0090-9556/99/2706-0695–700$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 6
Printed in U.S.A.
EFFECT OF LEPTIN ON CYTOCHROME P-450, CONJUGATION, AND ANTIOXIDANT
ENZYMES IN THE OB/OB MOUSE
ANGELA M. WATSON, SAMUEL M. POLOYAC, GEORGETTE HOWARD,
AND
ROBERT A. BLOUIN
Graduate Center for Toxicology (R.A.B.), and College of Pharmacy, Division of Pharmaceutical Sciences (A.M.W., S.M.P., G.H., R.A.B.),
University of Kentucky, Lexington, Kentucky
(Received October 30, 1998; accepted March 12, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
however, leptin exposure significantly increased ethoxyresorufin
activity in lean mice (14%) and decreased the activity in ob/ob mice
(36%). UDP-glucuronosyl-transferase and glutathione S-transferase activities were not altered. The antioxidant enzymes, catalase (11%) and glutathione peroxidase (26%), as well as glutathione reductase (17%), were lower in the ob/ob mice and leptin
treatment corrected these alterations. The results of this study
demonstrate alterations in constitutive expression of CYP2B,
CYP2E, CYP2A, catalase, glutathione peroxidase, and glutathione
reductase in ob/ob mice that were restored to lean control values
following leptin treatment. Additionally, CYP3A activity was increased following leptin treatment in ob/ob mice. The mechanism
for the observed alterations may be due to direct leptin effects or
via indirect alterations in insulin, corticosterone, and/or growth
hormone.
Leptin, a 167-amino acid peptide, is a newly discovered hormone
that regulates food intake and energy balance (Zhang et al., 1994). The
leptin mutation in the ob/ob mouse is characterized by obesity, hyperglycemia, hyperphagia, hyperinsulinemia, and infertility, which
can be corrected by leptin replacement (Pelleymounter et al., 1995).
However, in the db/db mouse and the obese Zucker rat (fa/fa), there
exists a leptin receptor mutation (Campfield et al., 1996). Although
most human obesity is not related to leptin deficiency but to leptin
resistance (Considine, 1996), the mechanisms by which leptin signals
changes in these metabolic parameters in the ob/ob mouse may lend
insight into these alterations with human obesity.
Leptin is mainly expressed in the adipose tissue with receptors
located in the central nervous system and in peripheral tissues (Chen
et al., 1996). The leptin receptor is a class I cytokine receptor, a family
that includes interleukin-2 receptor, the interferon receptor, and the
growth hormone (GH) receptor (Ihle, 1996). Leptin binding to recep-
tors in the hypothalamus leads to activation of Jak kinases, a class of
cytoplasmic tyrosine kinases and signal transducers and activators of
transcription (STAT)1, which results in the stimulation of transcription of responsive target genes.
The drug metabolizing and antioxidant enzyme system plays a
major role in the detoxification and activation of many xenobiotics
and reactive oxygen species. Therefore, changes in these hepatic
enzymes associated with obesity is of great interest. A study by
Zannikos et al. (1994) showed no alterations in cytochrome P-450
(CYP) 3A and CYP2C11 activities in the overfed rat, whereas Bandyapadhyay et al. (1993) showed significant alterations in CYP2C11
expression in the obese Zucker rat. Roe et al. (1999), Barnett et al.
(1992a), and Stengard et al. (1987) showed no significant changes in
CYP1A, CYP2B, CYP2E, and CYP3A isoforms in the ob/ob mouse
when compared with lean littermates. Additionally, Rouer and Leroux
(1980) showed a reduction in aniline hydroxylase activity (indicative
of CYP2E1 activity) in the ob/ob mouse, which is in direct contrast to
work by Raucy et al. (1990) in which enhanced catalytic activities
associated with CYP2E1 was seen in obese overfed rats.
In addition to the changes in hepatic CYP-mediated reactions in
obese rodent models, alterations in hepatic conjugative enzymes also
occur. Barnett et al. (1992a), Roe et al. (1999), and Prohaska et al.
This research was supported in part by the Kentucky Spinal Cord and Head
Injury Research Trust Grant BB9502 and also in part by National Institute of
Diabetes and Digestive and Kidney Diseases Grant 9785. A. W. was supported by
the Lyman T. Johnson Postdoctoral Fellowship. S. M. P. was supported by The
American Foundation for Pharmaceutical Education.
Send reprint requests to: Dr. Robert A. Blouin, College of Pharmacy, 907
Rose St., University of Kentucky, Lexington, KY 40536-0082. E-mail:
[email protected]
1
Abbreviations used are: STAT, signal transducers and activators of transcription; CYP, cytochrome P-450; GSHPx, glutathione peroxidase; GSHRed, glutathione reductase; GST, glutathione S-transferase; GH, growth hormone; PPAR,
peroxisome proliferator activated receptor.
695
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Leptin is a hormone that is secreted by adipocytes and regulates
body weight through its effect on satiety and energy metabolism.
The ob/ob mouse is deficient in this protein and is characterized by
obesity and other metabolic disorders. This study investigated the
alterations of several hepatic cytochrome P-450 (CYP), conjugation, and antioxidant enzymes in lean and ob/ob mice and the role
leptin plays in the modulation of these enzymes. Lean and ob/ob
male mice were injected with leptin (100 mg) or PBS for 15 days.
Liver microsomes from ob/ob mice, when compared with lean
controls, displayed significantly reduced chlorzoxazone 6-hydroxylation activity (27%); however, 7a- and 16a- testosterone
hydroxylation and pentoxyresorufin O-dealkylation activities were
significantly higher (47%, 22%, and 39%, respectively). Leptin administration corrected alterations seen with all P-450 activities.
Dealkylation of ethoxyresorufin and v-hydroxylation of lauric acid
activities from ob/ob and lean mice were not statistically different;
696
WATSON ET AL.
Materials and Methods
Chemicals and Instrumentation. All chemicals were purchased from
Sigma Chemical Co. (St. Louis, MO). All spectrophotometric analyses were
done using a Shimadzu UV2100U spectrophotometer, an EL340 Bio Kinetic
microplate reader or a Shimadzu HPLC.
Animals and Treatment. Twelve-week-old male ob/ob mice (57g) and
their lean littermates (32g; obtained from Jackson Laboratories, Bar Harbor,
ME) were acclimated to our laboratory conditions (12 h light/dark cycle) for
several days before experiments. The animals had free access to laboratory
rodent chow and tap water. Lean and ob/ob mice were injected with 100 mg
leptin i.p. (Amgen Biologicals, Thousand Oaks, CA) in PBS for 15 days.
Control mice (lean and ob/ob) received an equal volume of PBS. There were
nine mice for each treatment group. Initial blood glucose levels were determined from blood obtained from tail sticks. Ending glucose, insulin, and
corticosterone levels were determined from trunk blood. Liver tissue was
removed, perfused with ice-cold 0.9% saline and immediately frozen in liquid
nitrogen. All procedures were previously approved by the University of Kentucky Institutional Animal Care and Use Committee.
Blood Glucose Levels. Blood glucose levels were determined at the beginning and the end of the experiment using an AccuCheck blood glucose monitor
and Chemstrip BG test strips (Boehringer Mannheim, Indianapolis, IN).
Serum Insulin and Corticosterone Levels. Serum insulin levels were
determined using a rat insulin radioimmunoassay kit (Linco Research, St.
Charles, MO), which is cross-reactive with mouse insulin (100%). Corticosterone levels were determined using an Immuchem Double Antibody corticosterone radioimmunoassay kit (ICN Biomedicals, Costa Mesa, CA) for mice.
Microsomal Preparation. Livers were excised and perfused with cold
0.9% saline. Liver samples were homogenized with 154 mM KCl/250 mM
potassium phosphate buffer, pH 7.4 with the addition of butylated hydroxy
toluene as an antioxidant before homogenization. Livers were homogenized
using a Teflon grinder and spun to separate the microsomal and cytosolic
fractions. The resulting pellet was resuspended in 0.25 M sucrose/0.02 M Tris
buffer, pH 7.4. The cytosolic fraction and the resuspended microsomal pellet
was stored at 280°C until use.
Protein Concentration. Total protein concentration in mouse liver microsomes and cytosol were determined by the method of Lowry et al. (1951) using
BSA as the standard.
Spectral Analysis of Total CYP. Microsomal fractions were used in the
determination of total CYP levels. The concentration of CYP in these fractions
were determined spectrophotometrically by the method of Omura and Sato
(1964) and based on an extinction coefficient of 91 mM21 cm21.
Analysis of CYP Activity. The formation of monohydroxylated products
from testosterone was determined according to Sonderfan et al. (1987).
CYP2E1 activity was determined using the 6-hydroxylation of chlorzoxazone
according to the method of Peter et al. (1990). CYP1A and CYP2B activities
were determined using the dealkylation of ethoxyresorufin and pentoxyresorufin, respectively, to resorufin according to Burke et al. (1985). CYP4A activity
was determined using the v-oxidation of lauric acid according to Giera and van
Lier (1991).
Analysis of Conjugation and Antioxidant Enzymes. GST activity was
determined in liver cytosol by the method of Habig et al. (1974) using
chloro-2,4-dinitrobenzene in ethanol as the substrate. UDP-glucuronosyltransferase activity was determined in liver microsomes by the method of Bock et
al. (1979) using p-nitrophenol as the substrate. Total glutathione content was
determined according to the enzymatic recycling method of Tietze (1969)
using 59,59-dithiobis (2-nitrobenzoic acid) as the substrate on a microtiter plate.
GSHRed activity was determined in liver cytosol according to the method of
Carlberg and Mannervik (1975). GSHPx activity was assayed by the method
of Lawrence and Burk (1976) using hydrogen peroxide as the substrate.
Catalase activity was determined according to the method of Cohen et al.
(1970) using potassium permanganate as the substrate.
The following denote the enzymes evaluated in this study followed by the
substrates used to determine their activity. CYP2E1, chlorzoxazone 6-hydroxylation; CYP4A, lauric acid v-hydroxylation; CYP1A, ethoxyresorufin
dealkylation; CYP2B, pentoxyresorufin dealkylation; CYP2A, testosterone
7a-hydroxylation; CYP3A, testosterone 6b-hydroxylation; GST, chloro-2,4nitrobenzene; UDPGT, p-nitrophenol; total glutathione, 59,59-dithiobis(2nitrobenzoic acid); GSHPx, hydrogen peroxide; and catalase, potassium permaganate.
Statistical Analysis. To establish statistical differences between control and
leptin treated animals, a two-way ANOVA was performed. The Fischer leastsignificant difference (LSD) post hoc analysis was used to determine statistical
differences for each time point. Percentages are reported as the change due to
phenotype or to leptin treatment. For phenotype comparisons this percentage is
calculated as the observed change divided by the lean untreated values 3 100.
For leptin treatment comparisons the percentage is calculated as the observed
change divided by the untreated values 3 100.
Results
Effects of Leptin Administration on Body Weight, Blood Glucose, Serum Insulin, and Corticosterone Levels. To verify the effect
of leptin in the ob/ob mouse model, several parameters were monitored (Table 1). PBS-treated animals did not show any significant
weight changes; however, leptin administration caused weight reduction in both the ob/ob mice and their lean littermates. PBS treatment
caused no significant changes in glucose levels in the control animals.
Before leptin treatment, the ob/ob mice were not hyperglycemic
(.300 mg glucose/dl); however leptin administration caused a statistically significant decrease in this level. Leptin did not alter glucose
levels in the lean mice. Circulating insulin was about 5-fold higher in
ob/ob mice compared with their lean littermates; however, following
leptin administration, a decrease in insulin levels was seen in ob/ob
mice. Corticosterone levels were about 6-fold higher in ob/ob mice.
Leptin administration had similar effects on corticosterone levels in
both the lean and ob/ob mice, lowering corticosterone levels 2-fold.
Effects of Leptin Administration on Total Hepatic P-450 levels
and Microsomal CYP Activities. Total P-450 levels were statistically lower (22%) in ob/ob mice when compared with lean mice;
however, leptin administration had no significant effect on total P-450
in hepatic microsomes from lean or ob/ob mice (data not shown). The
dealkylation of ethoxyresorufin and pentoxyresorufin were used as
markers reflective of CYP1A and CYP2B activities, respectively. No
significant change was seen in CYP1A activity between lean and
ob/ob mice (Fig. 1A). There was a significant increase in this activity
(14%) following leptin administration in lean mice; however, the
ob/ob mice showed a significant decrease (36%) following leptin
exposure. ob/ob mice displayed significantly greater CYP2B activity
(39%) than the lean mice (Fig. 1B). Leptin treatment caused a significant decrease in this activity (63%) in ob/ob mice but no change in
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(1988) showed a 75%, 65%, and 55% decrease, respectively, in
glutathione S-transferase (GST) activity in the ob/ob mouse. However, Chaudhary et al. (1993), using obese Zucker rats, showed
significant increases in UDP-glucuronosyltransferase activity, which
is consistent with other dietary rodent models of obesity (Corcoran
and Wang, 1987).
Changes in the antioxidant defense mechanisms have been seen in
the obese rodent model. A study by Capel and Dorrell (1984) indicated lower levels of glutathione peroxidase (GSHPx) activities in
ob/ob mice when compared with their lean littermates, as well as
lower glutathione reductase (GSHRed) activity and total GSH levels.
Additionally, Prohaska et al. (1988) showed that hepatic GSHPx
activity of ob/ob mice was 70% of that in control lean mice and
copper-zinc superoxide dismutase activity was 30% lower in obese
mice.
Many of the drug metabolizing enzymes are altered in the obese
rodent model; however, whether leptin plays a role in the regulation
of these enzymes is unclear. Therefore, the objective of this study was
to investigate whether leptin could alter or correct changes in the
expression of several drug metabolizing and antioxidant enzymes in
ob/ob mice.
697
LEPTIN9S EFFECT ON PHASE I, PHASE II, AND ANTIOXIDANT ENZYMES
TABLE 1
Effect of leptin treatment on weight, blood glucose, insulin, and corticosterone in obese (ob/ob) and lean (Ob/?) mice
Data are expressed as means of nine replications for each treatment. Numbers in parentheses represent S.E.M. For weight and glucose levels, similar lowercase letters represent means
within a treatment that were not statistically different by LSD (P # .05). For insulin and corticosterone levels, similar lowercase letters represent means within a parameter that were not
statistically different by LSD (P # .05). Mice were i.p. injected with PBS (100/ml/mouse) or leptin (100/mg/mouse) for 15 days before euthanasia. LC and OC represent lean and ob/ob mice
injected with PBS, and LL and OL represent lean and ob/ob mice injected with leptin.
LC
Weight (g)
Initial
Ending
Change
Glucose (mg/dl)
Initial
Ending
Change
Insulin (ng/ml)
Corticosterone (ng/ml)
LL
31.3 (1.9)a
31.0 (2.2)a
20.33a
30.2 (2.3)a
28.8 (2.7)b
21.36b
131.2
151.3
20.1a
1.57
47.3
142.6 (25.6)
149.0 (20.4)
6.4a,b
2.58 (1.20)a,b
22.2 (4.53)a
(13.2)
(30.6)
(0.58)a
(11.9)a
Data are expressed as means 6 S.E. of nine replications for each treatment. A
and B, CYP1A1 and CYP2B1 activities are expressed as picomoles per minute per
milligram protein. C and D, CYP2E1 and CYP4A activities are expressed as
nanomoles per minute per milligram protein. Bars followed by a different letter are
significantly different by LSD (P # .05). Mice were i.p. injected with PBS (100
ml/mouse) or leptin (100 mg/mouse) for 15 days before euthanasia. LC and OC
represent lean and ob/ob mice injected with PBS and LL and OL represent lean and
ob/ob mice injected with leptin
the activity from the lean mice. The hydroxylation of chlorzoxazone
was used as a marker for CYP2E1 activity. ob/ob mice displayed
significantly lower CYP2E1 activity (27%; Fig. 1C). Following leptin
exposure, no significant effect was seen with this activity from lean
mice but the ob/ob mice displayed a significant increase in activity.
The v-hydroxylation of lauric acid was used as a marker for the
57.4 (2.6)b
58.9 (3.6)c
1.5c
181.5 (80.2)
173.4 (90.7)
28.1a,b
7.88 (2.68)b
278.1 (53.52)b
OL
56.4 (2.6)b
42.0 (2.3)d
214.4d
192.0 (86.9)
102.0 (8.76)
290b
5.95 (2.51)a,b
113.5 (35.82)a
activity of CYP4A. Although this activity was greater in the ob/ob
mice when compared with their lean littermates, no statistically significant differences were seen (Fig. 1D). Leptin exposure caused a
significant increase in the activity in the lean mice but did not increase
CYP4A activity further in the ob/ob mice.
Effects of Leptin Administration on Hydroxylation of Testosterone in Liver Microsomes. Several CYP activities can be monitored by the hydroxylation of testosterone. Higher activities of 7aand 16a-hydroxylation of testosterone were seen in the ob/ob mice
when compared with the lean mice (48% and 22%, respectively);
however, although higher, no statistically significant changes were
seen with the 6b-hydroxylation product (Table 2). Conversely, a
significantly lower activity was seen with the 6a-hydroxylation product (54%) in ob/ob mice when compared with lean controls. Leptin
exposure caused no significant changes with any of the hydroxylation
products in the lean mice. However, statistically significant increases
were seen with the 6a- and 6b-hydroxylation of testosterone (33%
and 16%, respectively) following leptin exposure with the ob/ob mice.
A statistically significant decrease was seen with the 7a-hydroxylation product (37%) following leptin treatment in ob/ob mice but no
change was seen in 16a-hydroxylation product.
Effects of Leptin Administration on Cytosolic GST Activity and
Microsomal UDP Glucuronidation. Figure 2A shows the changes in
cytosolic GST activity following leptin exposure in lean and ob/ob
mice. No change was seen in GST activity between the lean and ob/ob
mice. Leptin exposure caused no change in GST activity with the lean
mice; however, there was a trend toward a decrease with the ob/ob mice.
Figure 2B shows the effect leptin has on microsomal UDP-glucuronidation activity. No differences were seen between control lean and ob/ob
mice and leptin treatment had no affect.
Effects of Leptin Administration on Antioxidant Enzyme Activities. Figure 3 shows the effect leptin had on several antioxidant
enzymes and GSHRed. Catalase activity from ob/ob mice was statistically lower than activity (11%) from lean mice (Fig. 3A). Leptin
exposure had no effect on this activity in lean mice but there was a
specific correction (to lean control levels) in this activity (10%) in
ob/ob mice. Leptin can alter the observed defect in GSHPx activity in
ob/ob mice (Fig. 3B). GSHPx activity from ob/ob mice was statistically lower (26%) than activity from lean mice. A significant increase
in this activity in ob/ob mice (to control levels) was seen following
leptin exposure but no change was seen with the lean mice. GSHRed
activities from ob/ob mice was statistically lower (17%) than activity
from lean mice (Fig. 3C). In contrast to GSHPx, leptin exposure
caused a significant increase in GSHRed activity in both lean and
ob/ob mice. Figure 3D represents the changes in total glutathione
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FIG. 1. Activities of CYP1A1 (A), CYP2B1 (B), CYP2E1 (C), and CYP4A (D) in
liver microsomes from lean and ob/ob mice following leptin treatment.
OC
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WATSON ET AL.
TABLE 2
Effect of leptin on hydroxylation of testosterone in liver microsomes in obese (ob/ob) and lean (Ob/?) mice
Data are expressed as means of nine replications for each treatment. Activities are expressed as pmoles per minute per milligram protein. Numbers in parentheses represent S.E.M. Similar
lowercase letters represent means within an activity that are not statistically different by LSD ( p # .05). Mice were i.p. injected with PBS (100/ml/mouse) or leptin (100/mg/mouse) for 15 days
before euthanasia. LC and OC represent lean and ob/ob mice injected with PBS and LL and OL represent lean and ob/ob mice injected with leptin.
Hydroxylation
6a
6b
7a
16a
LC
LL
a
282.33 (11.34)
790.12 (31.66)a
419.90 (11.0)a
217.37 (9.91)a
OC
a
312.15 (18.63)
832.10 (71.64)a
411.80 (28.28)a
256.74 (24.44)a,b
OL
b
129.46 (8.12)
929.08 (51.26)a
799.15 (36.61)b
278.19 (11.51)b
193.20 (12.25)c
1105.87 (58.36b
501.58 (16.46)c
237.21 (9.83)a,b
Data are expressed as means 6 S.E. of nine replications for each treatment.
Activities are expressed as nanomoles per minute per milligram protein. Bars
followed by a different letter are significantly different by LSD (P # .05). Mice
were i.p. injected with PBS (100 ml/mouse) or leptin (100 mg/mouse) for 15 days
before euthanasia. LC and OC represent lean and ob/ob mice injected with PBS and
LL and OL represent lean and ob/ob mice injected with leptin.
FIG. 3. Catalase (A), GSHPx (B) and GSHRed (C), and total glutathione levels
(D) from lean and ob/ob mice following leptin treatment.
Discussion
Data are expressed as means 6 S.E. of nine replications for each treatment. A–C,
catalase, GSHPx, and GSHRed activities are expressed as nanomoles per minute
milligram protein. D, glutathione activity is expressed as nanomoles per milligram
protein. Bars followed by a different letter are significantly different by LSD (P #
.05). Mice were i.p. injected with PBS (100 ml/mouse) or leptin (100 mg/mouse) for
15 days before euthanasia. LC and OC represent lean and ob/ob mice injected with
PBS and LL and OL represent lean and ob/ob mice injected with leptin.
The metabolism of many xenobiotics by phases I and II enzymes
and antioxidant enzymes depends on the nature of these enzymes at
the time of exposure. Therefore, any factor, such as obesity, can
modulate the activities of these enzymes that could ultimately affect
the rate of xenobiotic metabolism. This study demonstrated that the
activity of several drug metabolizing and antioxidant enzymes are
altered in the ob/ob mouse and that leptin replacement effectively
corrects some of these alterations. Specifically, leptin treatment produced alterations in CYP2B, CYP2E, CYP2A, CYP3A, catalase,
GSHPx, and GSHRed. Leptin treatment produces alterations in several endogenous factors including insulin, corticosterone, and GH.
Any of these factors may alter the regulation of the affected enzymes.
The remainder of this discussion will focus on these results in relation
to known regulators of expression for each of the aforementioned
enzymes.
Several CYP-related activities were monitored in the ob/ob mouse
and their lean littermates using substrates for specific CYP isoforms.
Presently, as a result of a lack of specific antibodies and inhibitors for
mouse P-450, it is not known whether the activities for the individual
isoforms are the same as found in the rat model. However, activities
in mice will be assigned the same P-450 designations as the rat model.
levels in control lean and ob/ob mice following leptin exposure, where
there were no significant changes between control lean and ob/ob
mice.
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FIG. 2. GST (A) and UDP-glucuronosyltransferase (B) activities from lean and
ob/ob mice following leptin treatment.
LEPTIN9S EFFECT ON PHASE I, PHASE II, AND ANTIOXIDANT ENZYMES
lation of CYP3A transcription by a STAT factor, which is not consistent with our results. In our study, the ob/ob mice, characterized by
lower GH levels, exhibited a trend toward greater 6b-testosterone
hydroxylase activity (reflective of CYP3A activity). Irizar et al.
(1995) and Barnett et al. (1992a) showed greater CYP3A activity in
obese Zucker rats and ob/ob mice, respectively. ob/ob mice, following
leptin treatment, showed an increase in CYP3A activity (6b-testosterone hydroxylation), which may be a result of increased GH levels
in treated ob/ob mice. Increased GH levels may have caused activation of the STAT protein necessary for transcription of CYP3A
protein, resulting in higher activity. The different patterns of activity
for these isoforms suggest that they may be regulated by different
STAT proteins.
CYP4A activity was not altered significantly by phenotype but was
changed in the lean mice following leptin exposure but not in the
obese animals. The induction of CYP4A expression has been shown
to be a result of transcriptional activation, mediated possibly by a
peroxisome proliferator activated receptor (PPAR; Simpson, 1997).
The ob/ob mouse has elevated fatty acid levels (Enser and Ashwell,
1983), which has been suggested to be an activator of PPAR. Studies
by Robertson et al. (1999) showed that CYP4A10 mRNA was expressed at higher levels and CYP4A1 was lower in the ob/ob mice.
They also showed that in obese Zucker rats, CYP4A1 was unchanged,
CYP4A2 was elevated, and CYP4A3 was decreased when compared
with controls. In our study, the trend toward higher levels of CYP4A
activity in the ob/ob mouse may be a result of increased transcription
of this protein via fatty acids. Leptin treatment has been shown to
decrease triglyceride content in pancreatic islet from Wistar rats in the
presence of free fatty acids (Shimabukuro et al., 1997), which suggests a decrease in transcription rate of CYP4A. However, in our
study, there was an increase in CYP4A activity in the lean mice
following leptin exposure and no change in the ob/ob mice. This
suggests that, in addition to the activation of PPAR, other factors may
influence the regulation of the CYP4A gene.
With obesity comes changes in the CYP-mediated reactions as well
as conjugative enzymes such as GST that detoxify reactive substances
produced by CYPs. In our study, GST activity in ob/ob mice was not
significantly different when compared with lean controls. This is in
contrast to studies by Barnett et al. (1992a) who demonstrated a 75%
decrease in GST activity. This discordance may be explained by the
lower glucose levels in our study compared with those obtained by
Barnett and coworkers, who implicated hyperglycemia in the downregulation of GST.
Changes in the antioxidant defense mechanisms, which detoxify
reactive oxygen species, were also evaluated in the ob/ob mice. In our
study, similar decreases were seen in both GSHPx and catalase
activities in the ob/ob mice when compared with the lean mice. These
results are consistent with results reported by Capel and Dorrell
(1984). They suggest in their study that the decrease in GSHPx
activity in ob/ob mice may be caused by a deficiency in selenium,
which is needed for GSHPx when using H2O2 as the substrate. It has
also been suggested that increased b-oxidation in the peroxisomes
causes elevated peroxides (Murphy et al., 1979), which could also
result in decreases in these antioxidant enzymes. Some researchers
suggest that decreases in these antioxidant enzymes may be attributed
to an increase in CYP-mediated reactions, namely CYP2E1 (Lieber,
1997). However, in our study CYP2E1 activity was reduced in the
ob/ob mouse and therefore, may not have contributed substantially to
the observed decrease in these enzymes. It has been shown that
hyperinsulinemia also may play a role in intracellular H2O2 generation (Mukherjee and Lynn, 1977). It is possible that the increased
level of insulin in the ob/ob mouse may have contributed to the
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Many CYPs are influenced by a variety of factors, including inducers
and endogenous substances such as insulin, GH, testosterone, and
triglycerides. It is well known that ob/ob mice have higher levels of
insulin (Harris et al., 1998) when compared with their lean littermates
and insulin has been shown to differentially regulate CYP1A1/2 and
CYP2E1 isoforms. Barnett et al. (1992b) suggest that hyperinsulinemia
causes an increase in CYP1A activity in male Wistar albino rats. In our
study, CYP1A activity was not influenced by phenotype; however, leptin
treatment, lowering insulin levels in ob/ob mice, caused a decrease in
CYP1A activity. These results suggest that insulin, in addition to other
factors, may influence the activity of this isoform.
In addition to insulin’s effects on CYP1A, it has also been shown
to affect CYP2E1. A study by De Waizer et al. (1995) showed
down-regulation of CYP2E1 expression by insulin at the post-transcriptional level in a rat hepatoma cell line. Additionally, a study by
Woodcroft and Novak (1997) also showed decreased expression of
CYP2E1 in primary rat hepatocytes following insulin treatment. A
study by Roe et al. (1999) showed increased CYP2E1 activity in
diabetic ob/ob mice, which is in contrast to results seen in this study
in which the ob/ob mice were not diabetic. These results support
insulin’s role in the decreased expression of the CYP2E1 isoform
because insulin levels were elevated in ob/ob mice. Additionally, it is
known that the hyperglycemic state (Yamazoe et al., 1989), highenergy diets (Raucy et al., 1990) in obese rodents, and human obesity
(O’Shea et al., 1994) cause an increase in the expression of the
CYP2E1 isoform. This has been attributed to the presence of increased levels of ketone bodies secondary to the hyperglycemic state.
Although the levels of ketone bodies were not measured, it is assumed
that the ob/ob mice were not ketotic because they were not hyperglycemic; therefore, the ketotic state of the mice may not have played as
great a role in altering CYP2E1 activity. Leptin administration decreased insulin levels in the ob/ob mice, which resulted in an expected
increase in CYP2E1 activity. These results suggest that insulin and not
the ketotic state of the ob/ob mice played a greater role in the observed
CYP2E1 alterations in this study.
7a-Hydroxylation of testosterone (reflective of CYP2A activity),
was elevated 2-fold in the ob/ob mouse, which is in agreement with
the work of Thummel and Schenkman (1990) in streptozotocininduced diabetic rats. In Thummel’s study, GH administration did not
affect CYP2A activity but testosterone replacement reversed the
changes seen in this protein. In our study, leptin administration
corrected the 2-fold increase in 7a-hydroxylation of testosterone in
ob/ob mice. It is possible that leptin caused an increase in the secretion
of testosterone in the sterile ob/ob male mouse, which could have
affected the expression of CYP2A activity.
The ob/ob mouse model is characterized by a reduced level of GH
and this reduction in GH has been implicated in the changes seen with
several of the CYP isoforms. Waxman et al. (1995) showed that the
CYP genes CYP2C11 and CYP2C12 are dependent on GH and that
GH can activate several STAT proteins including 1, 3, and 5 (mainly
5) in the rat liver (Ram et al., 1996). These STAT-dependent signaling
pathways can target distinct genes and contribute to the diverse effect
of GH. GH has been shown to cause alterations in both the CYP2B and
CYP3A isoforms. In our study, there was a 2-fold greater activity of
CYP2B in ob/ob mice as evidenced by the dealkylation of pentoxyresorufin. Yamazoe et al. (1989) suggest that lower GH levels may trigger the
synthesis of CYP2 family of proteins, which is supportive of the results
seen in our study with ob/ob mice. However, following leptin administration, we observed a substantial decrease in pentoxyresorufin activity to
below control level, indicating a reduction in CYP2B expression, possibly via increased GH levels (Carro et al., 1997).
Subramanian et al. (1998) suggest that GH mediates the up-regu-
699
700
WATSON ET AL.
Acknowledgments. We are grateful to Amgen Inc. for supplying
Leptin and Kelly Babb for technical assistance.
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increased production of H2O2 and therefore, a reduction in these
antioxidant enzyme activities. Leptin administration caused both catalase and GSHPx activities in ob/ob mice to normalize (lean control
levels) but had no affect on activities in lean mice. Leptin administration causes a decrease in body weight by an increase in fat oxidation resulting in the loss of body fat (Pelleymounter et al., 1995). This
loss in body fat could potentially reverse the above-mentioned reactions occurring in the ob/ob mice, causing these activities to return to
normal. The normalization of insulin levels by leptin may have also
played a role in returning the antioxidant enzymes to normal levels.
Total GSH levels and GSHRed activity were monitored in lean and
ob/ob mice following leptin exposure. Total GSH levels were not
significantly altered with phenotype or with leptin administration;
however, the GSHRed activities were affected by both parameters.
The lower level of GSHRed activity found in the ob/ob mouse in our
study is supported by Capel and Dorrell (1984). It is possible that
reduced GSHRed reflects lower levels of reduced GSH, which are
needed for protection against reactive oxygen compounds.
In conclusion, the present study demonstrated that obesity does play
a role in altering some of the CYP-mediated reactions as well as
antioxidant enzymes in the ob/ob mouse that can be corrected by
leptin replacement. However, it is not known whether these changes
occur as a result of leptin’s effect on weight reduction, hormonal
changes, changes in the amount of nutrients as a result of decreased
food intake, or by activation of secondary messenger systems and
transcription factors. Therefore, further in vitro studies are necessary
to determine whether leptin treatment alterations occur as a result of
a direct second messenger event or whether the alterations are due to
indirect changes in insulin, cortisol, and/or GH