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The effect of experimental diabetes on drug metabolism

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Title:
The effect of experimental diabetes on drug metabolism
Creator:
Ackerman, Dennis M., 1946-
Publication Date:
Language:
English
Physical Description:
ix, 85 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Cytochromes ( jstor )
Diabetes ( jstor )
Diabetes complications ( jstor )
Enzymes ( jstor )
In vitro fertilization ( jstor )
Insulin ( jstor )
Liver ( jstor )
Metabolism ( jstor )
Rats ( jstor )
Type 1 diabetes mellitus ( jstor )
Diabetes Mellitus, Experimental ( mesh )
Dissertations, Academic -- Pharmacology -- UF ( mesh )
Pharmaceutical Preparations -- metabolism ( mesh )
Pharmacology thesis Ph.D ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1975.
Bibliography:
Includes bibliographical references (leaves 81-84).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Dennis M. Ackerman.

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University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. 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.
Resource Identifier:
022762885 ( ALEPH )
25764580 ( OCLC )
AEK7324 ( NOTIS )

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Full Text












TH:E _-FCT OF EXPERIMENTAL DIABETES ON DRUG METABOLISM











By


DENNIS M. ACKERMAN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL CF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIRE2MENTTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA




-Lil-Cj
EFFECT OF EXPERIMENTAL
DIABETES ON DRUG METABOLISM
By
DENNIS M. ACKERMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


ACKNOWLEDGMENTS
The author wishes foremost to thank his supervisory
committee chairman, Dr. Kenneth C. Leibman, for his sug
gestions, assistance, and most of all his patience
throughout the course of this study. Recognition is also
extended to the other members of his committee, Dr. Owen
M. Rennert, Dr. David M. Travis, and Dr. Betty p. Vogh.
Recognition is also extended to Dr. Ira Weinstein for his
assistance.
The author also wishes to acknowledge Mr. George
Wynns for his assistance in surgical procedures and is
grateful for the aid cf Dr. Robert J. Cohen xn assisting
with cyclic AMP assays.
The author would like to acknowledge and thank
Kathleen Ackerman tor her encouragement and support pro
vided during a large part of his tenure as a graduate
student. Sincere appreciation is also extended to the
author's parents for their everlasting support.
A special thanks is extended to Mrs. Elsa Couto for
her assistance, companionship, and warmth.
The author would also like to express his appreci
ation for the friendship and aid of Mrs. Theresa Fulford
as well as thanking her for typing this manuscript.
ii


TABLE OF CONTENTS
Page
Acknowledgments
List of Tables.......
List of Figures.
Abstract
Historical Review....
Goals
Materia1s and Methods
Results
Discussion...........
Summary
References.
Biographical Sketch..
ii
iv
vi
vii
.1
.19
.24
.34
-67
81
. b o
1 1X


LIST OF TABLES
Table Page
1. Summary of Literature 14
2. Hexobarbital Sleeping Times after Adminis
tration of Diabetogenic Agents 42
3. Effect of Streptozotocin on Kinetic Constants
for Hexobarbital and Aniline Hydroxylations....43
4. Effect of 6-Aminonicotinamide on Kinetic Con
stants for Hexobarbital and Aniline... 45
Hydroxylations
5. Effect of N-Methylacetamide on Kinetic Con
stants for Hexobarbital and Aniline
Hydroxylations 46
6. Hexobarbital and Aniline Hydroxylations One
Week after the Administration of
Streptozotocin 47
7. Effect of Alloxan on Hexobarbital and Aniline
Hydroxylations 49
S. Effect cf Streptozotocin on Hexobarbital and
Aniline Hydroxylations with Isolated
Microsomes as Enzyme Source 50
9.Effect of 6-Aminonicotinamide on Hexobarbital
and Aniline Hydroxylations with Isolated
Microsomes as Enzyme Source 51
10. Effect: of Glucose Infusion on Hexobarbital
Metabolism. .53
11. Effect of Diabetogenic Agents Added In Vitro
on Hexobarbital Metabolism 54
12. Effect of Diabetogenic Agents Added In Vitro
on Aniline Metabolism 55
iv


Table Page
13. Effect of Insulin In Vitro on Hexobarbital and
Aniline Metabolism Following Diabetogenic
Agents 57
14. Effect of Diabetogenic Agents on Hepatic Micro
somal Protein Concentration 58
15. Effect of Diabetogenic Agents on Cytochrome
P-450 Concentration in Liver Microsomes........60
16. Effect of Diabetogenic Agents on Cytochrome
P-450 Binding to Hexobarbital and
Aniline .61
17. Localization of Inhibitor of Hexobarbital
Metabolism Present in 100,000^ Super
natant Fraction of Rat Liver after
Pretreatment with Streptozotocin and
6-Amincnicot inamide 63
18. Localization of Inhibitor of Aniline Meta
bolism Present in 100,000a Supernatant
Fraction of Rat Liver after Pretreatment
with Streptozotocin and 6-Aminonicotin-
amide 6 4
19. Effect of Diabetogenic Agents on Hepatic c-AMP
Concentrations
66


LIST O1 FIGURES
Figure Page
1. Structures of Diabetogenic Agents 2 5
2. Relationship between Serum Glucose and Dose
of Streptozotccin 36
3. Relationship between Serum Glucose and Dose of
6-Aminonicotinamide 38
4. Relationship between Serum Glucose and Dose
of N-Methylacetamide .40
5. Metabolic Pathway of Hexobarbital 69
6. Metabolic Pathway of Aniline 70
vi


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF EXPERIMENTAL DIABETES ON DRUG METABOLISM
By
Dennis M. Ackerman
December, 1975
Chairman: Dr. Kenneth C. Leibman
Department: Pharmacology and Therapeutics
The purpose of this investigation was to examine the
effect of experimental diabetes on the hepatic microsomal
drug-metabolizing system of the liver in male Holtzman rats
and to elucidate a mechanism for these effects. Three dia
betogenic agents were used: streptozotocin, 6-aminonicotin-
arnide, and N-methylacetamide. Streptozotocin and 6-araino-
nicotinamide produce an insulin-deficient animal whereas
N-methylacetamide does not affect insulin production or
release but causes a condition in which insulin is incap
able of lowering blood glucose.
Both in vivo and in vitro experiments were performed
to investigate the effects of the diabetic state induced
by these three agents on hepatic drug metabolism. The
results of the in vivo experiments correlated with the
results of those in vitro in that inhibition of certain
pathways of drug metabolism was seen in animals pretreated
with streptozotocin and 6-aminonicotinamide but not in ani
mals pretreated with N-methy lacetamide even though ili
vii
A


treated animals were hyperglycemic.
The rate of hydroxylation of hexobarbital was not
significantly different in preparations from rats that had
been infused with glucose and those from saline-infused
control animals. This demonstrates conclusively that the
inhibition caused by diabetogenic agents was not due to
hyperglycemia.
Neither insulin nor any of the diabetogenic agents had
any direct effect on drug metabolism in vitro. Further
more, hepatic microsomal protein and cytochrome P-450
contents were not significantly different in any of the
diabetic animals than those of the control animals.
The binding of both hexobarbital and aniline to he
patic cytochrome P-450 was inhibited in animals pretreated
with streptczotocin but only that of hexobarbital was in
hibited in 6-aminonicotinamide-pretreated animals. The
fact that the affinity of aniline to cytochrome P-450 was
increased in microsomes from animals pretreated with N-
methylacetamide somewhat obscures the interpretation of
these results.
It had been shown previously that treatments that in
creased the endogenous levels of cyclic 35'-adenosine
monophosphate (c-AMP) in the liver were inhibitory to cer
tain drug metabolic pathways and that this was due to the
release of an inhibitor of drug metabolism into the liver
cytosol. The presence of an inhibitor of hexobarbital and
viii


aniline hydroxylation was shown to occur in the liver
cytosol of animals pretreated with streptozotocin and an
inhibitor only of hexobarbital hydroxylation in animals
pretreated with b-aminonieotinamide. This correlated with
elevated levels of hepatic c-AMP observed in animals pre
treated with streptozotocin and to a lesser degree in
animals pretreated with 6-aminonicotinamide. The levels
of c-AMP were not elevated in animals pretreated with N-
methylacetamide, as compared to control animals.
In conclusion, it appears that the inhibition of drug
metabolism in animals made diabetic with certain drugs is
not caused by hyperglycemia, but is rather the consequence
of insulin lack. It is proposed that this lack of insulin
causes an elevation of hepatic c-AMP which, in turn, causes
the release by liver microsomes of an inhibitor of the
metabolism of xenobiotics.
ix


HISTORICAL REVIEW
Many of the metabolic alterations due to diabetes are
well understood but the effect of the disease on hepatic
drug metabolism is not clear, even though this has been
studied for over 15 years. No definitive conclusions can
be drawn from the data accumulated because of the consid
erable amount of conflicting results that have appeared
in the literature.
In 1958 (1), the half-life of tolbutamide was deter
mined in both diabetic and nondiabetic humans of both
sexes. The half-life was found to be similar in both
groups (7.8 hours in the diabetic group and 8.0 hours in
the nondiabetic group). Prolonged use (2.5 5 months)
of tolbutamide in the diabetic patients was shown to
decrease the half-life of the drug in these patients
about 17% (7,3 hours to 6.5 hours). This decrease is
probably due to the fact that tolbutamide is an inducer
of the liver microsomal oxidase system that metabolizes
foreign compounds (2) and thus it stimulates its own
metabolism. Large deviations among diabetic and non
diabetic subjects as well as the small sample size pre
vented statistical analysis of these data.
1


2
It was shown in 1961 by Dixon et al. (3) that the
activities of drug-metabolizing enzymes of liver micro-
som.es were decreased by short-term alloxan diabetes (the
duracin of the diabetic state was never longer than three
days). In this study, microsomal liver preparations of
male Holtzman rats that had been pretreated with alloxan
had a diminished ability to metabolize hexobarbital (side
chain oxidation), chlorpromazine (oxidation of ring sulfur),
and codeine (O-demethylation). In vivo, hexobarbital
sleeping time was prolonged after alloxan pretreatment,
which correlated well with the reduced microsomal enzyme
activity toward hexobarbital. Both the in vitro and in
vivo effects could be reversed by treating the diabetic
animal with insulin. Neither alloxan nor insulin had any
effect when added to the incubation mixtures in vitro.
The authors concluded that the diabetic state had an
inhibitory effect on the ability of these animals to
metabolize drugs.
A follow-up study (4) in 1963 was done to see the
effect of long-term (1-3 month duration) alloxan dia
betes on certain pathways of the hepatic microsomal
mixed-function oxidase drug-metabolizing system. In this
study, male rats of the Long-Evans strain were used as
well as the Holtzman strain because the former appeared
to be more resistant to the toxic effect of alloxan
and the following diabetic condition. A decreased rate


3
of metabolism in vitro and a prolonging effect on hexo-
barbital action in vivo was again seen. Also, a decreased
rate of in vitro metabolism of aminopyrine (which is
demethylated) was observed, whereas an increased rate of
in vitro metabolism of aniline (which is hydroxylated)
was seen. Insulin treatment returned the rate of hexo-
barbital metabolism back to control levels but had no
effect on aminopyrine metabolism. The rate of aniline
metabolism was decreased below the control rate after
insulin treatment. From this study, the authors concluded
that the diabetic state had varying effects on drug meta
bolism which were substrate-dependent.
In the same year, it was reported by Gillette et al.
(5) that the administration of alloxan to immature female
rats did not alter zoxazolamine hydroxvlation or mono-
raethy1-4-aminopyrine demthylation. On the other hand,
the O-dealkyiation of phenacetin was stimulated by the
alloxan-diabetic state.
In 1965, Kato and Gillette (6) again demonstrated
that alloxan diabetes of three days' duration decreased
the activity of the hepatic microsomal enzyme system of
male Sprague-Dawley rats toward aminopyrine and hexobar-
bital but did not change the acitivity of the system
toward zoxazolamine. An enhancement cf aniline hydrox-
ylation was again seen. The effect of the alloxan dia
betic state in female Sprague-Dawley rats, though, was
much different. In these animals, the metabolism of


4
aminopyrine, aniline, and zoxazolamine was increased by
the diabetic state, but the activity of the hexobarbital-
metabolizing enzyme system was not significantly affected.
When female rats were castrated and given methyltestos-
terone, the results were different from those observed
with intact female rats, but were similar to those seen
with male rats. Also, alloxan diabetes did not produce a
further reduction in hexobarbital hydroxylation or amino
pyrine demethylation in male rats that had been castrated.
Castration of male diabetic rats, however, did not pre
vent the increased activity of the microsomal system
toward aniline that was also seen in the non-castrated
male diabetic rats. These results showed that certain
hepatic microsomal pathways in diabetic rats, such as
that of aminopyrine, were sex-dependent; whereas other
pathways, such as that for aniline, were not.
A cytochrome termed "P-450,found in the hepatic
microsomal fraction, is considered co be the final oxygen-
activating enzyme in many NADPH-dependent drug-metabolic
pathways. It catalyzes the reduction of an oxygen mole
cule with placement of one oxygen atom into water and
simultaneous insertion of the other oxygen atom into a
foreign substrate. This cytochrome can be measured quan
titatively by the difference spectrum observed when it is
reduced (usually with d.ithionite) and combined with carbon
monoxide.


5
Drugs combine with cytochrome P-450 (7) to produce
difference spectra of two general types, known as types I
and II. Type I compounds, exemplified by hexobarbital and
aminopyrine, give difference spectra with ^max in the range
of 385 390 ran and A in the range of 418 427 ran.
Type II compounds, exemplified by aniline, give ^max
between 425 435 nm and A between 390-405 (8). These
mm
spectral changes resulting from substrate interaction with
cytochrome P-450 have been presumed to represent the pri
mary binding of substrate for enzyme action and a change
in the degree of binding may be indicative of a change in
the amount of substrate being metabolized. The strength
of binding can be determined spectrophotometrically by
plotting the reciprocals of the spectra change vs. those
of substrate concentration in a Lineweaver-Burk type of
plot to give by extrapolation the spectral dissociation
constant (K^,)
In 1970, Kato et at. (9) studied the effect of alloxan
diabetes on Wistar rats and mice of the dd strain. In this
study, the effect of alloxan diabetes on the in vitro meta
bolism of several substrates and on the content of cyto
chrome P-450 and NADPH-cytochrome o reductase (the first
enzyme in the electron-transport chain involved in drug
metabolism in the liver) were examined. In male rats, a
decrease was again seen in the rates of both aminopyrine
N-demethylation and hexobarbital hydroxylation but in


female rats and male and female mice, an increase was
observed in the rates of metabolism of both of these sub
6
strates. An increase in the rate of aniline hydroxylation
was seen in both sexes of rats and mice.
The activity of NADPH-cytochrome e? reductase was not
altered by alloxan diabetes in either the male or female
rats but was increased by alloxan diabetes as compared to
controls in both sexes of mice. The cytochrome P-450 con
tent in liver microsomes from the diabetic animals was
slightly increased over that of controls in the female
rats but was not affected by the diabetes in male rats or
male or female mice. The authors concluded that the oxi
dative activities of the liver microsomes of the male rats
toward certain substrates such as hexobaroital and amino-
pyrine are enhanced by androgen and that this enhancement
is blocked by alloxan diabetes. In mice, this androgen-
dependent enhancement is not present. Aniline hyaroxyl-
ation is presumed not to be androgen-dependent and there
fore alloxan diabetes does not increase its rate of
hydroxylation. Why the rate of its hydroxylation is
increased, however, was not explained.
The same authors (10) studied the effect of alloxan
diabetes on cytochrome P-450 content, substrate inter
action with hepatic cytochrome P-450, and kinetics of
several drug-metabolic pathways in male and female Wistar
rats. Alloxan diabetes did not alter the amount of cyto
chrome P-450 in the livers of male rats but slightly


/
increased the cytochrome P-450 content in the livers of
the female rats of this species. The affinity of the
cytochrome P-450 for hexobarbital and aminopyrine v/as
lower in male diabetic rats than it was in normal male
rats. Also, a decrease in the rates of both hexobarbital
and aminopyrine oxidation was observed. Aniline, however,
was hydroxylated at a greater rate in the diabetic male
rats than in controls although there was no significant
increase in the binding of the cytochrome P-450 to aniline
from, the diabetic rats as compared to controls. An
increase in the hydroxylat on of zoxazolamine as well as
an increased affinity for cytochrome P-450 was also seen
in the diabetic male rats as compared to control animals.
In female diabetic rats, increases in the degree of binding
of hepatic microsomal cytochrome P-450 to both hexobarbital
and aniline, as compared to those of control rats were
seen.
Inasmuch as hexobarbital and aminopyrine, which are
both type I compounds, show both decreased binding to
cytochrome P-450 as compared to controls as well as
decreased rates of oxidation, whereas aniline, a type II
compound, shows no change in affinity to cytochrome P-450
compared to control but an increase over control animals
in the rate of its metabolism, it was concluded that the
effect of alloxan diabetes on hepatic drug-metabolic
enzymes may be related to the type of spectral change seen
whan the drug binds to cytochrome P-450. The results seen


8
with zoxazolarnine, a type I compound, in the male rats
were net explainable. Also, the effect of alloxan diabetes
does not appear to be related to the type of spectral
change a drug gives with cytochrome P-450 in female rats.
In 1972, Dajani and Kayyali (11) studied the meta
bolism of phenacetin in male New Zealand White rabbits
which had been made diabetic by the administration of
alloxan. They found that the urinary and plasma concen
trations of the unchanged drug were higher in the diabetic
animals than in the controls, and that the concentrations
of the major metabolite, acetaminophen, were lower. This
was suggestive of a decreased metabolism of this drug in
the diabetic animal. A decreased rate of in vitro metabo
lism of phenacetin in liver microsomes from alloxan-
diabetic rabbits proved that the metabolism of phenacetin
was inhibited in these animals. Both the in vivo and in
vitro inhibition of phenacetin metabolism were reversible
by the administration of insulin to the animals. To
attempt to explain this inhibition of metabolism of phen
acetin in alloxan-diabetic rabbits, the contents of
hepatic microsomal protein and hepatic NADPH, a necessary
cofactor for the hepatic microsomal drug-metabolizing sys
tem, were measured, and both were found to be lower in the
diabetic animals than in control animals. They concluded,
therefore, that the inhibition that occurred in the
alloxan-diabetic state in the male rabbit was probably due


to a deficiency of microsomal drug-metabolizing protein
and/or a deficiency in hepatic NADPH.
In a follow-up study the following year (12), the
metabolic transformation of phenylbutazone (which is
hydroxylated) was examined in the alloxan-diabetic Wistar
rat. As in the previous study, the rates of both the in
vivo" and in vitro metabolism of the drug were decreased
in alloxan-diabetic animals as compared to control animals
and treatment of the diabetic animals with insulin
returned the in vivo and in vitro metabolic rate to near
control levels. Again, this inhibition of metabolism in
the alloxan-diabetic animal was stated to probably be due
to a decreased hepatic microsomal protein and/or hepatic
NADPH content, which they showed to occur in the diabetic
rats.
In 1974, this same group (13) examined the metabolism
of phenacetin and acetaminophen by insulin-requiring dia
betic men and women and nondiabetic men and women. Aceta
minophen was used in this study to see if a diabetic state
in humans affects the conjugating enzyme system of the
liver. Acetaminophen, unlike phenacetin, already pos
sesses a center for conjugation and thus does not require
an initial biotransformation to form this center. The
diabetic group was deprived of insulin for 48 hours pre
ceding the administration of the drugs and during the 12
hours following the administration. With phenacetin, it


10
was shown that the diabetic group excreted six to ten times
the amount of nonmetabolized drug as did the nondiabetic
group. Also, the excretion of the principal metabolite,
acetaminophen, was from two to seven times greater in the
control than in the diabetic group. Treatment of the
diabetic group with insulin returned the excretion rate of
nonmetabolized phenacetin and acetaminophen nearly to con
trol values. It was also shown that there was a sub
stantial individual variation in the rate of excretion of
both nonmetabolized phenacetin and acetaminophen in the
diabetic group, which consisted of only five subjects, and
that this variation appeared to correlate with the serious
ness of the diabetic state as measured by blood glucose
level.
When acetaminophen was administered, twice as much
unchanged drug and 40% as much conjugated derivatives of
acetaminophen were found in the urine of the diabetic
group compared with that of the control group. From these
results, the investigators concluded that the inhibition
of metabolism of phenacetin and acetaminophen was due to
the diabetic state of the patients. They suggested that
the abnormality in these diabetic patients responsible for
this inhibition may be a reduction in hepatic NADPH.
The fundamental defects to which most of the wide
spread biochemical abnormalities seen in diabetes can be
traced are a reduced entry of glucose into various peri
pheral tissues and an increased release of glucose into


11
the circulation by the liver. The immediate result of
these defects is hyperglycemia. Several investigators
have studied the effect of a hyperglycemic state on drug
metabolism.
In 1951, Lamson et al. (14) showed that more than 25%
of dogs anesthetized with pentobarbital returned to sleep
when given an intravenous injection of glucose shortly
after awakening. This could not be correlated with blood
sugar leves and no explanation could be given why some dogs
did not respond at all. Other species were also investi
gated and the hamster, rabbit, pigeon, chicken, and guinea
pig showed this effect nearly 100% of the time whereas the
mouse, rat, goldfish, and tadpole did not show this effect.
They also found that certain metabolites of glucose, i.e. ,
lactate, pyruvate, levulose, and galactose, elicited a
similar response in guinea pigs. Moreover, they found
that lactate and pyruvate increased the rate of entrance
of barbital into the brain of guinea pigs whereas glucose
did not. These results indicated that glucose and some
of its metabolites potentiated the anesthetic action of
barbiturates in certain species and it was at least
partially due tc an increased penetration of the barbitu
rate into the brain.
In 1971, Strother et al. (15) studied the influence
of high sugar consumption by Swiss-Webster mice on the
duration of action of barbiturates and the in vitro meta-


12
holism of barbiturates, aniline, and p-nitroanisole.
They found that mice kept for two days on a diet of ground
chow ad libitum and a 35% glucose solution as their sole
source of fluid had an increased sleeping time after
administration of hexobarbital, pentobarbital, secobarbi
tal, amobarbital, or phenobarbital, compared to control
animals allowed ground chow ad libitum and water as their
sole source of fluid. The treated animals tended to com
pensate for the high level of glucose intake in that their
sleeping times returned to normal after three to four days
on this diet and then they apparently overcompensated
after five to eight days on this diet and actually slept
for a shorter length of time than did the control animals.
The mechanism of these changes in sleeping time is not
apparent because possible changes in the brain concen
trations of the barbiturates can not be ruled out, inas
much as they were not measured. The in vitro metabolism
of aniline was not affected under these conditions. None
of these effects could be correlated with blood glucose
level, as the treated animals never exhibited a hypergly
cemia and actually had a 20% lower mean blood sugar level
than did the control group.
There are a number of similar disturbances seen in
both starvation and diabetes, such as a negative nitrogen
balance and ketosis. Another apparent disturbance is an
alteration in hepatic drug metabolism. The effects of
starvation on drug metabolism, however, are also confusing


13
because of conflicting results reported by many researchers.
In 1970, Kato et at. were able to obtain results that
closely resembled those of Dixon et at. (16) and Kato and
Gillette (17). These results showed that starvation for
a 36- to 48-hour period increased the activity of the
hepatic drug-metabolizing enzymes in both sexes of mice and
female rats for nearly all substrates tested, including
aniline, whereas it decreased the activity of the system
in male rats toward hexobarbital and aminopyrine. These
results are similar to those seen in the alloxan-diabetic
state in that an inhibition in the drug-metabolizing sys
tem of male rats toward type I substrates occurs, whereas
either no inhibition or stimulation is seen with female
rats and male and female mice for types I and II
substrates.
Table I summarizes the work described so far in
animals in vitro.
From the previous discussion, it is apparent that
there is a high degree of uncertainty concerning the
effects of a diabetic state on the hepatic drug-metabo
lizing system and until recently, no investigation has
added any insight as to how diabetes may be affecting this
system.
The secretion of insulin from the pancreas is stimu
lated under conditions that raise intracellular concen
trations of cyclic 31,5'-adenosine monophosphate (c-AMP)


TABLE 1
SUMMARY OF LITERATURE
Year Investigator Species Sex Substrate
Hexobar- Chlorpro- Codeine Amino- Aniline
bital mazine pyrine
Alloxan Diabetes
1961
Dixon
e t
al.
(3)
Rat
M
Ia
i
1963
Dixon
. et
al.
(4)
Rat
M
1
i
A.
1965
Kato
e t
a l.
(6)
Rat
M
1
i
t
1965
Kato
et
a l.
(6)
Rat
F
N.E.
+
T
1965
Kato
et
a l.
(6)
Rat
Fb
1
i
t
1970
Kato
et
a l.
(9)
Rat
M
1
+
A
1970
Kato
et
a l.
(9)
Rat
+
i
+
Starvation
1970
Kato
et
al.
(9)
MiceC
M&F
N.E.
+
t
1970
Kato
et
a l.
(9)
Ratd
M
41
+
+
1970
Kato
et
a l.
(9)
Ratd
F
+
A
i
4-, decrease in rate of metabolism; t, increase in rate of metabolism; N.E., no effect.
V~)
These animals were castrated and treated with methyItesosterone.
Q
These animals were fasted for 24 hours.
These animals were fasted for 48 hours.


TABLE 1
continued
Year
Investigator
Species
Sex
Substrate
Zoxazol-
Mono-methy1-4-
Phena-
Phenyl-
amine
aminopyrine
cetin
butazone
All
oxan
Diabetes
1963
Gillette et at.
(5)
Rat
F
N.E.
N.E.
+
1965
Kato et at.
(6)
Rat
F
t
1965
Kato et at.
(6)
Rat
M
N.E.
1970
Kato et at.
(9)
Rat
M
1
1S72
Dajani et at.
(11)
Rabbit
M
4-
1973
Dajani et al.
(12)
Rat
M
1


16
in the pancreas (18). Diabetes is one of the conditions
f
that raise the intracellular level of c-AMP in the pan
creas (19,20) but because the islets of Langerhans are not
functional (in classical diabetes), no insulin release
occurs. Diabetes also has been shown to increase the
levels of c-AMP in other tissues, e.g. liver (21).
Weiner et al. (22) showed in 1972 that certain treat
ments that raised the endogenous levels of c-AMP, such
as the administration of glucagon and theophylline, were
inhibitory to the in vivo metabolism of hexobarbital
(as measured by sleeping time) and the in vitro metabolism
of both hexobarbital and p-chloro-N-methylaniline in male
Sprague-Dawley rats. Direct support for the involvement
of c-AMP in the metabolism of these substrates was pro
vided by the demonstration that the administration of
dibutyryl cyclic AMP increased hexobarbital sleeping time
and, in addition, inhibited the in vitro metabolism of
hexobarbital and p-chloro-N-methylaniline. The dibutyryl
derivative of c-AMP was used because it has been shown to
produce parallel effects caused by c-AMP at lower concen
trations, due to greater penetration through cellular
membranes and lower susceptibility to destruction by the
enzyme phosphodiesterase (23, 24). Weiner et al. concluded
that increasing endogenous c-AMP concentrations or admin
istering a c-AMP analogue is inhibitory to certain pathways
of drug metabolism and that this inhibition is mediated in
the liver.


17
Further studies by Weiner et at. (25, 26) into the
mechanism of inhibition of drug metabolism by cyclic
adenine nucleotides have shown that an inhibitor of the
in vitro metabolism of hexobarbital and of p-chloro-N-
methylanilina is released into the liver cytosol in
response to treatment with dibutyryl c-AMP. This inhi
bition was observed as early as ten minutes and as late as
24 hours after the administration of dibutyrl c-AMP. Pre
liminary characterization of this inhibitor has shown that
it has a molecular weight of less than 5,000 and that it
is probably not a protein.
Ross et at. (27) confirmed in 1973 the finding that
cyclic nucleotides are inhibitory to certain pathways of
the hepatic mixed-function oxidase system. They found
that male and female Holtzman rats pretreated with c-AMP
showed a decreased in vitro metabolism of aniline, whereas
in vitro ami.nopvrine demethylase activity was depressed in
the male but not in the female. Microsomal content of
hepatic cytochrome P-450 fell only in the male after pre
treatment with c-AMP. When dibutyryl cyclic AMP was used
at an equivalent dose, inhibition was seen in both the in
vitro metabolism of aniline and aminopyrine. Cytochrome
P-450 content was decreased in both sexes after pretreat
ment with dibutyryl cyclic AMP, Also, pretreatment with
dibutyryl cyclic AMP increased the spectral dissociation
constant (K ) of hexobarbital and cytochrome P-450 but had
s


18
no change in the K of aniline and cytochrome P-450.
They concluded that cyclic nucleotides exert a sex-
dependent inhibitory effect on the hepatic mixed-function
oxidase system and that this effect is at least partially
due to qualitative and quantitative changes in hepatic
cvtochrome P-450.


GOALS
The primary purpose of this investigation was to study
the effect of experimental diabetes on the microsomal drug-
metabolizing system of the liver and to elucidate a mecha
nism for this effect. Until now, the only work done in
studying the effect of diabetes on drug metabolism has
involved induction of the diabetic state with alloxan, a
chemical which destroys the insulin-producing beta-cells of
the islets of Langerhans in the pancreas. Alloxan, however,
has a number of unfavorable actions. First of all, it is
a highly toxic chemical and a mortality rate in Sprague-
Dawley rats as high as 40% has been observed with a normal
diabetogenic dose (28). Also, the nephrotoxicity of
alloxan is well established (29, 30). Finally, there is a
recovery of insulin production- in alloxan-diabetic animals
approximately three months after they receive the chemical
(31). A remission of the diabetes, consisting of an
increased body weight, decreased mean blood glucose level,
and a measurable rise in plasma insulin level as a response
to the administration of corticotropin, occurs concomi
tantly with this renewed production of insulin.
19


20
Because of these disadvantages, I have chosen three
other chemicals, streptozotocin, 6-aminonicotinamide, and
N-methylacetamide, to induce experimental diabetes.
Streptozotocin, an N-nitroso derivative of glucosamine (32)
produces diabetes by a mechanism similar to that of alloxan
(33, 34) but has some distinct advantages over alloxan.
The mortality rate in rats is as much as ten times lower
with streptozotocin than v/ith alloxan (28, 35). Also,
streptozotocin has a higher specificity as a beta-cytotoxic
agent in the rat than does alloxan (34, 36). Another
advantage is that it is much easier to achieve a graded
diabetogenic response with streptozotocin than with alloxan
(37). With alloxan, a graded diabetic response is nearly
impossible either the animal does not become diabetic or
it becomes severely diabetic and ketoacidotic. With strep
tozotocin, on the other hand, it is fairly easy to get a
graded diabetic response and the animal only becomes keto
acidotic at high doses. Finally, animals made diabetic by
streptozotocin do not recover from the diabetes (31).
The second agent used, 6-aminonicotinamide, an anti-
metabolite of NADP synthesis, produces diabetes by blocking
insulin release (38). It is hypothesized that NADPH, which
is formed in the pentose-phosphate shunt during the cata
bolism of glucose, is needed for the release of insulin,
and that 6-aminonicotinamide inhibits the synthesis of
NADPH by blocking the formation of its precursor, NADP.
The blocking of the in vitro release of insulin from an


21
individual islet by 6-aminonicotinamide can be reversed by
the addition of either NAD? or NADPK. The effects of 6-
aminonicotinamide are not irreversible and wear off with
time.
The third diabetogenic agent, N-methylacetamide, pro
duces diabetes by making the animal insulin-resistant (39).
Neither insulin synthesis nor release are affected, but
the insulin that is released is ineffective in lowering
the blood glucose level, even though the plasma insulin
level rises concomitantly with that of glucose. The
effects of this drug are irreversible and its toxicity is
such that a rat treated with it will usually be dead or
too moribund for further experimentation 43 hours after its
administration.
The administration of insulin to animals pretreated
with alloxan, streptozotocin, or 6-aminonicotinamide
reverses the diabetic effects caused by these agents but
has no effect on those in animals pretreated with N-methyl
acetamide .
To investigate the effect that the diabetes induced
by these three agents had on hepatic drug metabolism in
male Holtzman rats, both in vivo and in vitro experiments
were performed. The in vivo experiments involved measuring
the length of hypnosis after the administration of hexo-
barbital sodium in animals which were made diabetic. The
in vitro experiments were of several types. After the
diabetes had been induced with the three agents, the


22
kinetic constants for the metabolism of hexobarbital and
aniline were measured at several time periods. Also, the
metabolism of hexobarbital and aniline were measured one
week after the administration of streptozotocin to deter
mine if the effects seen on drug metabolism existed for
more than several days. To examine the effect of hyper
glycemia on drug metabolism, animals were infused intra
venously with glucose, after which the in vitro metabolism
of hexobarbital was measured. To rule out any direct
effect that the diabetogenic agents may have had, they
were added to in vitro incubation mixtures from control
animals in which hexobarbital and aniline hydroxylations
were measured. To rule out any direct effect that insulin
may have had, it was added to similar in vitro incubation
mixtures from control animals and those pretreated with
the diabetogenic agents. Hepatic microsomal protein and
cytochrome P-450 content were also measured. In addition,
the characteristics of binding of both hexobarbital and
aniline to hepatic cytochrome P-450 from the diabetic
animals were studied.
As was mentioned in the historical review, increasing
the endogenous levels of c-AMP or administering c-AMP de
rivatives to animals is inhibitory to certain hepatic drug
metabolic pathways. This increase in c-AMP content has
also been shown to be responsible for the release of an in
hibitor of drug metabolism into the liver cytosol. To
attempt to elucidate a mechanism for the effects seen


23
with experimental diabetes on drug metabolism, hepatic
c-AMP content under the different diabetic conditions
was determined and these were correlated with the appear
ance of an inhibitor of drug metabolism in the liver
cytosol.


MATERIALS AND METHODS
Materials -
6-Aminonicotinamide and N-methylacetamide were pur
chased from Aldrich Chemical Company, Inc., Milwaukee,
Wisconsin. The streptozotocin was a kind gift from Dr. W.
E. Duiin of The Upjohn Company, Kalamazoo, Michigan. The
structures of these three compounds are shown in Figure 1.
Alloxan was purchased from Eastman Kodak Company, Rochester,
New York.
Hexobarbital sodium was purchased from Winthrop
Laboratories, New York, New York and aniline hydrochloride
from Eastman Kodak Company, Rochester, New York. Both the
crystalline zinc insulin and the protamine zinc insulin
were purchased from Eli Lilly, Inc., Indianapolis, Indiana,
NADP, NADPH, glucose 6-phosphate, glucose 6-phosphate
dehydrogenase and Tris buffer were all purchased from
Sigma Chemical Company, St. Louis, Missouri.
The c-AMP radioimmunoassay kit was purchased from
Schwarz/Mann of Orangeburg, New York.
Ail other chemicals used were of reagent grade.
Animals
Adult male rats weighing 100-250 g were obtained
from Holtzman Laboratories, Madison, Wisconsin.
2 4
All animals


25
O
CH3-C-NH-CH3
N-!ETilYLACETMHDE
FIGURE 1
TRUCTURFS OF DIABETOGENIC AGENTS


26
were maintained on standard laboratory chow and water ad
libitum for a minimum of 48 hours in the Department of
Pharmacology and Therapeutics before experimentation. All
animals, except for those that were used in the glucose
infusion experiments were fasted overnight for 17 hours
preceding the experiment but were allowed water ad libitum.
Streptozotocin and 6-aminonicotinamide were dissolved
immediately before use, the former in a citrate buffer, pH
4.5 (26.5 ml of 0.1 M citric acid + 23 ml of 0.1 M sodium
citrate) and the latter in Krebs-Ringer bicarbonate solu
tion. These drugs were both administered intraperitoneally
Control animals used with streptozotocin-treated animals
were injected with the citrate buffer and those with 6-
aminonicotinamide were injected with the Krebs-Ringer
bicarbonate solution. N-Methylacetamide is a liquid and
was administered intragastrically. Control animals used
with N-methylacetamide animals were given normal saline
intragastrically. Alloxan was dissolved in normal saline
and injected subcutaneously at a dose of 115 mg/kg and
control animals used with alloxan-treated animals received
normal saline. Animals were considered diabetic only if
their blood glucose exceeded 200 mg/100 ml.
Enzyme Preparation
Animals were killed by a blow on the head followed by
decapitation. The livers were immediately removed, weighed
and homogenized in four volumes of ice-cold 0.1 M Tris
buffer, pH 7.5. The homogenate was centrifuged at 9000(7


27
for 15 minutes in a Serval.1 refrigerated centrifuge at
f
0 C. The supernatant fraction from this homogenate was
used for the enzyme assays. When microsomal protein deter
mination was necessary, part of this supernatant fraction
was centrifuged at 100,000# in aBeckman Model L ultracen
trifuge for one hour at 4 C. The microsomal pellet was
then resuspended in 0.15 M KC1 and recentrifuged at
100,000# for another hour. This microsomal pellet was
then resuspended and a protein determination was performed
(40) .
Isolated liver microsomes were also used in some
experiments. For these experiments, the liver was homog
enized in four volumes of ice-cold 0.25 M sucrose solution
containing 1 mM EDTA. The supernatant fraction obtained
from centrifugation at 9000# for 15 minutes at 0 C in a
Servall refrigerated centrifuge was then centrifuged at
100,000# in a Beckman Model L ultracentrifuge at 4 for one
hour. The microsomal pellet was then suspended in 0.15 M
KC1 and recentrifuged at 100,000# for one hour. This
microsomal pellet was then resuspended in 0.15 M KC1 con
taining 0.05 M Tris buffer, pH 7.5.
Incubation Conditions
The in vitro metabolism of hexobarbital was determined
by assay for unchanged hexobarbital by the method of Cooper
and Brodie (41). The incubation system used was as follows
2 ml of 9000# supernatant fraction equivalent to about 500
mg of liver, 1 micromole of hexobarbital, 30 micromoles of


28
nicotinamide, 25 micromoles of MgCl2, and 0.26 micromole of
NAD?. Tris buffer (0.1 M, pH 7.5) was added to bring the
volume to 4 ml in a 120-ml glass-stoppered bottle. The
incubation system used in microsomal experiments was the
same as above except that microsomes equivalent to 500 mg
of liver were used, and 1.8 micromoles of NADPH were sub
stituted for the NAD?. Also, the following were added:
25 micromoles of glucose 6-phosphate and 2 units of glucose
6-phosphate dehydrogenase. When either the 9000# fraction
or the microsomes were used as the enzyme source, the
mixtures were incubated with shaking at 37 C for 20 min
utes and at the end of the incubation period, all bottles
were packed in ice for five minutes to stop the reaction.
Then, in succession, 1.3 ml of phosphate buffer, pH 5.5
(96.5 ml of 0.2 N NaH2?04 + 3.5 ml of 0.2 N Na2HP04), 1.5
g NaCl, and 60 ml of heptane were added. The bottles were
then placed in a Burrei wrist-action shaker for 45 minutes,
after which the mixtures were centrifuged for 15 minutes
at room temperature in an International Centrifuge, Model
CS, at 40,000#. Then 40 ml of each organic solvent phase
was transferred to a 60-ml glass stoppered bottle containing
4 ml of phosphate buffer, pH 11 (8.5 ml 10 M NaOH + 200 ml
0.8 M Na2HOP4). This was then shaken for three minutes
after which 3 ml of the aqueous phase was used to determine
the spectrophotometric absorbance at 245 nm in a Gilford
spectrophotometer, Model 2400.


29
The in vitro metabolism of aniline was determined by
assay for p-aminophenol, the major metabolite formed in
rats, by the method of Brodie and Gillette (42) and modi
fied by Kato and Gillette (43). The incubation system
used was as follows: 2 ml of 9000^ supernatant fraction
equivalent to 500 mg of liver, 4 micromoles of aniline,
0.15 micromole of NADP, 15 micromoles of glucose 6-
phosphate, 15 micromoles MgSCh, and 0.5 ml of 0.3 M Tris
buffer, pH 7.5. Distilled water was used to bring the
final volume to 4 ml in a 120-ml glass-stoppered bottle.
The difference in conditions when microsomal experiments
were performed were the same as with the hexobarbital
experiments. In experiments with both 9000g supernatant
and microsomal fractions, the mixtures were incubated for
15 minutes with shaking at 37 C and then placed in an
ice-bath for five minutes. Then 2.5 g of NaCl and 50 ml
of ether were added, after which the bottles were shaken
in a Burrell wrist-action shaker for 15 minutes. Then 40
ml of each ether extract was removed and shaken with 3 ml
of a 0.1 M NaOH solution containing 1% phenol for three
minutes. This was allowed to stand for 30 minutes, after
which the ether was aspirated off and the absorbance of
the aqueous layer was determined at 620 nm in a Coleman
Junior spectrophotometer, Model 6A.


30
Petermination of Kinetic Constants
For these experiments, the 9000g supernatant fraction
was used as the enzyme source and the incubation conditions
were as previously described except that the concentrations
of hexobarbital and aniline were different. Hexobarbital
was added to the incubation mixtures so that the final con
centrations of hexobarbital were 0.5, 0.8, 1, 1.6, and 2 mM.
Aniline was added to the incubation mixtures so that the
final concentrations of aniline were 0.125, 0.167, 0.25,
0.5 and 1 mM. The kinetic constants were obtained by plot
ting the reciprocals of the concentrations vs. the recipro
cals of the initial velocities of the reaction.
Glucose Infusion
In these experiments, rats weighing between 200-250 g
were used and allowed laboratory chow throughout the exper
iment. Their right jugular veins were cannulated under light
ether anesthesia, after which the animals were allowed to
recover for 24-36 hours. They were then placed in plastic
restraining cages and were infused with 40% glucose solu
tion or normal saline solution for 24 hours with a Harvard
infusion/withdrawal pump at a rate of 0.074 ml/hour for a
total of 1.78 ml, equivalent to 710 mg of glucose.
Cytochrome P-450 Content
Cytochrome P-450 content was measured by the carbon
monoxide-induced difference spectrum of the reduced hemo-
protein according to the method of Omura and Sato (44),
Three ml of a microsomal suspension containing 2 mg/ml


31
protein were placed in quartz cuvettes and dithionite was
added to reduce the cytochrome P-450. Carbon monoxide was
then gently bubbled into the cuvette for one minute and the
change in absorbance at 450 nm minus that at 489 nm was
measured in an Aminco-Chance recording spectrophotometer
operated in the split-beam mode. Cytochrome P-450 content
was measured according to Beer's Law by the following
formula:
AA x 1000
c =
£ X d X (P)
where c is the concentration of cytochrome P-450
expressed as nanomoles per mg of protein
£ is the molar absorption coefficient
(91 mM 1 cm 1)
d is the optical path length (1 cm), and
(P) is the concentration of protein, expressed
as mg/mi
Drug Binding of Cytochrome P-450
The binding of drugs to cytochrome P-450 was measured
by the method of Remmer et at. (7). Three ml of a micro
somal suspension containing 2 mg of protein per ml were
placed in quartz cuvettes and either hexobarbitai or
aniline was added. Hexobarbitai was added in increments
which gave final concentrations of 0.125, 0.15, 0.187,
0.25, 0.375, and 0.75 mM. Aniline was added in increments
which gave final concentrations of 0.0625, 0.075, 0.094,


0.125, 0.188, and 0.375 roM. The change in absorbance after
each addition was measured and the reciprocal of this
change was plotted the reciprocal of the drug concen
tration; the apparent spectral dissociation constant (K )
was determined by extrapolation to the x-axis.
Determination of the Presence of an Inhibitor of Drug
Metabolism in Hepatic 100,000.? Supernatant Fraction
Microscmes were prepared as described previously
except that after the first one-hour centrifugation at
100,000^, the microsomal and supernatant fractions of
control and streptozotocin- and 6-aminonicotinamide-
pretreated rats were separated and various combinations
were prepared. The soluble fractions were added to the
microsomes immediately and were rehomogenized and then
kept on ice for 15 minutes. This homogenate was then
recentrifuged and the resultant microsomal pellet was
resuspended as previously described for the preparation
of the final microsomal suspension used for the enzyme
assays.
Miscellaneous
C-AMP levels were measured by the method of Steiner
et al. (45). The Schwarz/Mann c-AMP radioimmunoassay
kit was used for these determinations.
Blood samples for determination of blood glucose
were taken from the tail vein and were analyzed by the
o-toluidine reagent method (46).
Hexobarbital sleeping time was measured as the time


33
between the loss and recovery of the righting reflex after
intraperitoneal injection of 100 mg of hexobarbital sodium
per kg.
Statistics
The results are expressed as the mean standard
error of the mean (S.E.)- The p-values were calculated
by applying the Student t-test, which is a measure of the
probability that the differences observed are due to
chance (47).


RESULTS
Hyperglycemic Response to Diabetogenic Agents
Since the criterion chosen for a diabetic state was a
blood glucose concentration in excess of 200 mg/100 ml,
doses of the diabetogenic agents that would yield such a
blood glucose level had to be determined. The response to
streptozotocin at the dosages of 55 mg/kg and 65 mg/kg are
shown in Figure 2. The lower of the two doses was chosen
for further studies because there was little difference in
the hyperglycemic response and the higher dose was associ
ated with a greater toxicity (40% of those animals died
within 24 hours compared with no mortality with the lower
dose). Figure 3 shows the response to 6-aminonicotinamide
at dosages of 25 mg/kg and 35 mg/kg. No animals died
within 24 hours after each dose. The higher dose was
chosen hare because the lower dose did not produce a blood
glucose level that met the above criterion. Figure 4 shows
the response to N-methylacetamide at dosages of 6.75 ml/kg
and 10.0 ml/kg. The lower dose was chosen here because
the higher dose proved too toxic (60% died within 24 hours
vs. 0% at the lower dose).
34


FIGURE 2
RELATIONSHIP BETWEEN SERUM GLUCOSE AND DOSE OF STREPTOZOTCIN


Serum Glucose
mg/lOO ml
400
300
200
100
O 4 8 12 24
Hours
55mg/kg
LO


FIGURE 3
RELATIONSHIP BETWEEN SERUM CLUCOSE AND DOSE OF 6-AMINONICOTINAMIDE


rum Glucose
mg/l00 ml
400
300
200
100
0 4 8 12
Hours
A.
<3 5 mg/kg
LO
CO


FIGURE 4
RELATIONSHIP BETWEEN SERUM GLUCOSE AND DOSE OF N-METHYLACETAMIDE


Serum Glucose
rng/lOO ml
400
300
200
100
10.1ml/kg
6.2 5 rn l/kg
0
4
8 12
Hours
24


Hexobarbital Hypnosis
To ascertain if the diabetes induced by the diabeto
genic agents affected the in vivo sleeping time induced by
a hypnotic, 100 mg/kg of hexobarbital sodium was injected
intraperitoneally into rats. Table 2 shows the results
of these experiments; both streptozotocin and 6-aminonico-
tinamide increased the sleeping time, whereas N-methylace-
tamide had no effect.
Effect of Diabetogenic Agents on Kinetic Constants for
Hexobarbital and Aniline Hydroxylations
To determine if the diabetes induced by the three
diabetogenic agents affected the in vitro metabolism of
hexobarbital and aniline, the rates of hydroxylation of
these substrates were measured. Table 3 shows the kinetic
constants for both hexobarbital and aniline hydroxylation
in liver 9000^ supernatant fractions 24, 43, and 96 hours
after the administration of streptozotocin. The table
also shows the effect on these constants after the daily
subcutaneous injection of three units of protamine zinc
insulin for three days beginning 24 hours after the in
jection of streptozotocin. The results indicate that the
K values for both hexobarbital and aniline hydroxylations
were increased and the values of V decreased at all time
max
periods tested after the animal had been treated with
streptozotocin. The changes in these parameters for
aniline metabolism, although statistically significant,
were quite small. Treatment with insulin in vivo was able


TZiBLE 2
HEXOBARBITAL SLEEPING TIMES AFTER ADMINISTRATION OF DIABETOGENIC AGENTS
Pretreatment Duration of Hypnosis"1
(minutes)
Saline Control (n = 18)
V)
Streptozotocin (n = 6)
Q
Streptozotocin (n = 6)
6-Aminonicotinamide (n = 6)d
N-Methylacetamide (n = 7)^
39.4 1.1
68.7 1.3d
65.3 1.5d
59.2 3.0d
35.4 2.0e
a
Data are presented as means S.E.
kpretreated 24 hours before experiment.
Q
Pretreated 48 hours before experiment.
p < 0.01, compared with the appropriate control value.
0 ...
Not significantly different from control value.


TABLE 3
EFFECT OF STREPTOZOTOCIN ON KINETIC CONSTANTS
FOR HEXOBARBITAL AND ANILINE HYDROXYLATIONS
K
a
V
a
(iriH)
, IllCiA
(nanomoles/mg micros
;omal protein/min)
xobarbital
Control (n = 22)
0.57

0.01
7.65

0.09
Streptozotocin
V)
(n 6)D
1.01

0.03f
4.54

0.12f
Streptozotocin
(n = 6)
0.99

0.03f
5.21

0.22f
Streptozotocin
(n 6) d
0.97

0.03f
5.07

0.09f
Streptozotocin
+ Insulin
(n = 8 ) C
0.59

Cn
CN
o

o
7.63
Hr
0.15g
.iline
Control (n = 21)
0.090

0.001
0.79
+
0.01
Streptozotocin
(n = 6)b
0.105

0.004f
0.67

0.02f
Streptozotocin
(n = 6) C
0.09 9

0.003f
0.67

0.02f
Streptozotocin
(n = 6)d
0.101
J.
0.003f
0.71

0.03f
Streptozotocin
+ Insulin
(n = 6)e
0.088

0.002g
0.79

0.02g
a
Data are presented as means S.E.
Pretreated 24 hours before experiment.
Q
Pretreated 48 hours before experiment.
dPretreated 96 hours before experiment.
0 ,
Three units of protamine zinc insulin injected subcutaneously daily for 3 days
beginning 24 hours after streptozotocin was administered.
^p < 0.01, compared with the appropriate control value.
gNot significantly different from control value.


44
to reverse these effects and return the K and V for
m max
both hexobarbital and aniline hydroxylation to control
values.
Table 4 shows the kinetic constants for hexobarbital
and aniline hydroxylations 24 and 48 hours after the ad
ministration of 6-aminonicotinamide. The K values for
m
hexobarbital hydroxylation were increased and those of
V were decreased at both 24 and 48 hours. The K of
max m
aniline hydroxylation was increased 24 hours after admin
istration of the diabetogen, and the V was decreased.
These changes, although statistically significant, were
quite small. At 48 hours, neither of these parameters
was significantly different from those in preparations
from control animals.
Table 5 shows that 24 hours after the administration
of N-methylacetamide, there was no change in any of the
kinetic constants for either hexobarbital or aniline.
Effect of Streptozotocin Diabetes of One Week Duration on
Hexobarbital and Aniline Hydroxylations
To determine if the effects of streptozotocin diabe
tes on drug metabolism lasted for longer than three days,
hexobarbital and aniline hydroxylations were measured in
vitro one week after the administration of streptozotocin
Table 6 shows that the rates of both hexobarbital and ani
line metabolism were still below control values one week
after administration of streptozotocin.


TABLE 4
EFFECT OF 6-AMINONICOTINAMIDE ON KINETIC CONSTANTS
FOR HEXOBARBITAL AND ANILINE HYDROXYLATIONS
Hexobarbital
K
m
(mM)
V
max
(nanomoles/mg microsomal protein/min)
Control (n = 22)
0.57

0.01
7.65

0.09
6 Am inonicot. inamide
(n =
6)b
0.96
T
O
o
u>
4.33

0.0 id
6-Aminonicotinamide
(n =
6)
0.6 8
+
0.02d
6.15

0.12d
Aniline
Control (n = 21)
0.090

0.001
0.79
+
0.01
6-Aminonicotinamide
(n =
6)b
0.104

0.004d
0.70
+
d
0.01
6-Aminonicotinamide
(n =
6)C
0.089

0.003e
0.80

0.02e
dData are presented as means S.E.
bPretreated 24 hours before experiment.
Pretreated 48 hours before experiment.
p < 0.01, compared with tne appropriate control value.
0 ...
Not significantly different from control value.
Ln


TABLE 5
EFFECT OF N-METHYLACETAMIDE ON KINETIC CONSTANTS
FOR HEXOBARBITAL AND ANILINE HYDROXYLATIONS
K a V
m max
a
Hexobarbital
Control (n = 22)
N-Methylacetamide (n = 6)^*
(mM)
(nanomoles/mg microsomal protein/min)
0.57 0.01
7.65 0.09
0.59 0.02
7.47 0.10
Aniline
Control (n = 21)
N-Methylacetamide (n = 6)^
0.090 0.001
0.089 0.003
0.79 0.01
0.75 0.02
Data are presented as means S.E.
bPretreated 24 hours before experiment.
Q
Not significantly different from control value.


TABLE 6
HEXOBARBITAL
WEEK AFTER THE
AND ANILINE HYDROXYLATIONS ONE
ADMINISTRATION OF STREPTOZOTOCIN
Hexobarbitaj
micromoles metabolized/g livera
Control
(n = 5)
1.36 0.05
Treated
(n = 5)
0.90 0.05b
Aniline
micromoles p-aminophenol formed/g livera
Control
(n = 5)
0.40 0.02
Treated
(n = 5)
0.31 0.02b
aData are presented as means S.E.
bp < 0.01, compared with appropriate control value.


48
Effect of Alloxan Diabetes on Hexobarbital and Aniline
Hydroxylations
As was noted in the historical review, a number of
researchers had reported that the rates of metabolism of a
large number of drugs, including hexobarbital, were
decreased in alloxan-diabetic animals but those of a few
drugs, such as aniline, were enhanced. These results were
reexamined, as shown in Table 7. The metabolism of hexo
barbital in vitro was inhibited while that of aniline was
enhanced.
Effect of Diabetogenic Agents on Hexobarbital and Aniline
Hydroxyltiions Using Isolated Microsomes as Enzyme Source
To be sure that the inhibition observed in drug meta
bolism in animal preparations from rats pretreated with
streptozotocin and 6-aminonicotinamide was due to changes
in the microsomal enzyme system rather than to an altera
tion in the NADPH-generating system, isolated liver micro
somes were used as the enzyme source with an added NADPH-
generating system, to measure the metabolism of hexobarbi
tal and aniline after animals had been pretreated with
these diabetogens. N-methylacetamide was excluded in these
studies because no inhibition in drug metabolism was seen
when animals were pretreated with this agent. Tables 8
and 9 show that an inhibition in hexobarbital and aniline
metabolism occurred when preparations of liver microsomes
from animals that had been treated 24 hours in advance with
streptozotocin and 6-aminonicotinamide were used as the
enzyme source for the assay.


TABLE 7
EFFECT OF ALLOXAN ON HEXOBARBITAL AND ANILINE KYDROXYLATIONS
Hexobarbital micromoles metabolized/g liver3
Control (n =4) 1.41 0.05
Treated (n = 4)b 0.83 0.04c
Aniline micromoles p-aminophenol formed/g liver
Control (n = 4) 0.3810.02
Treated (n = 4)b 0.49 0.02i::i
cl
""Data are presented as means S.E.
bPretreated 24 hours before experiment,
p < 0.01, compared with the appropriate control value,
^p < 0.05, compared with the appropriate control value.


TABLE 8
EFFECT OF STREPTOZOTOCIN ON HEXOBARBITAL AND ANILINE
HYDROXYLATIONS WITH ISOLATED MICROSOMES AS ENZYME SOURCE
Plexobarbital
Control (n = 8)
Treated (n = 4)^
micromoles metabolized/g liver
1.11 0.3
0.59 0.06C
Aniline
Control (n = 8)
Treated (n = 4)^
micromoles p-aminophenol formed/g livera
0.26 0.01
0.20 0.01
Data are presented as means S.E.
^Pretreated 24 hours before experiment,
c
p < 0.01, compared with the appropriate control value.


TABLE 9
EFFECT OF 6-AMINONICOTINAMIDE ON HEXOBARBITAL AND ANILINE
HYDROXYLATIONS WITH ISOLATED MICROSOMES AS ENZYME SOURCE
Hexobarbital
Control (n = 8)
Treated (n = 4)
micromoles metabolized/g livera
1.11 0.03
0.81 0.01C
Aniline micromoles p-aminophenol formed/g livera
Control
(n =
8)
0.26
0.01
Treated
(n =
4) b
0.20
0.02d
a
Data are presented as means S.E.
kpretreated 24 hours before experiment.
Q
p < 0.01, compared with the appropriate control value,
p < 0.05, compared with the appropriate control value.


52
Effect of Glucose Infusion on Hexobarbital Hydroxylation
To test if hyperglycemia alone could be responsible
for the inhibition seen in drug metabolism, rats were
infused intravenously for 24 hours with glucose; each ani
mal received a total of 710 mg of glucose. All such ani
mals had blood glucose concentrations that exceeded 200 mg/
100 ml by the end of the infusion. Control animals were
similarly infused with normal saline solution. The data
in Table 10 demonstrate that hyperglycemia produced by
glucose infusion had no effect on the in vitro metabolism
of hexobarbital.
Effect of Diabetogenic Agents Added in Vitro on Hexobarbi-
tal and Aniline Hydroxylations
To rule out any direct effect that the diabetogenic
agents may have had on the in vitro metabolism of hexobar
bital and aniline, they were added at concentrations of
10 2, 10 3, and 10 4 M to incubation mixtures containing
preparations of livers from untreated animals. Tables 11
and 12 show that the in vitro addition of these agents had
no effect on either hexobarbital or aniline hydroxylation.
Effect of Insulin Added in Vitro on Hexobarbital and Aniline
Hydroxylations Following Treatment with Diabetogenic Agents
To determine whether addition of insulin in vitro
would reverse the effects of pretreatment with the diabeto
genic agents, crystalline zinc insulin ranging in amount
from 0.C01 to 1 unit was added to 4-ml incubation mixtures
containing preparations of livers from animals treated


TABLE 10
EFFECT OF GLUCOSE INFUSION ON HEXOBARBITAL METABOLISM
Micromoles metabolized/g
liver
a
Control (n = 5)
Treated (n = 5)
1.51 0.05
1.43 0.08
b
aData are presented as means
^Not significantly different
S.E.
from control value.


TABLE 11
EFFECT OF DIABETOGENIC AGENTS ADDED IN VITRO ON HEXOBARBITAL METABOLISM
Treatment micromoles metabolized/g livera
Control (n = 15)
1.50
+
0.01
Streptozotocin
10~2 M (n = 5)
1.51

0.0 ib
10~3 M (n = 5)
1.52

0.0 3b
10_4 M (n = 5)
1.52

0.0 lb
6-Aminonicotinamide
102 M (n = 5)
1.51

0.02b
10~3 M (n = 5)
1.52

0.02b
10~4 M (n = 5)
1.50

b
0.02
N-Methylacetamide
10 2 M (n = 5)
1.44

0.03b
LO
II
c-1
CO
1
o
11
1.46

0.03b
10'4 M (n = 5)
1.43

0.0 2b
aData are presented as means S.E.
bNot significantly different from control value.


TABLE 12
EFFECT OF DIABETOGENIC AGENTS ADDED IN VITRO ON ANILINE METABOLISM
Treatment micromoles p-aminophenol formed/g liver
Control (n = 15)
0.43

0.01
Streptozotocin
ID
II
£
CM
1
O
11
0.39

0.02b
103 M (n = 5)
0.39

0.02b
10"" M (n = 5)
0.40

0.02b
6-Aminonicotinamide
10_2 M (n = 5)
0.44

0.03b
10~3 M (n = 5)
0.42

0.02b
10~4 M (n = 5)
0.42

0.02b
N-Methylacetamide
10~2 M (n = 5)
0.40

0.03b
10~3 M (n = 5)
0.41

0.02b
10-4 M (n = 5)
0.41

0.0 2b
aData are presented as means S.E.
Not significantly different from control value.


56
with streptozotocin and 6-aminonicotinamide. Livers from
N-methylacetamide-treated animals were not used because
inhibition of neither hexobarbital nor aniline was seen
when animals were treated with this diabetogenic agent.
Table 13 shows that insulin had no direct effect on the in
vitro metabolism of hexobarbital or aniline after the ani
mal had been treated with streptozotocin or 6-aminonico-
tinamide.
Effect of Diabetogenic Agents on Hepatic Microsomal Protein
To see if the alterations in hepatic drug metabolism
may be due to changes in hepatic microsomal protein con
tent, the effect of the diabetogenic agents on hepatic
microsomal protein concentration was determined. Table 14
shows that protein concentration was not affected 24, 48,
or 96 hours after the administration of streptozotocin, 24
or 48 hours after the administration of 6-aminonicotinamide,
or 24 hours after N-methylacetamide. Also, the administra
tion of three units of protamine zinc insulin for three
days beginning 24 hours after the animals had been treated
with streptozotocin had no effect on hepatic microsomal
protein concentration.
Effect of Diabetogenic Agents on Cytochrome P-45Q in Liver
Microsomes
To examine the possibility that the changes seen in
drug metabolism may be due to alterations in cytochrome
P-450 content in liver microsomes, the effect of the
diabetogenic agents on the microsomal concentration of this


TABLE 13
EFFECT OF INSULIN IN VITRO ON HEXOBARBITAL
AND ANILINE METABOLISM FOLLOWING DIABETOGENIC AGENTS
Hexobarbital
micromoles metabolized/g
liver
a
Control
1 unit
0.1 unit
0.01
unit
0.001 unit
Insulin
Insulin
Insulin
Insulin
Streptozotocin^1
0.85

0.02
u
o

o
+i
CO
o
0.87 0.05C
0.85
0.0 3C
. b
6-Aminonicotmamide
1.00
+
0.05
0.99 0.05C
o
o

o
+1
m
cn
o
0.97
0.02C
0.99 0.05c
Aniline
micromoles p
-aminophenol formed/g
livera
Streptozotocin
0.33

0.03
0.32 0.03
0.33 0.03
0.33
0.02C
0.33 0.03
6-Aminonicotinamide13
0.30
-j-
0.01
0.29 0.01C
0.29 0.01C
0.30
0.01
u
i1
O
o
+1
o
CO
o
aData are presented as means S.E.
kpretreated 24 hours before experiment
'Not significantly different from control value.
U1
^1


TABLE 14
EFFECT OF DIABETOGENIC AGENTS ON HEPATIC MICROSOMAL PROTEIN CONCENTRATION
Pretreatment
mg/g
livera
Control (n = 42)
27.9
i1
o
+1
Streptozotocin (n = 12)^
27.6
0.3f
Q
Streptozotocin (n = 12)
27.6
f
0.2
Streptozotocin (n = 12)^
28.1
0.3f
Streptozotocin + Insulin (n = 14)e
27.7
0.2f
6-Aminonicotinamide (n = 12)u
27.7
0.3f
c
6-Aminonicotinamide (n = 12)
27.9
0.2 f
N-Methylacetamide (n = 12) J
27.7
0.2f
aData are presented as means S.E.
kpretreated 24 hours before experiment.
cPretreated 48 hours before experiment.
dPretreated 72 hours before experiment.
0 .
Three units of protamine zinc insulin injected subcutaneously
daily for 3 days beginning 24 hours after streptozotocin was
injected,
f ...
Not significantly different from control value.
U1
CO


59
cytochrome was determined. Table 15 shows that pretreat
ment 24 hours previously with neither streptozotocin,
6-aminonicotinamide; nor N-methylacetamide had any effect
on the concentration of cytochrome P-450 in the liver
microsomes.
Effect of Diabetogenic Agents on Cytochrome P-450 Binding
to Hexobarbital and Aniline
To ascertain if the changes observed may be due to
changes in the strength of binding of hexobarbital and
aniline to cytochrome P-450, the spectral binding constants
(K ) were determined in liver microsomes from control ani-
s
mals and those that had been treated with the diabetogenic
agents. Table 16 shows that the affinity of cytochrome
P-450 for hexobarbital was significantly lower in micro
somes from streptozotocin- and 6-aminonicotinamide-diabetic
animals than in controls, whereas the binding to hexobarbi
tal was not affected in microsomes from N-methylacetamide-
treated animals. In the case of aniline, a decrease in
affinity of cytochrome P-450 was seen only in preparations
from animals pretreated with 6-aminonicotinamide, and the
affinity of cytochrome P-450 for aniline was increased in
microsomes from animals treated with N-methylacetamide.
Effect of Hepatic Homogenate Fractions on Hexobarbital and
Aniline Hydroxylation
To determine if an inhibitor of hexobarbital or aniline
metabolism may have been present in the 100,000g supernatant
fraction of livers of rats pretreated with streptozotocin or
6-aminonicotinamide, various combinations of microsomes and


TABLE 15
EFFECT OF DIABETOGENIC AGENTS ON CYTOCHROME P-450 CONCENTRATION IN LIVER MICROSOMFS
Pretreatment
nanomoles cytochrome
3.
P-450/mg protein/ml
Control (n = 6)
0.43
0.02
Streptozotocin (n = 5)^
0.47
O
o
u>
o
6-Aminonicotinamide (n = 5)
0.42
o
o
o
N-Methylacetamide (n = 6)^
0.47

04
O
o
dData are presented as means S.E.
kpretreated 24 hours before experiment,
c ...
Not significantly different from control value.


TABLE 16
EFFECT OF DIABETOGENIC AGENTS ON CYTOCHROME P-450 BINDING TO HEXOBARBITAL AND ANILINE
Pretreatment
Control (n = 11)
Streptozotocin (n = 5)^
6-Aminonicotinamide (n = 5)d
N-Methylacetamide (n = 6)^
Kg (mM) Hexobarbitala
0.062 0.001
0.084 0.005
0.077 0.006C
0.065 0.002d
Ks (mM) Anilinea
0.73 0.02
0.73 0.02d
0.84 0.03C
0.63 0.04C
aData are presented as means S.E.
dPretreated 24 hours before experiment
0
p 0.01, compared with the appropriate control value.
dNot significantly different from control value.


62
supernatant fractions from control and treated animals
were mixed together as described in the Methods section.
Table 17 shows that in vitvo inhibition of hexobarbital
hydroxylation was seen when enzyme preparations containing
microsomes from animals pretreated with either streptozo-
toein or 6-aminonicotinamide were preincubated with either
their own or with control supernatant fraction. More
important, however, is that enzyme preparations containing
microsomes from control animals which had been incubated
with supernatant fractions from animals pretreated with
either of the diabetogenic agents also showed an inhibi
tion of hexobarbital metabolism.
Table 18 shows the same experiment except that aniline
was the substrate. The in vitro inhibition was again seen
when enzyme preparations containing microsomes of animals
pretreated with both diabetogenic agents were added with
either their own supernatant fraction or with control
supernatant fraction. Microsomes from control animals
which were combined with supernatant fraction of animals
pretreated with streptozotocin did show an inhibition of
aniline metabolism but when microsomes from control animals
were added to supernatant fraction of animals pretreated
with 6-aminonicotinamide, no inhibition occurred.


TABLE 17
LOCALIZATION OF INHIBITOR OF HEXOBARBITAL METABOLISM PRESENT IN 100,0000 SUPERNATANT
FRACTION OF RAT LIVER AFTER PRETREATMENT WITH STREPTOZOTOCIN AND 6-AMINONICOTINAMIDE
Microsomes
Supernatant
Number
of Rats
micromoles meta-
bolized/q liver
% of Control
Control
Control
10
1.00
+
0.02

Control
Streptozotocin^
Pretreated
5
0.86

0.02C
86
Streptozotocin
Pretreated
Streptozotocin
Pretreated
5
0.76

o.oic
76
Streptozotocin
Pretreated
Control
5
0.88

0.02C
88
Control
6-Aminonicotinamide^
Pretreated
5
0.91

0.02
91
6-Aminonicotinamide
Pretreated
6-Aminonicotinamide
Pretreated
5
0.86

0.02C
86
6-Aminonicotinamide
Pretreated
Control
5
0.90

O
o
H
O
90
Data are presented as means S.E.
'Pretreated 2 4 hours before experiment.
'p < 0.01, compared with value obtained in experiment in which both microsomal and
supernatant fractions were derived from control animals.


TABLE 18
LOCALIZATION OF INHIBITOR OF ANILINE METABOLISM PRESENT IN 100,000^ SUPERNATANT
FRACTION OF RAT LIVER AFTER PRETREATMENT WITH STREPTOZOTOCIN AND 6-AMINONICOTINAMIDE
Homogenate Fractions and Treatments
Number micromoles p-amino-
Microsomes Supernatant of Rats phenol formed/g liver % of Control
Control
Control
10
0.26
+
0.01

Control
St rep tozo toe i.nD
Pretreated
5
0.22
+
0.02C
85
Streptozotocin
Pretreated
Streptozotocin
Pretreated
5
0.20

0.02d
77
Streptozotocin
Pretreated
Control
5
0.22

O
o
11
o
85
Control
6-Aminonicotinamide^
Pretreated
5
0.23

0)
pH
o
o
88
6-Aminonicotinamide
Pretreated
6-Aminonicotinamide
Pretreated
5
0.20
+
0.02d
77
6-Aminonicotinamide
Pretreated
Control
5
0.21
+
o.oid
81
3.
Data are presented as means S.E.
kpretreated 24 hours before experiment.
c
p < 0.05, compared with value obtained in experiment in which both microsomal and
supernatant fractions were derived from control animals.
dp < 0.01, compared with value obtained in experiment in which both microsomal and
supernatant fractions were derived from control animals.
^Not significantly different from control experiment in which both microsomal and
supernatant fractions were derived from control animals.
CTl


65
Effect of Diabetogenic Agents on Hepatic c-AMP Concen
trations
C-AMP concentrations were measured in the liver
following treatment with the diabetogenic agents to
determine whether a correlation existed between the type
of diabetic state produced and the hepatic content of
c-AMP, since it had already been shown that elevation of
hepatic c-AMP is inhibitory to certain drug-metabolic
pathways. Table 19 shows that streptozotocin and, to a
lesser extent, 6-aminonicotinamide, elevated hepatic
c-AMP concentrations, whereas N-methylacetamide had no
effect as compared to control values.


TABLE 19
EFFECT OF DIABETOGENIC AGENTS ON HEPATIC C-AMP CONCENTRATIONS
Pretreatment
Control (n = 8)
Streptozotocin (n = 6)
6-/uninonicotinamide (n = 6)d
N-Methylacetamide (n = 6)
nanomoles/g livera
0.49 0.03
0.80 0.04C
0.65 0.03C
0.48 0.03d
aData are presented as means S.E.
kpretreated 24 hours before experiment
Cp < 0.01
^Not significantly different from control value.
Oh
Oh


DISCUSSION
It has been shown previously that alloxan diabetes was
inhibitory to certain pathways of drug metabolism in several
species (3 6, 9 12, 17). This study was undertaken to
investigate the effect of three newer diabetogenic agents
on drug metabolism. Streptozotocin and 6-aminonicotinamide
produce an insulin-deficient animal while the third agent,
N-methylacetamide does not produce an insulin-deficient
animal but instead produces a condition whereby insulin is
produced and released by the pancreas but is incapable of
lowering blood glucose. The results of the present experi
ments with these three diabetogenic agents indicate that,
with male Holtzman rats, it was not hyperglycemia that was
responsible for the inhibition of certain drug-metabolic
pathways in diabetes but rather that the inhibition was a
result of the lack of insulin.
Hexobarbital and aniline were chosen for these studies
because they have both been used extensively as model sub
strates in studies of the mixed-function oxidase system in
the liver, and because their metabolism in normal rats is
well understood. Also, as mentioned in the Historical
Review, they differ in their binding to cytochrome P-450
in that the difference spectra produced when they are added
67


to liver microsomes are qualitatively different.
Figure 5 shows the major metabolic pathway of hexobar-
bital. In the rat, the major metabolite is 3'-hydroxy-
hexobarbital; 3'-ketohexobarbital is a very minor metabo
lite (48). As described in the Methods section, the meta
bolism of hexobarbital was measured by the disappearance
of the substrate rather than by the appearance of a meta
bolite .
Figure 6 shows the metabolic pathway for aniline. In
rats, there is six times more p-aminophenol formed than o-
aminophenol (49). The metabolism of aniline was measured
by the appearance of the metabolite, p-aminophenol.
Hexobarbital sleeping time has often been used to meas
ure hepatic drug metabolism for several reasons. First,
the substance is apolar enough to be rapidly absorbed and
distributed in the body. Second, its hypnotic activity is
such that convenient doses can be injected intraperitoneally
to produce a rapid sleep of moderate duration so that an
alteration in the length of sleep can be measured with
acceptable precision. Lastly, and most important, hexobar
bital is rapidly metabolized in animals so that there is
little time for its accumulation in body fat. Thus, the
duration of its hypnotic activity is a reflection of meta
bolism rather than of tissue redistribution (50). Table 2
shows that rats pretreated with both streptozotocin and 6-
arainonicotinamide slept significantly longer than controls


3'-KETOHEXOBARBITAL
FIGURE 5
METABOLIC PATHWAY OF HEXOBARBITAL


ANILINE
-AMINOPHENOL
o-AMINOPHENOL
FIGURE 6
METABOLIC PATHWAY OF ANILINE


71
while those animals pretreated with N-methylacetamide did
not ^leep any longer than the controls.
To see if the in vivo results just described could be
correlated with the in vitro experiments, the metabolism
of hexobarbital and aniline were measured in in vitro
incubation mixtures using 9000# supernatant fractions from
liver homogenates as the enzyme source. The fact that the
L, values increased and the V__v decreased for both hexo-
barbital and aniline 24, 48, and 96 hours after the
streptozotocin and 24 hours after 6-aminonicotinamide
indicated that the in vitro metabolism of these substrates
was inhibited after pretreatment with these diabetogenic
agents. The effects of 6-aminonicotinamide upon insulin
release are reversed with time as the agent is eliminated
from the body; this was reflected by the less intense inhi
bition of hexobarbital metabolism and the lack of inhibition
of aniline metabolism 48 hours after the administration of
6-aminonicotinamide. The fact that the kinetic constants
for both hexobarbital and aniline were unchanged in animals
24 hours after they were treated with N-methylacetamide
again showed that this diabetogenic agent does not affect
the metabolism of either of these substrates. These results
and the fact that hyperglycemia produced by infusion of
glucose did not cause inhibition of in vitro hexobarbital
metabolism (Table 10), showed that the hyperglycemia of
diabetes was not responsible for the inhibition of drug
metabolism caused by streptozotocin and 6-aminonicotinamide.


72
Since the diabetogenic effect of streptozotocin was
irreversible but not lethal at the dosage used, it was
important to determine if the inhibitory effect of strepto-
zotocin-diabetes on hexobarbital and aniline hydroxylations
lasted for longer than three days. Table 6 showed that
the in vitro metabolism of hexobarbital and aniline were
still well below control values one week after the animals
had received streptozotocin.
As was mentioned in the Historical Review, previous
researchers had noted that the in vitro metabolism of
hexobarbital was inhibited in animals pretreated with
alloxan but that the in vitro metabolism of aniline was
enhanced (3, 4, 6, 9, 10). Since in the present studies
with streptozotocin, a diabetogenic agent similar in mecha
nism of action to alloxan, no enhancement of aniline meta
bolism was observed, it was important to reexamine the
results with alloxan reported previously. The results
shown in Table 7 are qualitatively similar to those
reported by other investigators, although the extent of
enhancement of aniline hydroxylation was not as great as
that previously reported. Nevertheless, it would appear
that the enhancing effect of alloxan on the rate of aniline
hydroxylation is not related to the diabetogenic activity
of this agent.


73
Since both hexobarbital and aniline are metabolized
by liver microsomes and not by any nonmicrosomal hepatic
cells, it was important to determine if the inhibition that
was seen with the in vitro incubation mixtures using hepatic
9000g supernatant fraction as the enzyme source could be
duplicated when isolated liver microsomes were used as
the enzyme source. This was necessary to show that the
inhibition was not due to an inability of the drugs to
reach the microsomes, or to a deficiency in the NADPH-
generating system. Tables 8 and 9 demonstrated that the
metabolism of both substrates after treatment with either
streptozotocin or 6-aminonicotinamide was inhibited in
isolated liver microsomes.
Addition of streptozotocin, 6-aminonicotinamide, and
N-methylacetamide to incubation mixtures were made to
determine the direct effect these agents may have had on
in vitro enzyme activity. These diabetogenic agents at
concentrations ranging from 10 2 to 10 4 M had no direct
effect on either hexobarbital or aniline hydroxylation.
The highest of the concentrations of the diabetogenic agents
used in these experiments was greater than any of the con
centrations that could have occurred in vivo, assuming
complete absorption.
The direct effect of crystalline zinc insulin on hexo
barbital and aniline nydroxylations was also examined.
Table 13 shows that insulin added to incubation mixtures
that contained preparations of livers from control animals


or those treated with streptozotocin or 6-aminonicotinamide
had no effect on the in vitro metabolism of either of these
substrates. The concentrations of insulin added in these
experiments both mimicked and exceeded the concentrations
of insulin that would occur in vivo.
The inhibitory action of streptozotocin and 6-aminonico
tinamide on both hexobarbital and aniline metabolism could
not be attributed to an alteration in hepatic microsomal
protein (Table 13) or to an alteration in hepatic micro
somal cytochrome P-450 (Table 14). Also, N-methylacetamide
did not alter hepatic microsomal protein or cytochrome P-450
content.
The binding of hepatic microsomal cytochrome P-450 with
a substrate has been assumed to be an initial step for the
oxidation of drugs, and with certain drugs there is a simi
larity between the dissociation constant of this binding
and the Michaelis constant for the metabolism of the same
drugs (7, 51). Table 15 showed that the binding of hexo
barbital, a type I substrate, to hepatic microsomal cyto
chrome P-450 from animals pretreated with streptozotocin
and 6-aminonicotinamide was inhibited while the binding of
the hexobarbital to cytochrome P-450 from N-metnylacetamide-
treated animals was not inhibited. With aniline, a type II
substrate, however, binding was only inhibited in microsoir.es
from 6-aminonicotinamide-treated animals and was not
inhibited in those from streptozotocin-treated animals.
With N-methylacetamide, the binding of aniline to hepatic


75
cytochrome P-450 was actually enhanced. These results
indicate that the inhibition of hexobarbital metabolism in
animals pretreated with streptozotocin and 6-aminonicotin-
amide may be partially due to an interference in binding
of hexobarbital to the hepatic microsomal cytochrome P-450
from these animals. This cannot be the case with aniline,
however, inasmuch as inhibition in binding was seen only
in animals that were pretreated with 6-aminonicotinamide
and not in those treated with streptozotocin, even though
animals pretreated with streptozotocin demonstrated an
inhibition in their in vitro metabolism of aniline. Also,
the enhancement in binding of aniline to cytochrome P-450
from animals pretreated with N-methylacetamide is not
explainable because no enhancement of the metabolism of
aniline had been noted in preparations from animals pre
treated with this compound. It is apparent that the
strength of binding of substrate to hepatic cytochrome P-450
is not always an accurate measure of the degree of the
metabolism of the substrate. Indeed, it has become
increasingly obvious in recent years that the earlier
expectations that the spectral binding constants of drugs
with cytochrome P-450 are closely related to the kinetic
constants for microsomal metabolism have not been borne
out by further investigations (52).


As was mentioned in the Historical Review section,
intracellular levels of c-AMP in the liver are known to
be elevated in classical diabetes (21). Also, treatments
that increase endogenous levels of c-AMP in vivo are
inhibitory to certain pathways of drug metabolism; this is
thought to be due to the release of an inhibitor of drug
metabolism by liver microsomes into the cytosol (22, 25).
The presence of an inhibitor of hexobarbital hydroxylation
was shown in the present work to exist in preparations
from animals pretreated with streptozotocin and 6-amino-
nicotinamide, and an inhibitor only of hexobarbital
hydroxylation in those of animals pretreated with 6-amino-
nicotinamide. When hepatic c-AMP concentrations were
determined in animals pretreated with the diabetogenic
agents, it was found that the levels in streptozotocin-
pretreated animals were nearly twice those of control ani
mals, while those in 6-aminonicotinamide-pretreated animals
were increased to a lesser extent. The levels of c-AMP in
liver preparations from N-methylacetamide-pretreated ani
mals were not significantly different from those of control
animals. These results correlate with the fact that prep
arations from streptozotocin-pretreated animals always
showed a greater inhibition of both hexobarbital and aniline
hydroxylations than did those from animals treated with
6-am.inonicotinamide. The fact that no increase in hepatic
c-AMP content was observed in preparations from N-methyl-
acetamide-pretreated animals, which never showed an


77
inhibition of drug metabolism, further substantiates the
finding that levels of hepatic c-AMP higher than normal
are inhibitory to certain drug-metabolic pathways.


SUMMARY
Until now, all the work done in studying the effects
of experimental diabetes on the microsomal drug-metabo
lizing system of the liver has involved the induction of
diabetes with alloxan, a toxic chemical that destroys the
beta-cells of the pancreas. The purpose of this investi
gation was to study the effect of experimental diabetes on
drug metabolism with newer, less toxic diabetogenic agents
and to elucidate a mechanism for these effects. Two of
the diabetogenic agents, streptozotocin and 6-aminonico-
tinamide, produce an insulin-deficient animal while the
third agent, N-methylacetamide, does not produce an insulin
deficient animal but instead causes a condition whereby
insulin is produced and released but is incapable of
lowering blood glucose.
To investigate the effect that the diabetes induced by
these agents had on hepatic drug metabolism, experiments on
male Holtzman rats were performed both in vivo and in vitro
In the in vivo experiments, the duration of hypnosis was
measured after the administration of hexobarbital sodium
to animals that had been made diabetic. The results showed
that there was an inhibition, as compared to control ani
mals, of the in vivo metabolism of hexobarbital only in
animals pretreated with streptozotocin and 6-aminonico-
78


79
tinamide and not in those pretreated with N-methylacetamide.
The in vitro experiments were of several types. After the
animals had been treated with these three agents, the
kinetic constants for hexobarbital and aniline hydroxyl-
ations were determined with 9000g supernatant fraction as
the enzyme source. Again, only preparations from animals
pretreated with streptozotocin and 6-aminonicotinamide
showed an inhibition as compared to control animals. Simi
lar results were also obtained when isolated microsomes
were used as the enzyme source. Also, the in vitro meta
bolism of hexobarbital and aniline was still inhibited as
compared to preparations from control animals one week after
the animals had been treated with streptozotocin. To
examine the effect of hyperglycemia on hepatic drug meta
bolism, animals were infused intravenously with glucose,
after which the in vitro hydroxylation of hexobarbital was
measured and found not to be inhibited as compared to
saline-infused control animals.
Neither insulin nor any of the diabetogenic agents had
any direct effect on drug metabolism in vitro. Furthermore,
hepatic microsomal protein and cytochrome P-450 contents
were not significantly different in any of the diabetic
animals than those of the control animals.
The binding of both hexobarbital and aniline to cyto
chrome P-450 was inhibited in animals pretreated with
streptozotocin but only the binding of hexobarbital was
inhibited in 6-aminonicotinamide-pretreated animals. The


80
fact that the affinity of aniline to cytochrome P-450
was increased in microsomes from animals pretreated with
N-raetnylacetamide somewhat obscures the interpretation of
these results.
It had been shown previously that treatments that
increased the endogenous levels of c-AMP in the liver
were inhibitory to certain drug-metabolic pathways and
that this was due to the release of an inhibitor of drug
metabolism into the liver cytosol. The presence of an
inhibitor of hexobarbital and aniline hydroxylations
was shown to occur in animals pretreated with streptozoto-
cin and an inhibitor only of hexobarbital hydroxylation in
animals pretreated with 6-aminonicotinamide. This cor
related with elevated levels of c-AMP observed in livers
of animals pretreated with streptozotocin and to a lesser
degree in animals pretreated with 6-aminonicotinamide. C-
AMP levels were not elevated in animals pretreated with
N-methylacetamide as compared to control animals.
In conclusion, it appears that the inhibition of drug
metabolism in animals made diabetic with certain drugs is
not caused by hyperglycemia, but is rather the consequence
of insulin lack. It is proposed that this lack of insulin
causes an elevation of hepatic c-AMP which, in turn, causes
the release by liver microsomes of an inhibitor of the meta
bolism of xenobiotics.


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84
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BIOGRAPHICAL SKETCH
Dennis M. Ackerman was born in Baltimore, Maryland,
March 30, 1946. He attended Baltimore City College High
School and was graduated from there in June, 1963. In
February, 1969, he received the Bachelor of Science Degree
in Business Administration from the University of Maryland,-
and in June, 1970, he received the Bachelor of Science De
gree in Pharmacy from the same university.
Mr. Ackerman began his graduate education at the
University of Florida in September, 1970. He was sup
ported in his studies by a training grant from the Na
tional Institutes of Health.
85


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
Owen M. Rennert
Professor of Pediatrics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
yi.
David M. Travis
Professor of Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
P V)o
Betty P. vogh
Associate Professor of Pharmacology


This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council,
and was accepted as partial fulfillment of the require
ments for the degree of Doctor of Philosophy.
December, 1975
Dean, Graduate School


Full Text
UNIVERSITY OF FLORIDA
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EFFECT OF EXPERIMENTAL DIABETES ON DRUG METABOLISM
Xii-C.
By
DENNIS M. ACKERMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL CF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1975

ACKNOWLEDGMENTS
The author wishes foremost to thank his supervisory
committee chairman, Dr. Kenneth C. Leibman, for his sug¬
gestions, assistance, and most of all his patience
throughout the course of this study. Recognition is also
extended to the other members of his committee, Dr. Owen
M. Rennert, Dr. David M. Travis, and Dr. Betty p. Vogh.
Recognition is also extended to Dr. Ira Weinstein for his
assistance.
The author also wishes to acknowledge Mr. George
Wynns for his assistance in surgical procedures and is
grateful for the aid cf Dr. Robert J. Cohen in assisting
with cyclic AMP assays.
The author would like to acknowledge and thank
Kathleen Ackerman tor her encouragement and support pro¬
vided during a large part of his tenure as a graduate
student. Sincere appreciation is also extended to the
author’s parents for their everlasting support.
A special thanks is extended to Mrs. Elsa Couto for
her assistance, companionship, and warmth.
The author would also like to express his appreci¬
ation for the friendship and aid of Mrs. Theresa Fulford
as well as thanking her for typing this manuscript.
ii

TABLE OF CONTENTS
Page
Acknowledgments
List of Tables.......
List of Figures.
Abstract
Historical Review....
Goals
Materia1s and Methods
Results
Discussion...........
Summary
References.
Biographical Sketch..
. ii
. iv
. vi
vii
-1
.19
.24
.34
.67
.78
.81
x i x

LIST OF TABLES
Table Page
1. Summary of Literature , . 14
2. Hexobarbital Sleeping Times after Adminis¬
tration of Diabetogenic Agents - 42
3. Effect of Streptozotocin on Kinetic Constants
for Hexobarbital and Aniline Hydroxylations....43
4. Effect of 6-Am.inonicotinamide on Kinetic Con¬
stants for Hexobarbital and Aniline... 45
Hydroxylations
5. Effect of N-Methylacetamide on Kinetic Con¬
stants for Hexobarbital and Aniline
Hydroxylations 46
6. Hexobarbital and Aniline Hydroxylations One
Week after the Administration of
Streptozotocin 47
7. Effect of Alloxan on Hexobarbital and Aniline
Hydroxylations 49
S. Effect cf Streptozotocin on Hexobarbital and
Aniline Hydroxylations with Isolated
Microsomes as Enzyme Source 50
9.Effect of 6-Aminonicotinamida on Hexobarbital
and Aniline Hydroxylations with Isolated
Microsomes as Enzyme Source 51
10. Effect: of Glucose Infusion on Hexobarbital
Metabolism. .53
11. Effect of Diabetogenic Agents Added In Vitro
on Hexobarbital Metabolism 54
12. Effect of Diabetogenic Agents Added In Vitro
on Aniline Metabolism 55
iv

Table Page
13. Effect of Insulin In Vitro on Hexobarbital and
Aniline Metabolism Following Diabetogenic
Agents . 57
14. Effect of Diabetogenic Agents on Hepatic Micro¬
somal Protein Concentration 58
15. Effect of Diabetogenic Agents on Cytochrome
P-450 Concentration in Liver Microsornes. ...... .60
16. Effect of Diabetogenic Agents on Cytochrome
P-450 Binding to Hexobarbital and
Aniline 6.1
17. Localization of Inhibitor of Hexobarbital
Metabolism Present in 100,000^ Super¬
natant Fraction of Rat Liver after
Pretreatment with Streptozotocin and
6-Amincnicot inamide 63
18. Localization of Inhibitor of Aniline Meta¬
bolism Present in 100,000a Supernatant
Fraction of Rat Liver after Pretreatment
with Streptozotocin and 6-Aminonicotin-
amide 6 4
19. Effect of Diabetogenic Agents on Hepatic c-AMP
Concentrations
66

LIST Oí1 FIGURES
Figure Page
1. Structures of Diabetogenic Agents 2 5
2. Relationship between Serum Glucose and Dose
of Streptozotccin 36
3. Relationship between Serum Glucose and Dose of
6-Aminonicotinamide 38
4. Relationship between Serum Glucose and Dose
of N-Methylacetamide .40
5. Metabolic Pathway of Hexobarbital 69
6. Metabolic Pathway of Aniline 70
vi

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF EXPERIMENTAL DIABETES ON DRUG METABOLISM
By
Dennis M. Ackerman
December, 1975
Chairman: Dr. Kenneth C. Leibman
Department: Pharmacology and Therapeutics
The purpose of this investigation was to examine the
effect of experimental diabetes on the hepatic microsomal
drug-metabolizing system of the liver in male Holtzman rats
and to elucidate a mechanism for these effects. Three dia¬
betogenic agents were used: streptozotocin, 6-aminonicotin-
amide, and N-methylacetamide. Streptozotocin and 6-araino-
nicotinamide produce an insulin-deficient animal whereas
N-methylacetamide does not affect insulin production or
release but causes a condition in which insulin is incap¬
able of lowering blood glucose.
Both in vivo and in vitro experiments were performed
to investigate the effects of the diabetic state induced
by these three agents on hepatic drug metabolism. The
results of the in vivo experiments correlated with the
results of those in vitro in that inhibition of certain
pathways of drug metabolism was seen in animals pretreated
with streptozotocin and 6-aminonicotinamide but not in ani¬
mals pretreated with N-methy lacetamide even though cili
vii
A

treated animals were hyperglycemic.
The rate of hydroxylation of hexobarbital was not
significantly different in preparations from rats that had
been infused with glucose and those from saline-infused
control animals. This demonstrates conclusively that the
inhibition caused by diabetogenic agents was not due to
hyperglycemia.
Neither insulin nor any of the diabetogenic agents had
any direct effect on drug metabolism in vitro. Further¬
more, hepatic microsomal protein and cytochrome P-450
contents were not significantly different in any of the
diabetic animals than those of the control animals.
The binding of both hexobarbital and aniline to he¬
patic cytochrome P-450 was inhibited in animals pretreated
with streptczotocin but only that of hexobarbital was in¬
hibited in 6-aminonicotinamide-pretreated animals. The
fact that the affinity of aniline to cytochrome P-450 was
increased in microsomes from animals pretreated with N-
methylacetamide somewhat obscures the interpretation of
these results.
It had been shown previously that treatments that in¬
creased the endogenous levels of cyclic 35'-adenosine
monophosphate (c-AMP) in the liver were inhibitory to cer¬
tain drug metabolic pathways and that this was due to the
release of an inhibitor of drug metabolism into the liver
cytosol. The presence of an inhibitor of hexobarbital and
viii

aniline hydroxylation was shown to occur in the liver
cytosol of animals pretreated with streptozotocin and an
inhibitor only of hexobarbital hydroxylation in animals
pretreated with b-aminonieotinamide. This correlated with
elevated levels of hepatic c-AMP observed in animals pre¬
treated with streptozotocin and to a lesser degree in
animals pretreated with 6-aminonicotinamide. The levels
of c-AMP were not elevated in animals pretreated with N-
methylacetamide, as compared to control animals.
In conclusion, it appears that the inhibition of drug
metabolism in animals made diabetic with certain drugs is
not caused by hyperglycemia, but is rather the consequence
of insulin lack. It is proposed that this lack of insulin
causes an elevation of hepatic c-AMP which, in turn, causes
the release by liver microsomes of an inhibitor of the
metabolism of xenobiotics.
ix

HISTORICAL REVIEW
Many of the metabolic alterations due to diabetes are
well understood but the effect of the disease on hepatic
drug metabolism is not clear, even though this has been
studied for over 15 years. No definitive conclusions can
be drawn from the data accumulated because of the consid¬
erable amount of conflicting results that have appeared
in the literature.
In 1958 (1), the half-life of tolbutamide was deter¬
mined in both diabetic and nondiabetic humans of both
sexes. The half-life was found to be similar in both
groups (7.8 hours in the diabetic group and 8.0 hours in
the nondiabetic group). Prolonged use (2.5 - 5 months)
of tolbutamide in the diabetic patients was shown to
decrease the half-life of the drug in these patients
about 17% (7,3 hours to 6.5 hours). This decrease is
probably due to the fact that tolbutamide is an inducer
of the liver microsomal oxidase system that metabolizes
foreign compounds (2) and thus it stimulates its own
metabolism. Large deviations among diabetic and non¬
diabetic subjects as well as the small sample size pre¬
vented statistical analysis of these data.
1

2
It was shown in 1961 by Dixon et al. (3) that the
activities of drug-metabolizing enzymes of liver micro-
som.es were decreased by short-term alloxan diabetes (the
duration of the diabetic state was never longer than three
days). In this study, microsomal liver preparations of
male Holtzman rats that had been pretreated with alloxan
had a diminished ability to metabolize hexobarbital (side
chain oxidation), chlorpromazine (oxidation of ring sulfur),
and codeine (O-demethylation). In vivo, hexobarbital
sleeping time was prolonged after alloxan pretreatment,
which correlated well with the reduced microsomal enzyme
activity toward hexobarbital. Both the in vitro and in
vivo effects could be reversed by treating the diabetic
animal with insulin. Neither alloxan nor insulin had any
effect when added to the incubation mixtures in vitro.
The authors concluded that the diabetic state had an
inhibitory effect on the ability of these animals to
metabolize drugs.
A follow-up study (4) in 1963 was done to see the
effect of long-term (1-3 month duration) alloxan dia¬
betes on certain pathways of the hepatic microsomal
mixed-function oxidase drug-metabolizing system. In this
study, male rats of the Long-Evans strain were used as
well as the Holtzman strain because the former appeared
to be more resistant to the toxic effect of alloxan
and the following diabetic condition. A decreased rate

3
of metabolism in vitro and a prolonging effect on hexo-
barbital action in vivo was again seen. Also, a decreased
rate of in vitro metabolism of aminopyrine (which is
demathylated) was observed, whereas an increased rate of
in vitro metabolism of aniline (which is hydroxylated)
was seen. Insulin treatment returned the rate of hexo-
barbital metabolism back to control levels but had no
effect on aminopyrine metabolism. The rate of aniline
metabolism was decreased below the control rate after
insulin treatment. From this study, the authors concluded
that the diabetic state had varying effects on drug meta¬
bolism which were substrate-dependent.
In the same year, it was reported by Gillette et at.
(5) that the administration of alloxan to immature female
rats did not alter zoxazolamine hydroxvlation or mono-
raethy1-4-aminopyrine demthylation. On the other hand,
the O-dealkyiation of phenacetin was stimulated by the
alloxan-diabetic state.
In 1965, Kato and Gillette (6) again demonstrated
that alloxan diabetes of three days' duration decreased
the activity of the hepatic microsomal enzyme system of
male Sprague-Dawley rats toward aminopyrine and hexobar-
bital but did not change the acitivity of the system
toward zoxazolamine. An enhancement cf aniline hydrox-
ylation was again seen. The effect of the alloxan dia¬
betic state in female Sprague-Dawley rats, though, was
much different. In these animals, the metabolism of

4
aminopyrine, aniline, and zoxazolamine was increased by
the diabetic state, but the activity of the hexobarbital-
metabolizing enzyme system was not significantly affected.
When female rats we re castrated and given raethyltestos-
terone, the results were different from those observed
with intact female rats, but were similar to those seen
with male rats. Also, alloxan diabetes did not produce a
further reduction in hexobarbital hydroxylation or amino¬
pyrine demethylation in male rats that had been castrated.
Castration of male diabetic rats, however, did not pre¬
vent the increased activity of the microsomal system
toward aniline that was also seen in the non-castrated
male diabetic rats. These results showed that certain
hepatic microsomal pathways in diabetic rats, such as
that of aminopyrine, were sex-dependent; whereas other
pathways, such as that for aniline, were not.
A cytochrome termed "P-450,found in the hepatic
microsomal fraction, is considered co be the final oxygen-
activating enzyme in many NADPH-dependent drug-metabolic
pathways. It catalyzes the redaction of an oxygen mole¬
cule with placement of one oxygen atom into water and
simultaneous insertion of the other oxygen atom into a
foreign substrate. This cytochrome can be measured quan¬
titatively by the difference spectrum observed when it is
reduced (usually with d.ithionite) and combined with carbon
monoxide.

5
Drugs combine with cytochrome P-450 (7) to produce
difference spectra of two general types, known as types I
and II. Type I compounds, exemplified by hexobarbital and
aminopyrine, give difference spectra with ^max in the range
of 385 - 390 ran and A . in the range of 418 - 427 ran.
Type II compounds, exemplified by aniline, give ^max
between 425 - 435 ran and A . between 390-405 (8). These
mm
spectral changes resulting from substrate interaction with
cytochrome P-450 have been presumed to represent the pri¬
mary binding of substrate for enzyme action and a change
in the degree of binding may be indicative of a change in
the amount of substrate being metabolized. The strength
of binding can be determined spectrophotometrically by
plotting the reciprocals of the spectra change vs. those
of substrate concentration in a Lineweaver-Burk type of
plot to give by extrapolation the spectral dissociation
constant (K^).
In 1970, Kato et at. (9) studied the effect of alloxan
diabetes on Wistar rats and mice of the dd strain. In this
study, the effect of alloxan diabetes on the in vitro meta¬
bolism of several substrates and on the content of cyto¬
chrome P-450 and NADPH-cytochrome o reductase (the first
enzyme in the electron-transport chain involved in drug
metabolism in the liver) were examined. In male rats, a
decrease was again seen in the rates of both aminopyrine
N-demethylation and hexobarbital hydroxylation but in

female rats and male and female mice, an increase was
observed in the rates of metabolism of both of these sub¬
6
strates. An increase in the rate of aniline hydroxylation
was seen in both sexes of rats and mice.
The activity of NADPH-cytochrome e? reductase was not
altered by alloxan diabetes in either the male or female
rats but was increased by alloxan diabetes as compared to
controls in both sexes of mice. The cytochrome P-450 con¬
tent in liver microsomes from the diabetic animals was
slightly increased over that of controls in the female
rats but was not affected by the diabetes in male rats or
male or female mice. The authors concluded that the oxi¬
dative activities of the liver microsomes of the male rats
toward certain substrates such as hexobaroital and amino-
pyrine are enhanced by androgen and that this enhancement
is blocked by alloxan diabetes. In mice, this androgen-
dependent enhancement is not present. Aniline hydroxyl-
ation is presumed not to be androgen-dependent and there¬
fore alloxan diabetes does not increase its rate of
hydroxylation. Why the rate of its hydroxylation is
increased, however, was not explained.
The same authors (10) studied the effect of alloxan
diabetes on cytochrome P-450 content, substrate inter¬
action with hepatic cytochrome P-450, and kinetics of
several drug-metabolic pathways in male and female Wistar
rats. Alloxan diabetes did not alter the amount of cyto¬
chrome P-450 in the livers of male rats but slightly

/
increased the cytochrome P-450 content in the livers of
the female rats of this species. The affinity of the
cytochrome P-450 for hexobarbital and aminopyrine v/as
lower in male diabetic rats than it was in normal male
rats. Also, a decrease in the rates of both hexobarbital
and aminopyrine oxidation was observed. Aniline, however,
was hydroxylated at a greater rate in the diabetic male
rats than in controls although there was no significant
increase in the binding of the cytochrome P-450 to aniline
from the diabetic rats as compared to controls. An
increase in the hydroxy.lati on of zoxazolamine as well as
an increased affinity for cytochrome P-450 was also seen
in the diabetic male rats as compared to control animals.
In female diabetic rats, increases in the degree of binding
of hepatic microsomal cytochrome P-450 to both hexobarbital
and aniline, as compared to those of control rats were
seen.
Inasmuch as hexobarbital and aminopyrine, which are
both type I compounds, show both decreased binding to
cytochrome P-450 as compared to controls as well as
decreased rates of oxidation, whereas aniline, a type II
compound, shows no change in affinity to cytochrome P-450
compared to control but an increase over control animals
in the rate of its metabolism, it was concluded that the
effect of alloxan diabetes on hepatic drug-metabolic
enzymes may be related to the type of spectral change seen
when the drug binds to cytochrome P-450. The results seen

8
with zoxazolarnine, a type I compound, in the male rats
were net explainable. Also, the effect of alloxan diabetes
does not appear to be related to the type of spectral
change a drug gives with cytochrome P-450 in female rats.
In 1972, Dajani and Kayyali (11) studied the meta¬
bolism of phenacetin in male New Zealand White rabbits
which had been made diabetic by the administration of
alloxan. They found that the urinary and plasma concen¬
trations of the unchanged drug were higher in the diabetic
animals than in the controls, and that the concentrations
of the major metabolite, acetaminophen, were lower. This
was suggestive of a decreased metabolism of this drug in
the diabetic animal. A decreased rate of in vitro metabo¬
lism of phenacetin in liver microsomes from alloxan-
diabetic rabbits proved that the metabolism of phenacetin
was inhibited in these animals. Both the in vivo and in
vitro inhibition of phenacetin metabolism were reversible
by the administration of insulin to the animals. To
attempt to explain this inhibition of metabolism of phen¬
acetin in alloxan-diabetic rabbits, the contents of
hepatic microsomal protein and hepatic NADPH, a necessary
cofactor for the hepatic microsomal drug-metabolizing sys¬
tem, were measured, and both were found to be lower in the
diabetic animals than in control animals. They concluded,
therefore, that the inhibition that occurred in the
alloxan-diabetic state in the male rabbit was probably due

to a deficiency of microsomal drug-metabolizing protein
and/or a deficiency in hepatic NADPH.
In a follow-up study the following year (12), the
metabolic transformation of phenylbutazone (which is
hydroxylated) was examined in the alloxan-diabetic Wistar
rat. As in the previous study, the rates of both the in
vivo" and in vitro metabolism of the drug were decreased
in alloxan-diabetic animals as compared to control animals
and treatment of the diabetic animals with insulin
returned the in vivo and in vitro metabolic rate to near
control levels. Again, this inhibition of metabolism in
the alloxan-diabetic animal was stated to probably be due
to a decreased hepatic microsomal protein and/or hepatic
NADPH content, which they showed to occur in the diabetic
rats.
In 1974, this same group (13) examined the metabolism
of phenacetin and acetaminophen by insulin-requiring dia¬
betic men and women and nondiabetic men and women. Aceta¬
minophen was used in this study to see if a diabetic state
in humans affects the conjugating enzyme system of the
liver. Acetaminophen, unlike phenacetin, already pos¬
sesses a center for conjugation and thus does not require
an initial biotransformation to form this center. The
diabetic group was deprived of insulin for 48 hours pre¬
ceding the administration of the drugs and during the 12
hours following the administration. With phenacetin, it

10
was shown that the diabetic group excreted six to ten times
the amount of nonmetabolized drug as did the nondiabetic
group. Also, the excretion of the principal metabolite,
acetaminophen, was from two to seven times greater in the
control than in the diabetic group. Treatment of the
diabetic group with insulin returned the excretion rate of
nonmetabolized phenacetin and acetaminophen nearly to con¬
trol values. It was also shown that there was a sub¬
stantial individual variation in the rate of excretion of
both nonmetabolized phenacetin and acetaminophen in the
diabetic group, which consisted of only five subjects, and
that this variation appeared to correlate with the serious¬
ness of the diabetic state as measured by blood glucose
level.
When acetaminophen was administered, twice as much
unchanged drug and 40% as much conjugated derivatives of
acetaminophen were found in the urine of the diabetic
group compared with that of the control group. From these
results, the investigators concluded that the inhibition
of metabolism of phenacetin and acetaminophen was due to
the diabetic state of the patients. They suggested that
the abnormality in these diabetic patients responsible for
this inhibition may be a reduction in hepatic NADPH.
The fundamental defects to which most of the wide¬
spread biochemical abnormalities seen in diabetes can be
traced are a reduced entry of glucose into various peri¬
pheral tissues and an increased release of glucose into

11
the circulation by the liver. The immediate result of
these defects is hyperglycemia. Several investigators
have studied the effect of a hyperglycemic state on drug
metabolism.
In 1951, Lamson et al. (14) showed that more than 25%
of dogs anesthetized with pentobarbital returned to sleep
when given an intravenous injection of glucose shortly
after awakening. This could not be correlated with blood
sugar leves and no explanation could be given why some dogs
did not respond at all. Other species were also investi¬
gated and the hamster, rabbit, pigeon, chicken, and guinea
pig showed this effect nearly 100% of the time whereas the
mouse, rat, goldfish, and tadpole did not show this effect.
They also found that certain metabolites of glucose, i.e. ,
lactate, pyruvate, levulose, and galactose, elicited a
similar response in guinea pigs. Moreover, they found
that lactate and pyruvate increased the rate of entrance
of barbital into the brain of guinea pigs whereas glucose
did not. These results indicated that glucose and some
of its metabolites potentiated the anesthetic action of
barbiturates in certain species and it was at least
partially due tc an increased penetration of the barbitu¬
rate into the brain.
In 1971, Strother et al. (15) studied the influence
of high sugar consumption by Swiss-Webster mice on the
duration of action of barbiturates and the in vitro meta-

12
holism of barbiturates, aniline, and p-nitroanisole.
They found that mice kept for two days on a diet of ground
chow ad libitum and a 35% glucose solution as their sole
source of fluid had an increased sleeping time after
administration of hexobarbital, pentobarbital, secobarbi¬
tal, amobarbital, or phenobarbital, compared to control
animals allowed ground chow ad libitum and water as their
sole source of fluid. The treated animals tended to com¬
pensate for the high level of glucose intake in that their
sleeping times returned to normal after three to four days
on this diet and then they apparently overcompensated
after five to eight days on this diet and actually slept
for a shorter length of time than did the control animals.
The mechanism of these changes in sleeping time is not
apparent because possible changes in the brain concen¬
trations of the barbiturates can not be ruled out, inas¬
much as they were not measured. The in vitro metabolism
of aniline was not affected under these conditions. None
of these effects could be correlated with blood glucose
level, as the treated animals never exhibited a hypergly¬
cemia and actually had a 20% lower mean blood sugar level
than did the control group.
There are a number of similar disturbances seen in
both starvation and diabetes, such as a negative nitrogen
balance and ketosis. Another apparent disturbance is an
alteration in hepatic drug metabolism. The effects of
starvation on drug metabolism, however, are also confusing

13
because of conflicting results reported by many researchers.
In 1970, Kato et at. were able to obtain results that
closely resembled those of Dixon et at. (16) and Kato and
Gillette (17). These results showed that starvation for
a 36- to 48-hour period increased the activity of the
hepatic drug-metabolizing enzymes in both sexes of mice and
female rats for nearly all substrates tested, including
aniline, whereas it decreased the activity of the system
in male rats toward hexobarbital and aminopyrine. These
results are similar to those seen in the alloxan-diabetic
state in that an inhibition in the drug-metabolizing sys¬
tem of male rats toward type I substrates occurs, whereas
either no inhibition or stimulation is seen with female
rats and male and female mice for types I and II
substrates.
Table I summarizes the work described so far in
animals in vitro.
From the previous discussion, it is apparent that
there is a high degree of uncertainty concerning the
effects of a diabetic state on the hepatic drug-metabo¬
lizing system and until recently, no investigation has
added any insight as to how diabetes may be affecting this
system.
The secretion of insulin from the pancreas is stimu¬
lated under conditions that raise intracellular concen¬
trations of cyclic 31,5'-adenosine monophosphate (c-AMP)

TABLE 1
SUMMARY OF LITERATURE
Year Investigator Species Sex S ubs trate
Hexobar- Chlorpro- Codeine Amino- Aniline
bital mazine pyrine
Alloxan Diabetes
1961
Dixon
e t
al.
(3)
Rat
M
Ia
i
1963
Dixon
. et
â–  al.
(4)
Rat
M
i
A.
1965
Kato
e t
a l.
(6)
Rat
M
1
i
t
1965
Kato
et
a l.
(6)
Rat
F
N.E.
+
T
1965
Kato
et
a l.
(6)
Rat
Fb
1
i
+
1970
Kato
et
a l.
(9)
Rat
M
1
A
1970
Kato
e t
a l.
(9)
Rat
rr\
+
+
Starvation
1970
Kato
et
al.
(9)
MiceC
M&F
N.E.
+
t
1970
Kato
et
a l.
(9)
Ratd
M
+
+
1970
Kato
et
a l.
(9)
Ratd
F
+
A
i
4-, decrease in rate of metabolism; t, increase in rate of metabolism; N.E., no effect.
V~)
These animals were castrated and treated with methyItesosterone.
Q
These animals were fasted for 24 hours.
These animals were fasted for 48 hours.

TABLE 1
continued
Year
Investigator
Species
Sex
Substrate
Zoxazol-
Mono-methyl-4-
Phena-
Phenyl-
amine
aminopyrine
cetin
butazone
All
oxan
Diabetes
1963
Gillette et al.
(5)
Rat
F
N.E.
N.E.
t
1965
Kato st al.
(6)
Rat
F
t
1965
Kato et al.
(6)
Rat
M
N.E.
1970
Kato et al.
(9)
Rat
M
1
1S72
Dajani et al.
(11)
Rabbit
M
4-
1973
Dajani et al.
(12)
Rat
M
1

16
in the pancreas (18). Diabetes is one of the conditions
f
that raise the intracellular level of c-AMP in the pan¬
creas (19,20) but because the islets of Langerhans are not
functional (in classical diabetes), no insulin release
occurs. Diabetes also has been shown to increase the
levels of c-AMP in other tissues, e.g. liver (21).
Weiner et al. (22) showed in 1972 that certain treat¬
ments that raised the endogenous levels of c-AMP, such
as the administration of glucagon and theophylline, were
inhibitory to the in vivo metabolism of hexobarbital
(as measured by sleeping time) and the iyi vitro metabolism
of both hexobarbital and p-chloro-N-methylaniline in male
Sprague-Dawley rats. Direct support for the involvement
of c-AMP in the metabolism of these substrates was pro¬
vided by the demonstration that the administration of
dibutyryl cyclic AMP increased hexobarbital sleeping time
and, in addition, inhibited the in vitro metabolism of
hexobarbital and p-chloro-N-methylaniline. The dibutyryl
derivative of c-AMP was used because it has been shown to
produce parallel effects caused by c-AMP at lower concen¬
trations, due to greater penetration through cellular
membranes and lower susceptibility to destruction by the
enzyme phosphodiesterase (23, 24). Weiner et al. concluded
that increasing endogenous c-AMP concentrations or admin¬
istering a c-AMP analogue is inhibitory to certain pathways
of drug metabolism and that this inhibition is mediated in
the liver.

17
Further studies by Weiner ei at. (25, 26) into the
mechanism of inhibition of drug metabolism by cyclic
adenine nucleotides have shown that an inhibitor of the
in vitro metabolism of hexobarbital and of p-chloro-N-
methylanilina is released into the liver cytosol in
response to treatment with dibutyryl c-AMP. This inhi¬
bition was observed as early as ten minutes and as late as
24 hours after the administration of dibutyrl c-AMP. Pre¬
liminary characterization of this inhibitor has shown that
it has a molecular weight of less than 5,000 and that it
is probably not a protein.
Ross et at. (27) confirmed in 1973 the finding that
cyclic nucleotides are inhibitory to certain pathways of
the hepatic mixed-function oxidase system. They found
that male and female Holtzman rats pretreated with c-AMP
showed a decreased in vitro metabolism of aniline, whereas
in vitro ami.nopvrine demethylase activity was depressed in
the male but not in the female. Microsomal content of
hepatic cytochrome P-450 fell only in the male after pre¬
treatment with c-AMP. When dibutyryl cyclic AMP was used
at an equivalent dose, inhibition was seen in both the in
vitro metabolism of aniline and aminopyrine. Cytochrome
P-450 content was decreased in both sexes after pretreat¬
ment with dibutyryl cyclic AMP, Also, pretreatment with
dibutyryl cyclic AMP increased the spectral dissociation
constant (K ) of hexobarbital and cytochrome P-450 but had
s

18
no change in the K of aniline and cytochrome P-450.
They concluded that cyclic nucleotides exert a sex-
dependent inhibitory effect on the hepatic mixed-function
oxidase system and that this effect is at least partially
due to qualitative and quantitative changes in hepatic
cvtochrome P-450.

GOALS
The primary purpose of this investigation was to study
the effect of experimental diabetes on the microsomal drug-
metabolizing system of the liver and to elucidate a mecha¬
nism for this effect. Until now, the only work done in
studying the effect of diabetes on drug metabolism has
involved induction of the diabetic state with alloxan, a
chemical which destroys the insulin-producing beta-cells of
the islets of Langerhans in the pancreas. Alloxan, however,
has a number of unfavorable actions. First of all, it is
a highly toxic chemical and a mortality rate in Sprague-
Dawley rats as high as 40% has been observed with a normal
diabetogenic dose (28). Also, the nephrotoxicity of
alloxan is well established (29, 30). Finally, there is a
recovery of insulin production- in alloxan-diabetic animals
approximately three months after they receive the chemical
(31). A remission of the diabetes, consisting of an
increased body weight, decreased mean blood glucose level,
and a measurable rise in plasma insulin level as a response
to the administration of corticotropin, occurs concomi¬
tantly with this renewed production of insulin.
19

20
Because of these disadvantages, I have chosen three
other chemicals, streptozotocin, 6-aminonicotinamide, and
N-methylacetamide, to induce experimental diabetes.
Streptozotocin, an N-nitroso derivative of glucosamine (32)
produces diabetes by a mechanism similar to that of alloxan
(33, 34) but has some distinct advantages over alloxan.
The mortality rate in rats is as much as ten times lower
with streptozotocin than v/ith alloxan (28, 35). Also,
streptozotocin has a higher specificity as a beta-cytotoxic
agent in the rat than does alloxan (34, 36). Another
advantage is that it is much easier to achieve a graded
diabetogenic response with streptozotocin than with alloxan
(37). With alloxan, a graded diabetic response is nearly
impossible - either the animal does not become diabetic or
it becomes severely diabetic and ketoacidotic. With strep¬
tozotocin, on the other hand, it is fairly easy to get a
graded diabetic response and the animal only becomes keto¬
acidotic at high doses. Finally, animals made diabetic by
streptozotocin do not recover from the diabetes (31).
The second agent used, 6-aminonicotinamide, an anti¬
metabolite of NADP synthesis, produces diabetes by blocking
insulin release (38). It is hypothesized that NADPH, which
is formed in the pentose-phosphate shunt during the cata¬
bolism of glucose, is needed for the release of insulin,
and that 6-aminonicotinamide inhibits the synthesis of
NADPH by blocking the formation of its precursor, NADP.
The blocking of the in vitro release of insulin from an

21
individual islet by 6-aminonicotinamide can be reversed by
the addition of either NADP or NADPH. The effects of 6-
aminonicotinainide are not irreversible and wear off with
time.
The third diabetogenic agent, N-methylacetamide, pro¬
duces diabetes by making the animal insulin-resistant (39).
Neither insulin synthesis nor release are affected, but
the insulin that is released is ineffective in lowering
the blood glucose level, even though the plasma insulin
level rises concomitantly with that of glucose. The
effects of this drug are irreversible and its toxicity is
such that a rat treated with it will usually be dead or
too moribund for further experimentation 43 hours after its
administration.
The administration of insulin to animals pretreated
with alloxan, streptozotocin, or 6-aminonicotinamide
reverses the diabetic effects caused by these agents but
has no effect on those in animals pretreated with N-methyl¬
acetamide .
To investigate the effect that the diabetes induced
by these three agents had on hepatic drug metabolism in
male Holtzman rats, both in vivo and in vitro experiments
were performed. The in vivo experiments involved measuring
the length of hypnosis after the administration of hexo-
barbital sodium in animals which were made diabetic. The
in vitro experiments were of several types. After the
diabetes had been induced with the three agents, the

22
kinetic constants for the metabolism of hexobarbital and
aniline were measured at several time periods. Also, the
metabolism of hexobarbital and aniline were measured one
week after the administration of streptozotocin to deter¬
mine if the effects seen on drug metabolism existed for
more than several days. To examine the effect of hyper¬
glycemia on drug metabolism, animals were infused intra¬
venously with glucose, after which the in vitro metabolism
of hexobarbital was measured. To rule out any direct
effect that the diabetogenic agents may have had, they
were added to in vitro incubation mixtures from control
animals in which hexobarbital and aniline hydroxylations
were measured. To rule out any direct effect that insulin
may have had, it was added to similar in vitro incubation
mixtures from control animals and those pretreated with
the diabetogenic agents. Hepatic microsomal protein and
cytochrome P-450 content were also measured. In addition,
the characteristics of binding of both hexobarbital and
aniline to hepatic cytochrome P-450 from the diabetic
animals were studied.
As was mentioned in the historical review, increasing
the endogenous levels of c-AMP or administering c-AMP de¬
rivatives to animals is inhibitory to certain hepatic drug
metabolic pathways. This increase in c-AMP content has
also been shown to be responsible for the release of an in
hibitor of drug metabolism into the liver cytosol. To
attempt to elucidate a mechanism for the effects seen

23
with experimental diabetes on drug metabolism, hepatic
c-AMP content under the different diabetic conditions
was determined and these were correlated with the appear¬
ance of an inhibitor of drug metabolism in the liver
cytosol.

MATERIALS AND METHODS
Materials -
6-Aminonicotinamide and N-methylacetamide were pur¬
chased from Aldrich Chemical Company, Inc., Milwaukee,
Wisconsin. The streptozotocin was a kind gift from Dr. W.
E. Duiin of The Upjohn Company, Kalamazoo, Michigan. The
structures of these three compounds are shown in Figure 1.
Alloxan was purchased from Eastman Kodak Company, Rochester,
New York.
Hexobarbital sodium was purchased from Winthrop
Laboratories, New York, New York and aniline hydrochloride
from Eastman Kodak Company, Rochester, New York. Both the
crystalline zinc insulin and the protamine zinc insulin
were purchased from Eli Lilly, Inc., Indianapolis, Indiana,
NADP, NADPH, glucose 6-phosphate, glucose 6-phosphate
dehydrogenase and Tris buffer were all purchased from
Sigma Chemical Company, St. Louis, Missouri.
The c-AMP radioimmunoassay kit was purchased from
Schwarz/Mann of Orangeburg, New York.
Ail other chemicals used were of reagent grade.
Animals
Adult male rats weighing 100-250 g were obtained
from Holtzman Laboratories, Madison, Wisconsin.
24
All animals

25
0
CH3-C-NH-CH3
M-'1ETilVLACtTAMIPE
FIGURE 1
TRUCTURFS OF DIABETOGENIC AGENTS

26
were maintained on standard laboratory chow and water ad
libitum for a minimum of 48 hours in the Department of
Pharmacology and Therapeutics before experimentation. All
animals, except for those that were used in the glucose
infusion experiments were fasted overnight for 17 hours
preceding the experiment but were allowed water ad libitum.
Streptozotocin and 6-aminonicotinamide were dissolved
immediately before use, the former in a citrate buffer, pH
4.5 (26.5 ml of 0.1 M citric acid + 23 ml of 0.1 M sodium
citrate) and the latter in Krebs-Ringer bicarbonate solu¬
tion. These drugs were both administered intraperitoneally
Control animals used with streptozotocin-treated animals
were injected with the citrate buffer and those with 6-
aminonicotinamide were injected with the Krebs-Ringer
bicarbonate solution. N-Methylacetamide is a liquid and
was administered intragastrically. Control animals used
with N-methylacetamide animals were given normal saline
intragastrically. Alloxan was dissolved in normal saline
and injected subcutaneously at a dose of 115 mg/kg and
control animals used with alloxan-treated animals received
normal saline. Animals were considered diabetic only if
their blood glucose exceeded 200 mg/100 ml.
Enzyme Preparation
Animals were killed by a blow on the head followed by
decapitation. The livers were immediately removed, weighed
and homogenized in four volumes of ice-cold 0.1 M Tris
buffer, pH 7.5. The homogenate was centrifuged at 9000(7

27
for 15 minutes in a Serval.1 refrigerated centrifuge at
0° C. The supernatant fraction from this homogenate was
used for the enzyme assays. When microsomal protein deter¬
mination was necessary, part of this supernatant fraction
was centrifuged at 100,000# in aBeckman Model L ultracen¬
trifuge for one hour at 4° C. The microsomal pellet was
then resuspended in 0.15 M KC1 and recentrifuged at
100,000# for another hour. This microsomal pellet was
then resuspended and a protein determination was performed
(40) .
Isolated liver microsomes were also used in some
experiments. For these experiments, the liver was homog¬
enized in four volumes of ice-cold 0.25 M sucrose solution
containing 1 mM EDTA. The supernatant fraction obtained
from centrifugation at 9000# for 15 minutes at 0° C in a
Servall refrigerated centrifuge was then centrifuged at
100,000# in a Beckman Model L ultracentrifuge at 4° for one
hour. The microsomal pellet was then suspended in 0.15 M
KC1 and recentrifuged at 100,000# for one hour. This
microsomal pellet was then resuspended in 0.15 M KC1 con¬
taining 0.05 M Tris buffer, pH 7.5.
Incubation Conditions
The in vitro metabolism of hexobarbital was determined
by assay for unchanged hexobarbital by the method of Cooper
and Brodie (41). The incubation system used was as follows
2 ml of 9000# supernatant fraction equivalent to about 500
mg of liver, 1 micromole of hexobarbital, 30 micromoles of

28
nicotinamide, 25 micromoles of MgCl2, and 0.26 micromole of
NAD?. Tris buffer (0.1 M, pH 7.5) was added to bring the
volume to 4 ml in a 120-ml glass-stoppered bottle. The
incubation system used in microsomal experiments was the
same as above except that microsomes equivalent to 500 mg
of liver were used, and 1.8 micromoles of NADPH were sub¬
stituted for the NAD?. Also, the following were added:
25 micromoles of glucose 6-phosphate and 2 units of glucose
6-phosphate dehydrogenase. When either the 9000# fraction
or the microsomes were used as the enzyme source, the
mixtures were incubated with shaking at 37° C for 20 min¬
utes and at the end of the incubation period, all bottles
were packed in ice for five minutes to stop the reaction.
Then, in succession, 1.3 ml of phosphate buffer, pH 5.5
(96.5 ml of 0.2 N NaH2?04 + 3.5 ml of 0.2 N Na2HP04), 1.5
g NaCl, and 60 ml of heptane were added. The bottles were
then placed in a Burrei wrist-action shaker for 45 minutes,
after which the mixtures were centrifuged for 15 minutes
at room temperature in an International Centrifuge, Model
CS, at 40,000#. Then 40 ml of each organic solvent phase
was transferred to a 60-ml glass stoppered bottle containing
4 ml of phosphate buffer, pH 11 (8.5 ml 10 M NaOH + 200 ml
0.8 M Na2HOP4). This was then shaken for three minutes
after which 3 ml of the aqueous phase was used to determine
the spectrophotometric absorbance at 245 nm in a Gilford
spectrophotometer, Model 2400.

29
The in vitro metabolism of aniline was determined by
assay for p-aminophenol, the major metabolite formed in
rats, by the method of Brodie and Gillette (42) and modi¬
fied by Kato and Gillette (43). The incubation system
used was as follows: 2 ml of 9000^ supernatant fraction
equivalent to 500 mg of liver, 4 micromoles of aniline,
0.15 micromole of NADP, 15 micromoles of glucose 6-
phosphate, 15 micromoles MgSCh, and 0.5 ml of 0.3 M Tris
buffer, pH 7.5. Distilled water was used to bring the
final volume to 4 ml in a 120-ml glass-stoppered bottle.
The difference in conditions when microsomal experiments
were performed were the same as with the hexobarbital
experiments. In experiments with both 9000g supernatant
and microsomal fractions, the mixtures were incubated for
15 minutes with shaking at 37° C and then placed in an
ice-bath for five minutes. Then 2.5 g of NaCl and 50 ml
of ether were added, after which the bottles were shaken
in a Burrell wrist-action shaker for 15 minutes. Then 40
ml of each ether extract was removed and shaken with 3 ml
of a 0.1 M NaOH solution containing 1% phenol for three
minutes. This was allowed to stand for 30 minutes, after
which the ether was aspirated off and the absorbance of
the aqueous layer was determined at 620 nm in a Coleman
Junior spectrophotometer, Model 6A.

30
Petermination of Kinetic Constants
For these experiments, the 9000g supernatant fraction
was used as the enzyme source and the incubation conditions
were as previously described except that the concentrations
of hexobarbital and aniline were different. Hexobarbital
was added to the incubation mixtures so that the final con¬
centrations of hexobarbital were 0.5, 0.8, 1, 1.6, and 2 mM.
Aniline was added to the incubation mixtures so that the
final concentrations of aniline were 0.125, 0.167, 0.25,
0.5 and 1 mM. The kinetic constants were obtained by plot¬
ting the reciprocals of the concentrations vs. the recipro¬
cals of the initial velocities of the reaction.
Glucose Infusion
In these experiments, rats weighing between 200-250 g
were used and allowed laboratory chow throughout the exper¬
iment. Their right jugular veins were cannulated under light
ether anesthesia, after which the animals were allowed to
recover for 24-36 hours. They were then placed in plastic
restraining cages and were infused with 40% glucose solu¬
tion or normal saline solution for 24 hours with a Harvard
infusion/withdrawal pump at a rate of 0.074 ml/hour for a
total of 1.78 ml, equivalent to 710 mg of glucose.
Cytochrome P-450 Content
Cytochrome P-450 content was measured by the carbon
monoxide-induced difference spectrum of the reduced hemo-
protein according to the method of Omura and Sato (44),
Three ml of a microsomal suspension containing 2 mg/ml

31
protein were placed in quartz cuvettes and dithionite was
added to reduce the cytochrome P-450. Carbon monoxide was
then gently bubbled into the cuvette for one minute and the
change in absorbance at 450 nm minus that at 489 nm was
measured in an Aminco-Chance recording spectrophotometer
operated in the split-beam mode. Cytochrome P-450 content
was measured according to Beer's Law by the following
formula:
AA x 1000
c =
£ X d X (P)
where c is the concentration of cytochrome P-450
expressed as nanomoles per mg of protein
£ is the molar absorption coefficient
(91 mM 1 cm 1)
d is the optical path length (1 cm), and
(P) is the concentration of protein, expressed
as mg/mi
Drug Binding of Cytochrome P-450
The binding of drugs to cytochrome P-450 was measured
by the method of Remmer et at. (7). Three ml of a micro¬
somal suspension containing 2 mg of protein per ml were
placed in quartz cuvettes and either hexobarbitai or
aniline was added. Hexobarbitai was added in increments
which gave final concentrations of 0.125, 0.15, 0.187,
0.25, 0.375, and 0.75 mM. Aniline was added in increments
which gave final concentrations of 0.0625, 0.075, 0.094,

22
0.125, 0.188, and 0.375 mM. The change in absorbance after
each addition was measured and the reciprocal of this
change was plotted . the reciprocal of the drug concen¬
tration; the apparent spectral dissociation constant (K )
was determined by extrapolation to the x-axis.
Determination of the Presence of an Inhibitor of Drug
Metabolism in Hepatic 100,000.? Supernatant Fraction
Microscmes were prepared as described previously
except that after the first one-hour centrifugation at
100,000^, the microsomal and supernatant fractions of
control and streptozotocin- and 6-aminonicotinamide-
pretreated rats were separated and various combinations
were prepared. The soluble fractions were added to the
microsomes immediately and were rehomogenized and then
kept on ice for 15 minutes. This homogenate was then
recentrifuged and the resultant microsomal pellet was
resuspended as previously described for the preparation
of the final microsomal suspension used for the enzyme
assays.
Miscellaneous
C-AMP levels were measured by the method of Steiner
et ol. (45). The Schwarz/Mann c-AMP radioimmunoassay
kit was used for these determinations.
Blood samples for determination of blood glucose
were taken from the tail vein and were analyzed by the
o-toluidine reagent method (46).
Hexobarbital sleeping time was measured as the time

33
between the loss and recovery of the righting reflex after
intraperitoneal injection of 100 mg of hexobarbital sodium
per kg.
Statistics
The results are expressed as the mean ± standard
error of the mean (S.E.). The p-values were calculated
by applying the Student t-test, which is a measure of the
probability that the differences observed are due to
chance (47).

RESULTS
Hyperglycemic Response to Diabetogenic Agents
Since the criterion chosen for a diabetic state was a
blood glucose concentration in excess of 200 mg/100 ml,
doses of the diabetogenic agents that would yield such a
blood glucose level had to be determined. The response to
streptozotocin at the dosages of 55 mg/kg and 65 mg/kg are
shown in Figure 2. The lower of the two doses was chosen
for further studies because there was little difference in
the hyperglycemic response and the higher dose was associ¬
ated with a greater toxicity (40% of those animals died
within 24 hours compared with no mortality with the lower
dose). Figure 3 shows the response to 6-aminonicotinamide
at dosages of 25 mg/kg and 35 mg/kg. No animals died
within 24 hours after each dose. The higher dose was
chosen hare because the lower dose did not produce a blood
glucose level that met the above criterion. Figure 4 shows
the response to N-methylacetamide at dosages of 6.75 ml/kg
and 10.0 ml/kg. The lower dose was chosen here because
the higher dose proved too toxic (60% died within 24 hours
vs. 0% at the lower dose).
34

FIGURE 2
RELATIONSHIP BETWEEN SERUM GLUCOSE AND DOSE OF STREPTOZOTÓCIN

Serum Glucose
mg/100 ml
400
300
200
100
0
4
LO
CT\

FIGURE 3
RELATIONSHIP BETWEEN SERUM CLUCOSE AND DOSE OF 6-AMINONICOTINAMIDE

rum Glucose
mg/l00 ml
400
300
200
100
0 4 8 12
Hours
A,
<3 5 mg/kg
LO
CO

FIGURE 4
RELATIONSHIP BETWEEN SERUM GLUCOSE AND DOSE OF N-METHYLACETAMIDE

Serum Glucose
rng/lOO ml
400
300
200
100
10.1ml/kg
«6.25ml/kg
0
4
8 12
Hours
24

Hexobarbital Hypnosis
To ascertain if the diabetes induced by the diabeto¬
genic agents affected the in vivo sleeping time induced by
a hypnotic, 100 mg/kg of hexobarbital sodium was injected
intraperitoneally into rats. Table 2 shows the results
of these experiments; both streptozotocin and 6-aminonico-
tinamide increased the sleeping time, whereas N-methylace-
tamide had no effect.
Effect of Diabetogenic Agents on Kinetic Constants for
Hexobarbital and Aniline Hydroxylations
To determine if the diabetes induced by the three
diabetogenic agents affected the in vitro metabolism of
hexobarbital and aniline, the rates of hydroxylation of
these substrates were measured. Table 3 shows the kinetic
constants for both hexobarbital and aniline hydroxylation
in liver 9000^ supernatant fractions 24, 43, and 96 hours
after the administration of streptozotocin. The table
also shows the effect on these constants after the daily
subcutaneous injection of three units of protamine zinc
insulin for three days beginning 24 hours after the in¬
jection of streptozotocin. The results indicate that the
K values for both hexobarbital and aniline hydroxylations
were increased and the values of V decreased at all time
max
periods tested after the animal had been treated with
streptozotocin. The changes in these parameters for
aniline metabolism, although statistically significant,
were quite small. Treatment with insulin in vivo was able

TZiBLE 2
HEXOBARBITAL SLEEPING TIMES AFTER ADMINISTRATION OF DIABETOGENIC AGENTS
Pretreatment Duration of Hypnosis"1
(minutes)
Saline Control (n = 18)
• V)
Streptozotocin (n = 6)
Q
Streptozotocin (n = 6)
6-Aminonicotinamide (n = 6)d
N-Methylacetamide (n = 7)^
39.4 ± 1.1
68.7 ± 1.3d
65.3 ± 1.5d
59.2 ± 3.0d
35.4 ± 2.0e
a
Data are presented as means ± S.E.
kpretreated 24 hours before experiment.
Q
Pretreated 48 hours before experiment.
p < 0.01, compared with the appropriate control value.
0 ,
Not significantly different from control value.

TABLE 3
EFFECT OF STREPTOZOTOCIN ON KINETIC CONSTANTS
FOR HEXOBARBITAL AND ANILINE HYDROXYLATIONS
K
a
V
a
(iriM)
, , IllClA
(nanomoles/mg micros
:omal protein/min)
â– xobarbital
Control (n = 22)
0.57
±
0.01
7.65
±
0.09
Streptozotocin
V)
(n - 6)D
1.01
±
0.03f
4.54
±
0.12f
Streptozotocin
(n = 6)°
0.99
±
0.03f
5.21
±
0.22f
Streptozotocin
(n - 6) d
0.97
±
0.03f
5.07
±
0.09f
Streptozotocin
+ Insulin
(n = 8 ) C
0.59
±
Cn
CN
o
•
o
7.63
±
0.15g
.iline
Control (n = 21)
0.090
±
0.001
0.79
±
0.01
Streptozotocin
(n = 6)b
0.105
±
0.004f
0.67
±
0.02f
Streptozotocin
(n = 6) C
0.09 9
±
0.003f
0.67
±
0.02f
Streptozotocin
(n = 6)d
0.101
J.
0.003f
0.71
±
0.03f
Streptozotocin
+ Insulin
(n = 6)e
0.088
±
0.002g
0.79
±
0.02g
a
Data are presented as means ± S.E.
Pretreated 24 hours before experiment.
Pretreated 48 hours before experiment.
dPretreated 9 6 hours before experiment.
0 ,
Three units of protamine zinc insulin injected subcutaneously daily for 3 days
beginning 24 hours after streptozotocin was administered.
^p < 0.01, compared with the appropriate control value.
gNot significantly different from control value.

44
to reverse these effects and return the K and V for
m max
both hexobarbital and aniline hydroxylation to control
values.
Table 4 shows the kinetic constants for hexobarbital
and aniline hydroxylations 24 and 48 hours after the ad¬
ministration of 6-aminonicotinamide. The K values for
m
hexobarbital hydroxylation were increased and those of
V were decreased at both 24 and 48 hours. The K of
max m
aniline hydroxylation was increased 24 hours after admin¬
istration of the diabetogen, and the V was decreased.
These changes, although statistically significant, were
quite small. At 48 hours, neither of these parameters
was significantly different from those in preparations
from control animals.
Table 5 shows that 24 hours after the administration
of N-methylacetamide, there was no change in any of the
kinetic constants for either hexobarbital or aniline.
Effect of Streptozotocin Diabetes of One Week Duration on
Hexobarbital and Aniline Hydroxylations
To determine if the effects of streptozotocin diabe¬
tes on drug metabolism lasted for longer than three days,
hexobarbital and aniline hydroxylations were measured in
vitro one week after the administration of streptozotocin
Table 6 shows that the rates of both hexobarbital and ani
line metabolism were still below control values one week
after administration of streptozotocin.

TABLE 4
EFFECT OF 6-AMINONICOTINAMIDE ON KINETIC CONSTANTS
FOR HEXOBARBITAL AND ANILINE HYDROXYLATIONS
Hexobarbital
K
m
(mM)
V
max
(nanomoles/mg microsomal protein/min)
Control (n = 22)
0.57
±
0.01
7.65
±
0.09
6 - Am inonicot. inamide
(n =
6)b
0.96
T
O
o
u>
4.33
±
0.0 id
6-Aminon.i cot inamide
(n =
6)°
0.6 8
+
0.02d
6.15
±
0.12d
Aniline
Control (n = 21)
0.090
±
0.001
0.79
+
0.01
6-Aminonicotinamide
(n =
6)b
0.104
±
0.004d
0.70
+
d
0.01
6-Aminonicotinamide
(n =
6)C
0.089
±
0.003e
0.80
±
0.02e
dData are presented as means ± S.E.
bPretreated 24 hours before experiment.
Q
Pretreated 48 hours before experiment.
p < 0.01, compared with tne appropriate control value.
0 ...
Not significantly different from control value.
U1

TABLE 5
EFFECT OF N-METHYLACETAMIDE ON KINETIC CONSTANTS
FOR HEXOBARBITAL AND ANILINE HYDROXYNATIONS
K a V
m max
a
Hexobarbital
Control (n = 22)
N-Methylacetamide (n = 6)^
(mM)
(nanomoles/mg microsomal protein/min)
0.57 ± 0.01
7.65 ± 0.09
0.59 ± 0.02
7.47 ± 0.10
Aniline
Control (n = 21)
N-Methylacetamide (n = 6)^
0.090 ± 0.001
0.089 ± 0.003C
0.79 ± 0.01
0.75 ± 0.02G
Data are presented as means ± S.E.
bPretreated 24 hours before experiment,
c ...
'Not significantly different from control value.

TABLE 6
BEXOBARBITAL
WEEK AFTER THE
AND ANILINE HYDROXYLATIONS ONE
ADMINISTRATION OF STREPTOZOTOCIN
Hexobarbitaj
micromoles metabolized/g livera
Control
(n = 5)
1.36 ± 0.05
Treated
(n = 5)
0.90 ± 0.05b
Aniline
micromoles p-aminophenol formed/g livera
Control
(n = 5)
0.40 ± 0.02
Treated
(n = 5)
0.31 ± 0.02b
aData are presented as means ± S.E.
bp < 0.01, compared with appropriate control value.

48
Effect of Alloxan Diabetes on Hexobarbital and Aniline
Hydroxylations
As was noted in the historical review, a number of
researchers had reported that the rates of metabolism of a
large number of drugs, including hexobarbital, were
decreased in alloxan-diabetic animals but those of a few
drugs, such as aniline, were enhanced. These results were
reexamined, as shown in Table 7. The metabolism of hexo¬
barbital in vitro was inhibited while that of aniline was
enhanced.
Effect of Diabetogenic Agents on Hexobarbital and Aniline
Hydroxy ladiions Using Isolated Microsomes as Enzyme Source
To be sure that the inhibition observed in drug meta¬
bolism in animal preparations from rats pretreated with
streptozotocin and 6-aminonicotinamide was due to changes
in the microsomal enzyme system rather than to an altera¬
tion in the NADPH-generating system, isolated liver micro¬
somes were used as the enzyme source with an added NADPH-
generating system, to measure the metabolism of hexobarbi¬
tal and aniline after animals had been pretreated with
these diabetogens. N-methylacetamide was excluded in these
studies because no inhibition in drug metabolism was seen
when animals were pretreated with this agent. Tables 8
and 9 show that an inhibition in hexobarbital and aniline
metabolism occurred when preparations of liver microsomes
from animals that had been treated 24 hours in advance with
streptozotocin and 6-aminonicotinamide were used as the
enzyme source for the assay.

TABLE 7
EFFECT OF ALLOXAN ON HEXOBARBITAL AND ANILINE KYDROXYLATIONS
Hexobarbital micromoles metabolized/g liver3
Control (n =4) 1.41 ± 0.05
Treated (n = 4)b 0.83 ± 0.04c
Aniline micromoles p-aminophenol formed/g liver
Control (n = 4) 0.3810,02
Treated (n = 4)b 0.49 ± 0.02i::i
cl
""Data are presented as means ± S.E.
bPretreated 24 hours before experiment,
p < 0.01, compared with the appropriate control value,
^p < 0.05, compared with the appropriate control value.

TABLE 8
EFFECT OF STREPTOZOTOCIN ON HEXOBARBITAL AND ANILINE
HYDROXYLATIONS WITH ISOLATED MICROSOMES AS ENZYME SOURCE
Plexobarbital
Control (n = 8)
Treated (n = 4)^
micromoles metabolized/g liver
1.11 ± 0.Ü3
0.59 ± 0.06C
Aniline
Control (n = 8)
Treated (n = 4)^
micromoles p-aminophenol formed/g live
0.26 ± 0.01
0.20 ± 0.01°
Data are presented as means ± S.E.
^Pretreated 24 hours before experiment,
c
p < 0.01, compared with the appropriate control value.

TABLE 9
EFFECT OF 6-AMINONICOTINAMIDE ON HEXOBARBITAL AND ANILINE
HYDROXYLATIONS WITH ISOLATED MICROSOMES AS ENZYME SOURCE
Hexobarbital
Control (n = 8)
Treated (n = 4)13
micromoles metabolized/g livera
1.11 ± 0.03
0.81 ± 0.01C
Aniline micromoles p-aminophenol formed/g livera
Control
(n =
8)
0.26
± 0.01
Treated
(n =
4) b
0.20
± 0.02d
a
Data are presented as means ± S.E.
kpretreated 24 hours before experiment.
Q
p < 0.01, compared with the appropriate control value,
p < 0.05, compared with the appropriate control value.

52
Effect of Glucose Infusion on Hexobarbital Hydroxylation
To test if hyperglycemia alone could be responsible
for the inhibition seen in drug metabolism, rats were
infused intravenously for 24 hours with glucose; each ani¬
mal received a total of 710 mg of glucose. All such ani¬
mals had blood glucose concentrations that exceeded 200 mg/
100 ml by the end of the infusion. Control animals were
similarly infused with normal saline solution. The data
in Table 10 demonstrate that hyperglycemia produced by
glucose infusion had no effect on the in vitro metabolism
of hexobarbital.
Effect of Diabetogenic Agents Added in Vitro on Hexobarbi-
tal and Aniline Hydroxylations
To rule out any direct effect that the diabetogenic
agents may have had on the in vitro metabolism of hexobar¬
bital and aniline, they were added at concentrations of
10 2, 10 3, and 10 4 M to incubation mixtures containing
preparations of livers from untreated animals. Tables 11
and 12 show that the in vitro addition of these agents had
no effect on either hexobarbital or aniline hydroxylation.
Effect of Insulin Added in Vitro on Hexobarbital and Aniline
Hydroxylations Following Treatment with Diabetogenic Agents
To determine whether addition of insulin in vitro
would reverse the effects of pretreatment with the diabeto¬
genic agents, crystalline zinc insulin ranging in amount
from 0.C01 to 1 unit was added to 4-ml incubation mixtures
containing preparations of livers from animals treated

TABLE 10
EFFECT OF GLUCOSE INFUSION ON HEXOBARBITAL METABOLISM
Micromoles metabolized/g
liver
a
Control (n = 5)
Treated (n = 5)
1.51 ± 0.05
1.43 ± 0.08
b
aData are presented as means
^Not significantly different
± S.E.
from control value.

TABLE 11
EFFECT OF DIABETOGENIC AGENTS ADDED IN VITFO ON HEXOBARBITAL METABOLISM
Treatment micromoles metabolized/g livera
Control (n = 15)
1.50
+
0.01
Streptozotocin
10_2 M (n = 5)
1.51
±
0.0 ib
10“3 M (n = 5)
1.52
±
0.0 3b
10“4 M (n = 5)
1.52
±
0.0 lb
6-Aminonicotinamide
If)
II
3
CM
1
o
1—1
1.51
±
0.02b
10“3 M (n = 5)
1.52
±
0.02b
10“4 M (n = 5)
1.50
±
b
0.02
N-Methylacetamide
10 2 M (n = 5)
1.44
±
0.03b
LO
II
»c-1
CO
1
o
1—i
1.46
±
&
ro
o
o
10'4 M (n = 5)
1.43
±
0.0 2b
aData are presented as means ± S.E.
bNot significantly different from control value.

TABLE 12
EFFECT OF DIABETOGENIC AGENTS ADDED IN VITRO ON ANILINE METABOLISM
Treatment micromoles p-aminophenol formed/g liver
Control (n = 15)
0.43
±
0.01
Streptozotocin
ID
II
£
s
CM
1
O
1—1
0.39
±
0.02b
10“3 M (n = 5)
0.39
±
0.02b
10"" M (n = 5)
0.40
±
0.02b
6-Aminonicotinamide
10_2 M (n = 5)
0.44
±
0.03b
10~3 M (n = 5)
0.42
±
0.02b
10~4 M (n = 5)
0.42
±
0.02b
N-Methylacetamide
1(T2 M (n = 5)
0.40
±
0.03b
10~3 M (n = 5)
0.41
±
0.02b
10-4 M (n = 5)
0.41
±
0.0 2b
aData are presented as means ± S.E.
Not significantly different from control value.

56
with streptozotocin and 6-aminonicotinamide. Livers from
N-methylacetamide-treated animals were not used because
inhibition of neither hexobarbital nor aniline was seen
when animals were treated with this diabetogenic agent.
Table 13 shows that insulin had no direct effect on the in
vitro metabolism of hexobarbital or aniline after the ani¬
mal had been treated with streptozotocin or 6-aminonico-
tinamide.
Effect of Diabetogenic Agents on Hepatic Microsomal Protein
To see if the alterations in hepatic drug metabolism
may be due to changes in hepatic microsomal protein con¬
tent, the effect of the diabetogenic agents on hepatic
microsomal protein concentration was determined. Table 14
shows that protein concentration was not affected 24, 48,
or 96 hours after the administration of streptozotocin, 24
or 48 hours after the administration of 6-aminonicotinamide,
or 24 hours after N-methylacetamide. Also, the administra¬
tion of three units of protamine zinc insulin for three
days beginning 24 hours after the animals had been treated
with streptozotocin had no effect on hepatic microsomal
protein concentration.
Effect of Diabetogenic Agents on Cytochrome P-45Q in Liver
Microsomes
To examine the possibility that the changes seen in
drug metabolism may be due to alterations in cytochrome
P-450 content in liver microsomes, the effect of the
diabetogenic agents on the microsomal concentration of this

TABLE 13
EFFECT OF INSULIN IN VITRO ON HEXOBARBITAL
AND ANILINE METABOLISM FOLLOWING DIABETOGENIC AGENTS
Hexobarbital
micromoles metabolized/g
liver
a
Control
1 unit
0.1 unit
0.01
unit
0.001
unit
Insulin
Insulin
Insulin
Insulin
Streptozotocin^1
0.85
±
0.02
0
o
•
o
+i
CO
o
0.87 ± 0.05C
0.85
i+
o
o
U)
o
. . b
6-Aminonicotmamide
1.00
+
0.05
0.99 ± 0.05C
o
o
•
o
+1
m
cn
o
0.97
± 0.02c
0.99
± 0.05c
Aniline
micromoles p
-aminophenol formed/g
livera
Streptozotocin
0.33
±
0.03
0.32 ± 0.03°
0.33 ± 0.03°
0.33
± 0.02C
0.33
± 0.03°
6-Aminonicotinamide13
0.30
-j-
0.01
0.29 ± 0.01C
0.29 ± 0.01C
0.30
± 0.01C
0.30
u
i—1
O
•
o
+1
aData are presented as means ± S.E.
kpretreated 24 hours before experiment
'“Not significantly different from control value.
U1
•^1

TABLE 14
EFFECT OF DIABETOGENIC AGENTS ON HEPATIC MICROSOMAL PROTEIN CONCENTRATION
Pretreatment
mg/g
livera
Control (n = 42)
27.9
i—1
o
+1
Streptozotocin (n = 12)^
27.6
± 0.3f
Q
Streptozotocin (n = 12)
27.6
4-Í
CM
O
Streptozotocin (n = 12)^
28.1
± 0.3f
Streptozotocin + Insulin (n = 14)e
27.7
± 0.2f
6-Aminonicotinamide (n = 12)u
27.7
± 0.3f
c
6-Aminonicotinamide (n = 12)
27.9
± 0.2 f
T_
N-Methylacetamide (n = 12) J
27.7
± 0.2f
aData are presented as means ± S.E.
kpretreated 24 hours before experiment.
cPretreated 48 hours before experiment.
dPretreated 72 hours before experiment.
0 . .
Three units of protamine zinc insulin injected subcutaneously
daily for 3 days beginning 24 hours after streptozotocin was
injected,
f ...
Not significantly different from control value.
U1
CO

59
cytochrome was determined. Table 15 shows that pretreat¬
ment 24 hours previously with neither streptozotocin,
6-aminonicotinamide; nor N-methylacetamide had any effect
on the concentration of cytochrome P-450 in the liver
microsomes.
Effect of Diabetogenic Agents on Cytochrome P-450 Binding
to Hexobarbital and Aniline
To ascertain if the changes observed may be due to
changes in the strength of binding of hexobarbital and
aniline to cytochrome P-450, the spectral binding constants
(K ) were determined in liver microsomes from control ani-
s
mals and those that had been treated with the diabetogenic
agents. Table 16 shows that the affinity of cytochrome
P-450 for hexobarbital was significantly lower in micro¬
somes from streptozotocin- and 6-aminonicotinamide-diabetic
animals than in controls, whereas the binding to hexobarbi¬
tal was not affected in microsomes from N-methylacetamide-
treated animals. In the case of aniline, a decrease in
affinity of cytochrome P-450 was seen only in preparations
from animals pretreated with 6-aminonicotinamide, and the
affinity of cytochrome P-450 for aniline was increased in
microsomes from animals treated with N-methylacetamide.
Effect of Hepatic Homogenate Fractions on Hexobarbital and
Aniline Hydroxylation
To determine if an inhibitor of hexobarbital or aniline
metabolism may have been present in the 100,000g supernatant
fraction of livers of rats pretreated with streptozotocin or
6-aminonicotinamide, various combinations of microsomes and

TABLE 15
EFFECT OF DIABETOGENIC AGENTS ON CYTOCHROME P-450 CONCENTRATION IN LIVER MICROSOMFS
Pretreatment
nanomoles cytochrome
3.
P-450/mg protein/ml
Control (n = 6)
0.43 ±
0.02
Streptozotocin (n = 5)^
0.47 ±
O
o
u>
o
6-Aminonicotinamide (n = 5)°
0.42 ±
o
o
o
N-Methylacetamide (n = 6)^
0.47 ±
Ü
Ol
o
o
dData are presented as means ± S.E.
kpretreated 24 hours before experiment,
c ...
Not significantly different from control value.

TABLE 16
EFFECT OF DIABETOGENIC AGENTS ON CYTOCHROME P-450 BINDING TO HEXOBARBITAL AND ANILINE
Pretreatment
Control (n = 11)
Streptozotocin (n = 5)^
6-Aminonicotinamide (n = 5)d
N-Methylacetamide (n = 6)^
Ks (mM) - Hexobarbitala
0.062 ± 0.001
0.084 ± 0.005°
0.077 ± 0.006C •
0.065 ± 0.002d
Ks (mM) - Anilinea
0.73 ± 0.02
0.73 ± 0.02d
0.84 ± 0.03C
0.63 ± 0.04C
aData are presented as means ± S.E.
dPretreated 24 hours before experiment
0
p 0.01, compared with the appropriate control value.
dNot significantly different from control value.

62
supernatant fractions from control and treated animals
were mixed together as described in the Methods section.
Table 17 shows that in vitro inhibition of hexobarbital
hydroxylation was seen when enzyme preparations containing
microsones from animals pretreated with either streptozo-
toein or 6-aminonicotinamide were preincubated with either
their own or with control supernatant fraction. More
important, however, is that enzyme preparations containing
microsomes from control animals which had been incubated
with supernatant fractions from animals pretreated with
either of the diabetogenic agents also showed an inhibi¬
tion of hexobarbital metabolism.
Table 18 shows the same experiment except that aniline
was the substrate. The in vitro inhibition was again seen
when enzyme preparations containing microsomes of animals
pretreated with both diabetogenic agents were added with
either their own supernatant fraction or with control
supernatant fraction. Microsomes from control animals
which were combined with supernatant fraction of animals
pretreated with streptozotocin did show an inhibition of
aniline metabolism but when microsomes from control animals
were added to supernatant fraction of animals pretreated
with 6-aminonicotinamide, no inhibition occurred.

TABLE 17
LOCALIZATION OF INHIBITOR OF HEXOBARBITAL METABOLISM PRESENT IN 100,0000 SUPERNATANT
FRACTION OF RAT LIVER AFTER PRETREATMENT WITH STREPTOZOTOCIN AND 6-AMINONICOTINAMIDE
Microsomes
Supernatant
Number
of Rats
micromoles meta-
bolized/q liver
% of Control
Control
Control
10
1.00
+
0.02
—
Control
Streptozotocin^
Pretreated
5
0.86
±
0.02C
86
Streptozotocin
Pretreated
Streptozotocin
Pretreated
5
0.76
±
o.oic
76
Streptozotocin
Pretreated
Control
5
0.88
±
0.02C
88
Control
6-Aminonicotinamide^
Pretreated
5
0.91
±
0.02°
91
6-Aminonicotinamide
Pretreated
6-Aminonicotinamide
Pretreated
5
0.86
Hr
0.02C
86
6-Aminonicotinamide
Pretreated
Control
5
0.90
±
O
o
H
O
90
Data are presented as means ± S.E.
'’Pretreated 2 4 hours before experiment.
'p < 0.01, compared with value obtained in experiment in which both microsomal and
supernatant fractions were derived from control animals.

TABLE 18
LOCALIZATION OF INHIBITOR OF ANILINE METABOLISM PRESENT IN 100,000^ SUPERNATANT
FRACTION OF RAT LIVER AFTER PRETREATMENT WITH STREPTOZOTOCIN AND 6-AMINONICOTINAMIDE
Homogenate Fractions and Treatments
Number micromoles p-amino-
Microsomes Supernatant of Rats phenol formed/g liver % of Control
Control
Control
10
0.26
+
0.01
—
Control
St rep tozo toe i.nD
Pretreated
5
0.22
+
o
O
ro
o
85
Streptozotocin
Pretreated
Streptozotocin
Pretreated
5
0.20
±
0.02d
77
Streptozotocin
Pretreated
Control
5
0.22
±
0 . oic
85
Control
6-Aminonicotinamide^
Pretreated
5
0.23
±
0)
pH
o
o
88
6-Aminonicotinamide
Pretreated
6-Aminonicotinamide
Pretreated
5
0.20
+
0.02d
77
6-Aminonicotinamide
Pretreated
Control
5
0.21
+
o.oid
81
3.
Data are presented as means ± S.E.
kpretreated 24 hours before experiment.
c
p < 0.05, compared with value obtained in experiment in which both microsomal and
supernatant fractions were derived from control animals.
dp < 0.01, compared with value obtained in experiment in which both microsomal and
supernatant fractions were derived from control animals.
^Not significantly different from control experiment in which both microsomal and
supernatant fractions were derived from control animals.
CTl

65
Effect of Diabetogenic Agents on Hepatic c-AMP Concen¬
trations
C-AMP concentrations were measured in the liver
following treatment with the diabetogenic agents to
determine whether a correlation existed between the type
of diabetic state produced and the hepatic content of
c-AMP, since it had already been shown that elevation of
hepatic c-AMP is inhibitory to certain drug-metabolic
pathways. Table 19 shows that streptozotocin and, to a
lesser extent, 6-aminonicotinamide, elevated hepatic
c-AMP concentrations, whereas N-methylacetamide had no
effect as compared to control values.

TABLE 19
EFFECT OF DIABETOGENIC AGENTS ON HEPATIC C-AMP CONCENTRATIONS
Pretreatment
Control (n = 8)
Streptozotocin (n = 6)°
6-/uninonicotinamide (n = 6)d
N-Methylacetamide (n = 6)°
nanomoles/g livera
0.49 ± 0.03
0.80 ± 0.04C
0.65 ± 0.03C
0.48 ± 0.03d
aData are presented as means ± S.E.
kpretreated 24 hours before experiment
Cp < 0.01
^Not significantly different from control value.
Oh
Oh

DISCUSSION
It has been shown previously that alloxan diabetes was
inhibitory to certain pathways of drug metabolism in several
species (3 - 6, 9 - 12, 17). This study was undertaken to
investigate the effect of three newer diabetogenic agents
on drug metabolism. Streptozotocin and 6-aminonicotinamide
produce an insulin-deficient animal while the third agent,
N-methylacetamide does not produce an insulin-deficient
animal but instead produces a condition whereby insulin is
produced and released by the pancreas but is incapable of
lowering blood glucose. The results of the present experi¬
ments with these three diabetogenic agents indicate that,
with male Holtzman rats, it was not hyperglycemia that was
responsible for the inhibition of certain drug-metabolic
pathways in diabetes but rather that the inhibition was a
result of the lack of insulin.
Hexobarbital and aniline were chosen for these studies
because they have both been used extensively as model sub¬
strates in studies of the mixed-function oxidase system in
the liver, and because their metabolism in normal rats is
well understood. Also, as mentioned in the Historical
Review, they differ in their binding to cytochrome P-450
in that the difference spectra produced when they are added
67

to liver microsomes are qualitatively different.
Figure 5 shows the major metabolic pathway of hexobar-
bital. In the rat, the major metabolite is 3'-hydroxy-
hexobarbital; 3'-ketohexobarbital is a very minor metabo¬
lite (48). As described in the Methods section, the meta¬
bolism of hexobarbital was measured by the disappearance
of the substrate rather than by the appearance of a meta¬
bolite .
Figure 6 shows the metabolic pathway for aniline. In
rats, there is six times more p-aminophenol formed than o-
aminophenol (49). The metabolism of aniline was measured
by the appearance of the metabolite, p-aminophenol.
Hexobarbital sleeping time has often been used to meas¬
ure hepatic drug metabolism for several reasons. First,
the substance is apolar enough to be rapidly absorbed and
distributed in the body. Second, its hypnotic activity is
such that convenient doses can be injected intraperitoneally
to produce a rapid sleep of moderate duration so that an
alteration in the length of sleep can be measured with
acceptable precision. Lastly, and most important, hexobar¬
bital is rapidly metabolized in animals so that there is
little time for its accumulation in body fat. Thus, the
duration of its hypnotic activity is a reflection of meta¬
bolism rather than of tissue redistribution (50). Table 2
shows that rats pretreated with both streptozotocin and 6-
aminonicotinamide slept significantly longer than controls

—a*
3'-KETOHEXOBARBITAL
FIGURE 5
METABOLIC PATHWAY OF HEXOBARBITAL

ANILINE
-AMI NOPHENOL
o-AMINOPHENOL
FIGURE 6
METABOLIC PATHWAY OF ANILINE

71
while those animals pretreated with N-methylacetamide did
not ^leep any longer than the controls.
To see if the in vivo results just described could be
correlated with the in vitro experiments, the metabolism
of hexobarbital and aniline were measured in in vitro
incubation mixtures using 9000# supernatant fractions from
liver homogenates as the enzyme source. The fact that the
L, values increased and the decreased for both hexo-
barbital and aniline 24, 48, and 96 hours after the
streptozotocin and 24 hours after 6-aminonicotinamide
indicated that the in vitro metabolism of these substrates
was inhibited after pretreatment with these diabetogenic
agents. The effects of 6-aminonicotinamide upon insulin
release are reversed with time as the agent is eliminated
from the body; this was reflected by the less intense inhi¬
bition of hexobarbital metabolism and the lack of inhibition
of aniline metabolism 48 hours after the administration of
6-aminonicotinamide. The fact that the kinetic constants
for both hexobarbital and aniline were unchanged in animals
24 hours after they were treated with N-methylacetamide
again showed that this diabetogenic agent does not affect
the metabolism of either of these substrates. These results
and the fact that hyperglycemia produced by infusion of
glucose did not cause inhibition of in vitro hexobarbital
metabolism (Table 10), showed that the hyperglycemia of
diabetes was not responsible for the inhibition of drug
metabolism caused by streptozotocin and 6-aminonicotinamide.

72
Since the diabetogenic effect of streptozotocin was
irreversible but not lethal at the dosage used, it was
important to determine if the inhibitory effect of strepto-
zotocin-diabetes on hexobarbital and aniline hydroxylations
lasted for longer than three days. Table 6 showed that
the in vitro metabolism of hexobarbital and aniline were
still well below control values one week after the animals
had received streptozotocin.
As was mentioned in the Historical Review, previous
researchers had noted that the in vitro metabolism of
hexobarbital was inhibited in animals pretreated with
alloxan but that the in vitro metabolism of aniline was
enhanced (3, 4, 6, 9, 10). Since in the present studies
with streptozotocin, a diabetogenic agent similar in mecha¬
nism of action to alloxan, no enhancement of aniline meta¬
bolism was observed, it was important to reexamine the
results with alloxan reported previously. The results
shown in Table 7 are qualitatively similar to those
reported by other investigators, although the extent of
enhancement of aniline hydroxylation was not as great as
that previously reported. Nevertheless, it would appear
that the enhancing effect of alloxan on the rate of aniline
hydroxylation is not related to the diabetogenic activity
of this agent.

73
Since both hexobarbital and aniline are metabolized
by liver microsomes and not by any nonmicrosomal hepatic
cells, it was important to determine if the inhibition that
was seen with the in vitro incubation mixtures using hepatic
9000g supernatant fraction as the enzyme source could be
duplicated when isolated liver microsomes were used as
the enzyme source. This was necessary to show that the
inhibition was not due to an inability of the drugs to
reach the microsomes, or to a deficiency in the NADPH-
generating system. Tables 8 and 9 demonstrated that the
metabolism of both substrates after treatment with either
streptozotocin or 6-aminonicotinamide was inhibited in
isolated liver microsomes.
Addition of streptozotocin, 6-aminonicotinamide, and
N-methylacetamide to incubation mixtures were made to
determine the direct effect these agents may have had on
in vitro enzyme activity. These diabetogenic agents at
concentrations ranging from 10 2 to 10 4 M had no direct
effect on either hexobarbital or aniline hydroxylation.
The highest of the concentrations of the diabetogenic agents
used in these experiments was greater than any of the con¬
centrations that could have occurred in vivo, assuming
complete absorption.
The direct effect of crystalline zinc insulin on hexo¬
barbital and aniline nydroxylations was also examined.
Table 13 shows that insulin added to incubation mixtures
that contained preparations of livers from control animals

or those treated with streptozotocin or 6-aminonicotinamide
had no effect on the in vitro metabolism of either of these
substrates. The concentrations of insulin added in these
experiments both mimicked and exceeded the concentrations
of insulin that would occur in vivo.
The inhibitory action of streptozotocin and 6-aminonico¬
tinamide on both hexobarbital and aniline metabolism could
not be attributed to an alteration in hepatic microsomal
protein (Table 13) or to an alteration in hepatic micro¬
somal cytochrome P-450 (Table 14). Also, N-methylacetamide
did not alter hepatic microsomal protein or cytochrome P-450
content.
The binding of hepatic microsomal cytochrome P-450 with
a substrate has been assumed to be an initial step for the
oxidation of drugs, and with certain drugs there is a simi¬
larity between the dissociation constant of this binding
and the Michaelis constant for the metabolism of the same
drugs (7, 51). Table 15 showed that the binding of hexo¬
barbital, a type I substrate, to hepatic microsomal cyto¬
chrome P-450 from animals pretreated with streptozotocin
and 6-aminonicotinamide was inhibited while the binding of
the hexobarbital to cytochrome P-450 from N-met’nylacetamide-
treated animals was not inhibited. With aniline, a type II
substrate, however, binding was only inhibited in microsoir.es
from 6-aminonicotinamide-treated animals and was not
inhibited in those from streptozotocin-treated animals.
With N-methylacetamide, the binding of aniline to hepatic

75
cytochrome P-450 was actually enhanced. These results
indicate that the inhibition of hexobarbital metabolism in
animals pretreated with streptozotocin and 6-aminonicotin-
amide may be partially due to an interference in binding
of hexobarbital to the hepatic microsomal cytochrome P-450
from these animals. This cannot be the case with aniline,
however, inasmuch as inhibition in binding was seen only
in animals that were pretreated with 6-aminonicotinamide
and not in those treated with streptozotocin, even though
animals pretreated with streptozotocin demonstrated an
inhibition in their in vitro metabolism of aniline. Also,
the enhancement in binding of aniline to cytochrome P-450
from animals pretreated with N-methylacetamide is not
explainable because no enhancement of the metabolism of
aniline had been noted in preparations from animals pre¬
treated with this compound. It is apparent that the
strength of binding of substrate to hepatic cytochrome P-450
is not always an accurate measure of the degree of the
metabolism of the substrate. Indeed, it has become
increasingly obvious in recent years that the earlier
expectations that the spectral binding constants of drugs
with cytochrome P-450 are closely related to the kinetic
constants for microsomal metabolism have not been borne
out by further investigations (52).

As was mentioned in the Historical Review section,
intracellular levels of c-AMP in the liver are known to
be elevated in classical diabetes (21). Also, treatments
that increase endogenous levels of c-AMP in vivo are
inhibitory to certain pathways of drug metabolism; this is
thought to be due to the release of an inhibitor of drug
metabolism by liver microsomes into the cytosol (22, 25).
The presence of an inhibitor of hexobarbital hydroxylation
was shown in the present work to exist in preparations
from animals pretreated with streptozotocin and 6-amino-
nicotinamide, and an inhibitor only of hexobarbital
hydroxylation in those of animals pretreated with 6-amino-
nicotinamide. When hepatic c-AMP concentrations were
determined in animals pretreated with the diabetogenic
agents, it was found that the levels in streptozotocin-
pretreated animals were nearly twice those of control ani¬
mals, while those in 6-aminonicotinamide-pretreated animals
were increased to a lesser extent. The levels of c-AMP in
liver preparations from N-methylacetamide-pretreated ani¬
mals were not significantly different from those of control
animals. These results correlate with the fact that prep¬
arations from streptozotocin-pretreated animals always
showed a greater inhibition of both hexobarbital and aniline
hydroxylations than did those from animals treated with
6-aminonicotinamide. The fact that no increase in hepatic
c-AMP content was observed in preparations from N-methyl-
acetamide-pretreated animals, which never showed an

77
inhibition of drug metabolism, further substantiates the
finding that levels of hepatic c-AMP higher than normal
are inhibitory to certain drug-metabolic pathways.

SUMMARY
Until now, all the work done in studying the effects
of experimental diabetes on the microsomal drug-metabo¬
lizing system of the liver has involved the induction of
diabetes with alloxan, a toxic chemical that destroys the
beta-cells of the pancreas. The purpose of this investi¬
gation was to study the effect of experimental diabetes on
drug metabolism with newer, less toxic diabetogenic agents
and to elucidate a mechanism for these effects. Two of
the diabetogenic agents, streptozotocin and 6-aminonico-
tinamide, produce an insulin-deficient animal while the
third agent, N-methylacetamide, does not produce an insulin
deficient animal but instead causes a condition whereby
insulin is produced and released but is incapable of
lowering blood glucose.
To investigate the effect that the diabetes induced by
these agents had on hepatic drug metabolism, experiments on
male Holtzman rats were performed both in vivo and in vitro
In the in vivo experiments, the duration of hypnosis was
measured after the administration of hexobarbital sodium
to animals that had been made diabetic. The results showed
that there was an inhibition, as compared to control ani¬
mals, of the in vivo metabolism of hexobarbital only in
animals pretreated with streptozotocin and 6-aminonico-
78

79
tinamide and not in those pretreated with N-methylacetamide.
The in vitro experiments were of several types. After the
animals had been treated with these three agents, the
kinetic constants for hexobarbital and aniline hydroxyl-
ations were determined with 9000g supernatant fraction as
the enzyme source. Again, only preparations from animals
pretreated with streptozotocin and 6-aminonicotinamide
showed an inhibition as compared to control animals. Simi¬
lar results were also obtained when isolated microsomes
were used as the enzyme source. Also, the in vitro meta¬
bolism of hexobarbital and aniline was still inhibited as
compared to preparations from control animals one week after
the animals had been treated with streptozotocin. To
examine the effect of hyperglycemia on hepatic drug meta¬
bolism, animals were infused intravenously with glucose,
after which the in vitro hydroxylation of hexobarbital was
measured and found not to be inhibited as compared to
saline-infused control animals.
Neither insulin nor any of the diabetogenic agents had
any direct effect on drug metabolism in vitro. Furthermore,
hepatic microsomal protein and cytochrome P-450 contents
were not significantly different in any of the diabetic
animals than those of the control animals.
The binding of both hexobarbital and aniline to cyto¬
chrome P-450 was inhibited in animals pretreated with
streptozotocin but only the binding of hexobarbital was
inhibited in 6-aminonicotinamide-pretreated animals. The

80
fact that the affinity of aniline to cytochrome P-450
was increased in microsomes from animals pretreated with
N-raetnylacetamide somewhat obscures the interpretation of
these results.
It had been shown previously that treatments that
increased the endogenous levels of c-AMP in the liver
were inhibitory to certain drug-metabolic pathways and
that this was due to the release of an inhibitor of drug
metabolism into the liver cytosol. The presence of an
inhibitor of hexobarbital and aniline hydroxylations
was shown to occur in animals pretreated with streptozoto-
cin and an inhibitor only of hexobarbital hydroxylation in
animals pretreated with 6-aminonicotinamide. This cor¬
related with elevated levels of c-AMP observed in livers
of animals pretreated with streptozotocin and to a lesser
degree in animals pretreated with 6-aminonicotinamide. C-
AMP levels were not elevated in animals pretreated with
N-methylacetamide as compared to control animals.
In conclusion, it appears that the inhibition of drug
metabolism in animals made diabetic with certain drugs is
not caused by hyperglycemia, but is rather the consequence
of insulin lack. It is proposed that this lack of insulin
causes an elevation of hepatic c-AMP which, in turn, causes
the release by liver microsomes of an inhibitor of the meta¬
bolism of xenobiotics.

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BIOGRAPHICAL SKETCH
Dennis M. Ackerman was born in Baltimore, Maryland,
March 30, 1946. He attended Baltimore City College High
School and was graduated from there in June, 1963. In
February, 1969, he received the Bachelor of Science Degree
in Business Administration from the University of Maryland,-
and in June, 1970, he received the Bachelor of Science De¬
gree in Pharmacy from the same university.
Mr. Ackerman began his graduate education at the
University of Florida in September, 1970. He was sup¬
ported in his studies by a training grant from the Na¬
tional Institutes of Health.
85

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
Owen M. Rennert
Professor of Pediatrics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
íyi.
David M. Travis
Professor of Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality
as a dissertation for the degree of Doctor of Philosophy.
P V)o
Betty P. vogh
Associate Professor of Pharmacology

This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council,
and was accepted as partial fulfillment of the require¬
ments for the degree of Doctor of Philosophy.
December, 1975
Dean, Graduate School

UNIVERSITY OF FLORIDA
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