 ). COX activity in skeletal muscle from of CIT horses was significantly higher after the exercise bout compared to CTR group ( p= 0.045 : Fig. 4 ) The data suggest that horses supplemented with CIT but not the horses in the control group, were able to increase COX activity dur ing or following an exercise bout Fig. 3 Citrate Synthase activity in the individual hor ses ( mol/min/mg protein). A. Control horses: CS activity remained stable after supplementati on with placebo (d14) and slightly decrease d after exercise (d15) but not significantly B. Citrulline horses: CS activity remained stable after supplementation and exercise. The responders (solid lines) and non responders (dashed lines) described in results for Cytochrome c Oxidase activity ( Fig. 5 ) did not show significant differences in Citrate Synthase activity and therefore mitochondrial content. A. Con trol B Citrulline a a c b c b Fig. 4 Enzyme activity of Cytochrome c Oxidase ( mol/min/mg protein) in control (light blue bars) and CIT horses (dark blue bars). CIT supplementation was associated with an increase in Cytochrome c Oxidase (COX) activity after exercise (d15). COX activity in controls was elevated on d14, but this increase was not observed after the exercise bout. Same letter index denotes values significantly different from each other; a: p = 0.007; b: p = 0.013; c: p = 0.045. Data are shown as mean SD.

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Maria Guzman Biology Honors Thesis 10 COX activities for in dividual horses in the C TR and CIT groups are depicted in Fig. 5 A and Fig. 5 B respectively The horses in the control group displayed a similar pattern from d0 to d14, and after exercise ( Fig. 5 A ). However, w ithin the CIT group a pattern emerged in which some horses had significantly lower COX a ctivities at day 0 than others ( Fig. 5 B solid lines versus dashed lines ) Following this observation, we evaluated the responses of these two distinct groups within the CIT horses Those with lower COX activity on d0 exhibited a significant response to CIT treatment (responders ; solid lines ), and those with higher COX activity on d0 exhibited a minor response to CIT treatment (non responders ; d ashed lines ). On average, mean COX activ ity in skeletal muscle from CIT responders ( Fig. 6 citrulline A ) increased significantly compared to d0 after supplementation (d14; p= 0.027 ), and after exercise (d15; p= 0.001 ) On the other hand, COX activity in skeletal muscle from a b c, d b e a e d f f c Fig. 6 Enzyme activity of skeletal muscle Cytochrome c Oxidase ( mol/min/mg protein). CIT supplemented horses were grouped into on their different baseline COX activity (d0; A. index c) and on their different course of response to CIT treatment. COX activity in CIT responders was significantly increased on d14 and after exercise (d15), while it was not significantly changed in CIT non responders. Control horses displayed an increase simil ar to CIT responders on d14, but exercise did not induce a further increase. Same letter index denotes values significantly different from each other; a: p = 0.027; b: p < 0.001; c: p = 0.025; d: p = 0.049; e: p = 0.005; f: p = 0.001 Fig. 5 Cytochrome c Oxidase activity for individual horses ( mol/min/mg protein). a. Control Samples b. C itrulline samples COX activity of responders (indicated by solid lines) and non responders (indicated by dashed lines). A. Control B Citrulline

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Maria Guzman Biology Honors Thesis 11 CIT non responders ( Fig. 6 citrulline B ) did not differ after supplementation (d14; p= 0.344 ) or exercise (d15; p= 0.503 ) compared to d0 Moreover, although CTR horses had an increased COX activity after 14 days of placebo supplementation ( Fig 6, c ontrol ) the mean COX activity after exercise was not significantly different from either d14 or d0. In fact COX activity tended to be decrease d by nearly 25% after exercise (p=0.069). We then calculated the percent change in COX activity from d0 to d14 and from d 0 to d15 for each individual horse and averaged among the groups ( Fig. 7 ). Th is representation of the data for responders and non r esponders emphasizes the different response to supplementation and exercise between CIT responders and CIT non responders Remarkably, COX activity in CIT responders ( citrulline A) was doubled after 14 days of CIT supplementation and further doubled after the exercise routine. In contrast, non responders ( citrulline B ) did not change significantly after supplementation or exercise. These results suggest that CIT in combination with exercise may be beneficial in increasing COX activity in horses with endogenously low COX activities ( Fig. 6) COX activity normalized to CS activity We were also interested in evaluating the COX activity normalized to mitochondrial content using CS activity as the normalizer ( Fig. 8 ) We thereby eliminated any effects on our data due to differen t mitochondrial content in each horse Normalized COX activity between CIT responders and CIT non responders did not differ significantly on d0 (p= 0.163) However, normalized COX activity in responders was significantly increased on d14 and after exercise compared to d0; normalized COX activity in non responders did not change significantly from d0 to d14 and d15, respectively; and COX activity after exercise was still significantly higher in CIT responders compared to CIT non responders ( Fig. 8 ) Interestingly, normalized COX activity in control horses increased similarly compared to CIT Fig. 7 Percent c hange in COX activity from d0 baselin e. Percent change expressed as a percentage of baseline activity. CIT supplementation was associated with an increase in COX activity in responders after exercise (d15). COX activity in controls was elevated on d14, but this increase was not observed after the exercise bout. Same letter index denotes values significantly different from each other; a: p = 0.020; b: p < 0.001; c: p < 0.001; d: p < 0.001; e: p = < 0.001; f: p = 0.020. Data are shown as mean SD. d a b b c, d e a c f f e

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Maria Guzman Biology Honors Thesis 12 responders; and although COX activity after the exercise bout tended to be lower in controls compared to CIT responders, this difference was not quite significant (p= 0.058 ). COX activity in CIT non responders and controls was not different between the groups at any ti me point In summary, we found that CIT supplementation had an effect on mitochondrial fun ction characterized by increased COX activity in horse skeletal muscle predominantly following an exercise bout However, this effect was mostly seen in CIT responders, which exhibited significantly low COX activity at d0 compared to non responders ( Fig. 6 ). Following CIT supplementation, responders exhibited a 2 fold increase in COX activity after exercise ( Fig 7 ), whereas no effect was observed in non responders ( Fig 7 ) These results may suggest that CIT treatment could have a beneficial effect on mitochondrial function in horses that, although phenotypically inconspicuous, show a minor mitochondrial impairment. a b a c b c, d e f d e g Fig. 8 Enzyme activity of skeletal muscle Cytochrome c Oxidase normalized to CS activity (activity expressed as a percentage of CS activity). COX activity after exercise was significantly greater in CIT responders compared to CIT non responders. Control horses displayed an increase in normalized COX activity from d0 to d14 and after exercise (d15) similar to CIT responders. Same letter index denotes values significantly different from each other; a: p = 0.035; b: p < 0.001; c: p < 0.001; d: p = 0.043; e: p = 0.046; f: p < 0.001; g: p = 0.049. Data are shown as mean SD. f g

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Maria Guzman Biology Honors Thesis 13 DISCUSSION In the present study we investigate d the effect of citrulline malate supplementation on mitochondrial function and biogenesis in equine skeletal muscle. Citr ate synthase and Cytochrome c Oxidase activities were used as markers to assess mitochondrial content and function, respectively, in muscle tissues from horses that were either fed a citrulline malate supplement or an isonitrogenous placebo We compared the activity of these enzymes at baseline (d0), after 14 days of supplementation (d14), and one hour after a medium intensity, two hour exercise bout on Day 15 (d15) We hypothesized that citrulline supplementation would induce mitochondrial biogenesis and improve mitochondrial function ; thereby supporting and potentially enhan cing exercise performance. Our findings suggest that citrulline malate supplementation does not have a significant effect on mitochondrial content in skeletal muscle at any time point However we found evidence that CIT supplementation may play a role in increasing COX activity significantly in horses with endogenously low COX activity especially after exercise stimulation Citrate Synthase Activity Citrate Synthase activity is stron gly associated with mitochondrial content and is therefore a good biomarker for mitochondrial number 9,10 CS activity in skeletal muscle from horses enrolled in the citrulline supplementation study was measured to assess the effect of CIT and/or medium intensity exercise on mitochondrial content in horse skeletal muscle. We found that t here was no significant difference in mean CS activity ( Fig. 2 ) between the treatments or across time points. Statis tical analysis indicat ed a significant time effect of both treatments from d0 to d15 (p=0.036) These results indicate that mitochondrial content is not affected by CIT supplementation applied in this study, and does not change significantly in muscle samp les collected 1 hour after a medium intensity exercise bout. We conclude that the CIT supplementation regimen in this study does not have a significant effect on mitochondrial content in horse skeletal muscle. Cytochrome c Oxidase Activity The enzyme activity of complex IV (COX) of the mitochondrial electron transport chain is an appropriate marker of muscle oxidative capacity 9 It was assessed in this study to evaluate the effect of CIT supplementation on mitochondrial function. One interes ting finding of this study was that despite random assignment of the horses to either control or CIT group, horses in the CIT group were distinct from each other and could be separated in two groups ( Fig. 6 ): Horses with lower COX activity on d0 but signi ficant response to CIT treatment (responders), and

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Maria Guzman Biology Honors Thesis 14 horses with higher COX activity on d0 but minor response to CIT treatment (non responders). The difference in COX activity on d0 in the responders and non responders ( Fig. 6 ) was significant (p=0.025) and justified examining these groups separately due to their significantly different responses to treatment ( Fig. 7 ). While responders and controls exhibited a similar increase in COX activity on d14, responders had a significant response to exercise which was not seen in controls. Activity in non responders did not change over time ( Fig. 6 ). Following the exercise bout, COX activity of the CIT responders, which started out with a significantly lower on d0 compared to the non responders, reached similar COX ac tivity values to that of non responders on d14 ( Fig. 6 ) This could be an indication that the non responders already had high endogenous COX activity and did not need to increase activity to sustain exercise performance at medium intensity. On the other ha nd, the responders with lower endogenous COX activity at baseline perhaps had greater need and capacity to increase activity after supplementation and exercise. Different endogenous COX activities in these two groups would explain their different respons es to CIT supplementation and exercise. It should be pointed out that we did not detect this heterogeneity in the control group. The percent change in COX activity ( Fig. 7 ) highlights the substantially different responses to supplementation and exercise among the CIT responders and non responders. Interestingly, COX activity in CIT responders doubled after supplementation and further doubled after the medium intensity exerci se bout. In contrast, non responders did not change significantly after supplementation or exercise. CTR horses exhibited increased COX activity after placebo supplementation; however, activity tended to decrease by nearly 25% after exercise (p=0.069). The se results suggest that CIT combined with exercise may increase COX activity in horses with endogenously low activities. We then normalized COX activity to mitochondrial content using CS activity, thereby eliminating any effects due to different mitochondr ial content ( Fig. 8 ). This would be a better indicator of changes in COX activity between the groups by accounting for horses that may have higher endogenous mitochondrial content. In essence, a higher number of mitochondria could naturally be associated w ith a higher overall COX activity. By normalizing COX activity to mitochondrial content, we were able to distinguish true differences in the enzym atic activity of COX. For example, in theory the samples with the higher mean COX activities ( non responder s, Fig. 6 ) could have a higher mitochondrial content, rather than higher COX activity per mitochondria. However in this study we did not find differences in mitochondrial content between these two group using CS activity as a marker ( Fig. 3B )

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Maria Guzman Biology Honors Thesis 15 W e found that non responders had lower COX activities per mitochondrial content compared to responders significantly on d15 ( Fig. 8 ) This is an interesting observation and suggests that higher endogenous COX activities observed for the non responders might actua lly be due to higher mitochondrial content rather than higher enzyme activity in each mitochondrion Measuring COX protein content using Western blots would help support the conclusion that the non responders may have higher COX content, as opposed to high er enzyme activity. Further analyses have to investigate whether the increase in COX activity after exercise was due to true activity increase or increased COX enzyme content within already present mitochondria Our results showed that normalized COX activ ity after the exercise bout was significantly higher in responders than non responders (p=0.043) but not significantly different from control ( Fig. 8) These results suggest that CIT supplementation, in combination with an exercise bout, can have a benefi cial effect on mitochondrial function in horses that show a minor mitochondrial impairment, characterized by lower endogenous COX activity. A possible explanation for the different levels of endogenous COX content and activity in the CIT responders and non responders could be due to individual horse differences. Although the horses were randomly selected for the study, some horses were inevitab the personality or level of activity of responders vs. non responders (study PI, personal communication) However, the great variation in enzyme activities in among the h orses was likely due to such individual differences. Strangely, the overall mean COX activity in CTR horses decreased after exercise ( Fig. 6 ), whereas COX activity normalized per mitochondrial content was actually higher after exercise ( Fig. 8 ). It is well known that exercise induces mitochondrial function, and it is therefore not surprising that control horses display increased COX activities post exercise Our results suggest that mitochondrial content in CTR horses decreased after exercise, perhaps due t o oxidative damage to mitochondrial components which would explain the lower average COX activity. This observation is not supported by CS activity ( Fig. 2 ), which does not significantly change in the CTR group from d14 to d15, suggesting that mitochondrial content remains the same before and after exercise. Further analysis is needed to explain why normalized COX activity increased after exercise, while mea n COX activity decreased. Interestingly, mean COX activity ( Fig. 4 ) in the CTR horses was significantly higher after the supplementation period (d14) and similarly tended to be elevated in CIT horses at this time point. It is interesting that COX activit y in the CTR and CIT horses had a similar response on d14, after supplementation with urea and citrulline malate,

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Maria Guzman Biology Honors Thesis 16 respectively. We reason that the similar response may be due to study related horse management rather than treatment. In conjunction with anot her study, t he horses needed to be fasted and were confined 24 hours prior to the muscle biopsy on d14 The confinement in the stall could have caused tension on the musculature as well as stress, which in turn could have induced changes to skeletal muscl e mitochondria However, after a medium intensity exercise bout on d15 COX activity was significantly elevated in CIT horses compared to d0 (p=0.007 : Fig 4 ) and compared to controls at the same time point (p= 0.045: Fig 4 ) On the other hand, a decrease in mean COX activity was observed in CTR horses after the exercise bout. This suggests that CIT supplementation combined with exercise might play a role in increasing COX activity and thereby enhancing mitochondrial function. Limitations and further studies The major limitation of the study is the small sample population. With only six experimental and six control horses, it is difficult to determine whether any variation in the results was due to random factors such as indivi dual personalities or horse breed For instance, we found significant differences in the baseline COX activity between the CIT responders and CIT non responders. However, with such a small sample size any significant differences could be due to random err or rather than a true difference between these groups A larger scale study would add more statistical power to our observations, and thereby be helpful to determine with more confidence whether the trends and significant differences that we observed are t rue or occurred by chance. In addition, the skeletal muscle specimen collected at the three different time points were relatively small and prevented the determination of additional and comprehensive parameters that would be helpful to support our results. If skeletal muscle specimen size allows, f urther analyses of our samples have to investigate whether the increase in COX activity after exercise was due to true activity increase or increased COX en zyme content. Analysis of oxidatively damaged proteins in CIT versus control tissue could help explain the more modest increase in COX activity after exercise in control horses. Additionally, assessment of protein expression of the energy sensing enzyme AM P activated protein kinase (AMPK) and downstream effectors could help to characterize the differences between responders and non responders in the CIT group. Finally, a dditional analysis of these samples is necessary to conclude with certainty the effect o f CIT 14 Another study found that

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Maria Guzman Biology Honors Thesis 17 both PGC 1 mRNA and P GC 1 protein increased 2 fold after a single bout of exercise and the change was evident 18 hours after exercise 15 Measuring these markers in our horse samples would help us draw conclusions about changes in mitochondrial number. However, it must be noted that muscle samples in the present study were collected shortly after the exercise bout, perhaps not allowing enough time for new mitochondria to form. Future studies should collect muscle samples at different time points after an exercise bout to observe changes in mitochondrial number with more confidence.

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Maria Guzman Biology Honors Thesis 18 CONCLUSION In conclusion, in the present study we found that citrulline malate supplementation does not have a significant effect on mitochondrial content in skeletal muscle collected 1 hour after a 2 h medium intensity exercise bout However CIT supplementation played a role in increasing COX activity significantly in horses with endogenously low COX activity. These results may suggest that CIT treatment could have a beneficial effect on mitochondr ial function in horses that, although phenotypically inconspicuous, show a minor mitochondrial impairment. CIT supplementation also resulted in a significant increase in COX activity after exercise whereas activity in the CTR group dropped nearly 25% after exercise. This further suggests that CIT supplementation combined with exercise plays a role in increasing COX activity and thereby enhancing mitochondrial function. Our results support data in the literature which reported that citrulline malate supplementation ha d an effect on mitochondrial function and enhance d exercise performance. Although this study did not specifically evaluate exercise performance, it was apparent that CIT supplementation had its most significant effects after the e xercise bout on d15. Our results show that CIT supplementation ha d no significant effect on skeletal muscle mitochondrial function under normal, resting conditions Studies have suggested that citrulline malate enhances mitochondrial function through the e ffects of malate on the citric acid cycle 8 However, an increase in Arg availability 4 would also explain increased mitochondrial function and therefore greater energy supply during exercise. Additional evaluation of the biochemical pathways of citrulline m alate supplementation is needed to gain a true understanding of its role in increasing mitochondrial function.

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Maria Guzman Biology Honors Thesis 19 REFERENCES 1. Curis, E., Nicolis, I., Moinard, C., Osowska, S., Zerrouk, N., Bnazeth, S., & Cynober, L. (2005). Almost all about citrulline in ma mmals. Amino acids 29 (3), 177 205. 2. Giannesini B Le Fur Y, Cozzone PJ, Verleye M, Le Guern ME, Bendahan D. (2011) citrulline malate supplementation increases muscle efficiency in rat skeletal muscle. Eur J Pharmacol 667: 100 104. 3. Takeda K, Machida M, Kohara A, Omi N, Takemasa T. (2011) Effects of citrulline supplementation on fatigue and exercise performance in mice. J Nutr Sci Vitaminol57: 246 250 4. Sureda A, Cordova A, Ferrer MD, Tauler P, Perez G, Tur JA, Pons A. (2009) Effects of L citrulline oral s upplementation on polymorphonuclear neutrophils oxidative burst and nitric oxide production after exercise. Free Rad Res 43 (9): 828 835. 5. Barbul, A. (1986). Arginine: biochemistry, physiology, and therapeutic implications. Journal of Parenteral and Enteral Nutrition, 10(2), 227 238. Barbul, A. (1986). Arginine: biochemistry, physiology, and therapeutic implications. Journal of Parenteral and Enteral Nutrition 10 (2), 227 238. 6. Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, Bracale R, Valer io A, Francolini L, Moncada S, O. Carruba M (2003) Mitochondrial Biogenesis in Mammals: The Role of Endogenous Nitric Oxide. Science 299: 896 899. 7. Callis, A., Magnan, D. B. B., Serrano, J. J., Bellet, H., & Saumade, R. (1991). Activity of citrulline malate on acid base balance and blood ammonia and amino acid levels. Study in the animal and in man. Arzneimittel Forschung 41 (6), 660. 8. Bendahan, D., Mattei, J. P., Ghattas, B., Confort Gouny, S., Le Guern, M. E., & Cozzone, P. J. (2002). citrulline /malate promotes aerobic energy production in human exercising muscle. British journal of sports medicine 36 (4), 282 289. 9. Larsen, S., Nielsen, J., Hansen, C. N., Nielsen, L. B., Wibrand, F., Stride, N., ... & Hey Mogensen, M. (2012). Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. The Journal of Physiology 590 (14), 3349 3360.

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Maria Guzman Biology Honors Thesis 20 10. Reichmann, H., Hoppeler, H., Mathieu Costello, O., Bergen, F. V., & Pette, D. (1985). Biochemical and ultrastructural changes of skeletal muscle mito chondria after chronic electrical stimulation in rabbits. Pflgers Archiv European Journal of Physiology 404 (1), 1 9. 11. Adhihetty, P. J., Uguccioni, G., Leick, L., Hidalgo, J., Pilegaard, H., & Hood, D. A. (2009). The role of PGC on mitochondrial functio n and apoptotic susceptibility in muscle. American Journal of Physiology Cell Physiology 297 (1), C217 C225. 12. Smith L. (1955). Spectrophotometric assay of cytochrome c oxidase. Methods Biochem Anal 2 ; pp. 427 434 13. Kuznetsov A.V., Lassnig B. Gnaiger E. (2010). Laboratory protocol: Citrate synthase. Mitochondrial marker enzyme. Mitochondrial Physiology Network. 08.14(1 10). 14. Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti S., Lowell, B., Scarpulla R. C., & Sp iegelman, B. M. (1999). Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC 1. Cell 98, 115 124. 15. Baar, K., Wende, A. R., Jones, T. E., Marison, M., Nolte, L. A., Chen, M., Kelly D. P. & Holloszy, J. O. ( 2002). Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC 1. The FASEB journal 16(14), 1879 1886.


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