Am J Physiol Endocrinol Metab 289: E1015–E1022, 2005.
First published July 19, 2005; doi:10.1152/ajpendo.00169.2005.
Topiramate treatment causes skeletal muscle insulin sensitization
and increased Acrp30 secretion in high-fat-fed male Wistar rats
Jason J. Wilkes, M. T. Audrey Nguyen, Gautam K. Bandyopadhyay,
Elizabeth Nelson, and Jerrold M. Olefsky
Division of Endocrinology and Metabolism, Department of Medicine, University of
California, San Diego; and The Whittier Diabetes Institute, La Jolla, California
Submitted 18 April 2005; accepted in final form 18 July 2005
high fat; insulin sensitization; adenosine monophosphate-activated
protein kinase; adiponectin
showed that Topiramate caused insulin sensitization when
added to fat tissue, but not muscle.
Adiponectin (Acrp30) is synthesized and secreted exclusively by adipocytes (16), and many studies have shown that
high levels of adiponectin are associated with insulin sensitivity whereas low levels are found in insulin resistance (8, 9, 17).
Furthermore, direct experiments have demonstrated that adiponectin can mediate insulin-sensitizing effects, most likely by
activating AMP-activated protein kinase (AMPK).
Adiponectin circulates in a low-molecular-weight (LMW)
hexameric form and a high-molecular-weight (HMW) form
consisting of trimer hexamers, and it is the HMW form of
circulating adiponectin that may contain most of the in vivo
insulin-sensitizing activity (10, 11, 21). This adipokine appears
to exert its biological effects by binding to its recently identified cell surface R1 and R2 receptors (24) followed by activating of AMPK (25). Recently, we have shown that adenoviral mediated ectopic transgenic expression of adiponectin
improves whole body insulin sensitivity in Wistar rats, completely protects these animals from high-fat diet (HFD)-induced insulin resistance, and leads to activation of AMPK
activity in skeletal muscle, but not liver.
On this basis, we speculated that Topiramate-induced insulin
sensitization may be mediated, at least in part, through adipocyte-derived adiponectin. The current studies in HFD Wistar
rats treated with Topiramate provide strong support for this
hypothesis.
MATERIALS AND METHODS
is a characteristic feature of type 2 diabetes
and is the defining abnormality of syndrome X, or the metabolic syndrome. Given the epidemic proportion of these disorders, development of new insulin-sensitizing therapeutics
could be of great importance. Topiramate is a clinically approved antiseizure compound, and clinical experience as well
as limited animal studies have suggested that this agent may
lead to decreased insulin resistance and modest weight loss. In
earlier studies (23), we showed that treatment of female Zucker
fatty (ZDF) rats with Topiramate led to improved insulin
sensitivity, independently of weight loss, and that the primary
drug effects were exerted on adipose tissue. Thus overall in
vivo insulin sensitivity was improved, but when tissues were
excised and studied ex vivo, adipose tissue insulin sensitization
was maintained, but skeletal muscle insulin action was no
longer enhanced. Consistent with this, direct in vitro studies
Materials. Anti-AMPK␣ and phospho-AMPK (Thr172) antibodies
were purchased from Cell Signaling Technology (Beverly, MA).
PVDF membrane was purchased from Millipore (Bedford, MA). HFD
was purchased from DyEts (Bethlehem, PA). General reagents were
purchased from Sigma Chemical (St. Louis, MO). Johnson & Johnson
(Raritan, NJ) kindly provided us with Topiramate (TPM).
General use of animals. Male Wistar rats (Charles River, Wilmington, MA) weighing between 350 and 375 g were used. To induce
obesity, we divided rats into two groups and provided one with chow
and the other an HFD. Animals on HFD were given a diet that
contained saturated fat as the primary source of calories (55% hydrogenated coconut oil, 15% casein, 15% sucrose, 4% soybean oil),
whereas rats in the low-fat group were given normal rodent chow with
⬍5% dietary fat. HFD rats were permitted to consume their diet freely
for 5– 6 wk. Thereafter, pair feeding was implemented to control for
the food restriction effects of TPM. Thus rats on HFD were matched
by body weight and allotted to pair-fed HFD ⫹ placebo, regular fed
Address for reprint requests and other correspondence: J. Wilkes, Dept. of
Medicine (0673), UCSD, 9500 Gilman Dr., La Jolla, CA 92093 (e-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INSULIN RESISTANCE
http://www.ajpendo.org
0193-1849/05 $8.00 Copyright © 2005 the American Physiological Society
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Wilkes, Jason J., M. T. Audrey Nguyen, Gautam K. Bandyopadhyay, Elizabeth Nelson, and Jerrold M. Olefsky. Topiramate
treatment causes skeletal muscle insulin sensitization and increased
Acrp30 secretion in high-fat-fed male Wistar rats. Am J Physiol
Endocrinol Metab 289: E1015–E1022, 2005. First published July 19,
2005; doi:10.1152/ajpendo.00169.2005.—We show that Topiramate
(TPM) treatment normalizes whole body insulin sensitivity in high-fat
diet (HFD)-fed male Wistar rats. Thus drug treatment markedly
lowered glucose and insulin levels during glucose tolerance tests and
caused increased insulin sensitization in adipose and muscle tissues as
assessed by euglycemic clamp studies. The insulin-stimulated glucose
disposal rate increased twofold (indicating enhanced muscle insulin
sensitivity), and suppression of circulating FFAs increased by 200 to
300%, consistent with increased adipose tissue insulin sensitivity.
There were no effects of TPM on hepatic insulin sensitivity in these
TPM-treated HFD-fed rats. In addition, TPM administration resulted
in a three- to fourfold increase in circulating levels of total and
high-molecular-weight (HMW) adiponectin (Acrp30). Western blot
analysis revealed normal AMPK (Thr172) phosphorylation in liver
with a twofold increased phospho-AMPK in skeletal muscle in TPMtreated rats. In conclusion, 1) TPM treatment prevents overall insulin
resistance in HFD male Wistar rats; 2) drug treatment improved
insulin sensitivity in skeletal muscle and adipose tissue associated
with enhanced AMPK phosphorylation; and 3) the tissue “specific”
effects are associated with increased serum levels of adiponectin,
particularly the HMW component.
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TOPIRAMATE IS INSULIN SENSITIZING IN HF-FED WISTAR RATS
Table 1. Food record of HFD rats on TPM treatment
or placebo
Food Intake, g/24 h
Time Before GTT, h
TPM Treated
Pair Fed
Regular Fed
64
40
13.4⫾0.8
13.8⫾0.7
13.4⫾0.8
13.8⫾0.7
14.3⫾0.6
16.8⫾1.8
Values are means ⫾ SE. Column 1 shows the times when 24-h food intake
data were collected; columns 2– 4 show amount of food (g) consumed over a
24-h period by Topiramamte (TPM)-treated, pair-fed (⫹placebo), and regularfed (⫹placebo) rats, respectively. Data show that all high-fat diet (HFD)-fed
rats tested by glucose tolerance test (GTT) had consumed an equal number of
grams of diet over the final 2–3 day period before testing (P ⬎ 0.1).
Fig. 1. Glucose tolerance is normalized in Topiramate (TPM)-treated high-fat-diet (HFD)-fed obese Wistar rats. A: HFD feeding increased body weights of
Wistar rats. Pair feeding normalized body weights of TPM- and placebo-treated rats. All experimental groups of rats underwent glucose tolerance testing (3 g/kg
ip) on day 16. B: body weights of HFD-fed rats were increased by day 16; *P ⬍ 0.05 vs. chow-fed rats. C and D: consumption of HFD impaired glucose tolerance,
and TPM treatment normalized glucose-challenged plasma glucose (C) and plasma insulin (D) concentrations in basal and postprandial states; ¶P ⬍ 0.05 vs.
placebo-treated controls. Bars represent means ⫾ SE (n ⫽ 8).
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HFD ⫹ TPM or untreated regular feeding HFD group. Pair-fed rats
consumed equal portions of food as TPM-treated rats daily. Single
daily doses of TPM (100 mg/kg) were given by oral gavage to
TPM-treated rats. Drug-treated (HFD regular fed ⫹ TPM) and placebo treated (HFD pair fed ⫹ placebo) and normal chow-fed animals
all underwent glucose tolerance testing on day 16 to generate data on
general aspects of insulin sensitivity (i.e., glucose and insulin levels)
of experimental groups. More detailed assessments of muscle and
liver insulin sensitivity [i.e., glucose disposal rate (GDR), hepatic
glucose production (HGP)] were made in fully recovered catheterized
rats that were pair fed and untreated or regular fed and treated with
and without TPM.
Glucose tolerance tests. Glucose tolerance tests (GTT) were performed in overnight-fasted rats on HFD and treated with or without
Topiramate, as well as in untreated chow-fed animals. First, unchallenged blood draws were performed for basal glucose and insulin
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TOPIRAMATE IS INSULIN SENSITIZING IN HF-FED WISTAR RATS
Fig. 2. Reversal of diet-induced whole body insulin resistance as determined
by glucose infusion rate (GINF). HFD decreased GINF measured in
mg 䡠 kg⫺1 䡠 min⫺1; *P ⬍ 0.05 vs. chow. TPM-treated rats on HFD demonstrated
reversal of whole body insulin resistance, as shown by reversal of GINF. The
GINF of rats on TPM treatment was less than those on placebo (¶P ⬍ 0.05) and
equal to those on chow. Bars represent means ⫾ SE (n ⫽ 6).
AMPK measurement in muscle and liver tissues. EDL and liver
samples underwent homogenization to generate tissue lysates for
phospho-AMPK Western blotting, as described in a previous paper
(22). Western blotting was performed using acrylamide-based gels.
Visualization of Western blots was made possible by an enhanced
chemiluminescence system (Pierce). Bands were quantified using a
Macintosh computer connected to an ARCUS scanner by way of NIH
Image 1.6 software.
Muscle triglyceride analysis. Lipids were extracted from muscle by
a method adapted from Frayn and Maycock (7). Total intramuscular
triglyceride (IMTG) content was determined in lipid extracts by use of
a kit from Thermo DMA (Arlington, TX).
Calculations and statistical analysis. HGP and GDR were calculated using Steele’s equation (18). All data were analyzed using
two-way analysis of variance and t-tests as appropriate. Data calculation and statistical analysis were performed using StatView (Abacus
Concepts, Berkley, CA). All data are reported as means ⫾ SE.
RESULTS
GTTs. TPM has a transient effect to suppress food intake in
rats, resulting in transient weight loss (23). Therefore, to study
rats at an equal body weight, we first induced obesity (i.e.,
before dosing) by HFD and then matched food intakes of TPMand placebo-treated groups by pair feeding. Fig. 1A shows that
the HFD caused rats to gain weight and that pair feeding
effectively normalized body weights of placebo- and TPMtreated rats. Table 1 shows that rats treated with TPM and those
that were pair fed consumed equal amounts of food over the
final 2–3 days of treatment. As seen in Fig. 1B, both groups of
rats on HFD (HFD ⫹ placebo and HFD ⫹ TPM) showed
Table 2. Metabolic characteristics of catheterized male Wistar rats on a regular chow diet or HFD and receiving a 100
mg/kg daily dose of TPM (HFD ⫹ TPM) or placebo (HFD ⫹ PL) for 7–9 days
Basal Period
Clamped Period
Group
Body Weight,
kg
Glucose,
mmol/l
Insulin, ng/ml
FFA, mmol/l
FFA,
%2
FFA, mmol/l
Glucose,
mmol/l
Lactate,
mmol/l
Insulin, ␮U/ml
Chow
HFD ⫹ PL
HFD ⫹ TPM
0.437⫾0.18
0.490⫾0.13*
0.485⫾0.17*
6.6⫾0.2
8.1⫾0.3*
8.4⫾0.4*
1.09⫾0.08
2.67⫾0.38*
1.96⫾0.37*
0.96⫾0.04
0.90⫾0.04
1.21⫾0.05
80
55
85
0.19⫾0.01
0.39⫾0.07*
0.18⫾0.02†
8.2⫾0.1
8.1⫾0.2
7.9⫾0.4
2.66⫾0.23
2.80⫾0.11
2.63⫾0.13
2,815⫾216
3,113⫾301
3,184⫾706
Values are means ⫾ SE. FFA, free fatty acids. *P ⬍ 0.05 vs. chow; †P ⬍ 0.05 vs. HFD ⫹ PL.
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measurements. This was achieved by nipping tails and collecting
blood from tail veins (⬃100 ␮l per rat) in heparinized capillary tubes.
Glucose concentrations were determined immediately using a portable
blood glucose analyzer (Hemocue, Mission Viejo, CA). Plasma was
spun out of remaining samples and stored at ⫺80°C. Plasma insulin
was determined at a later time. After basal sampling, glucose loads
were delivered (3 g/kg body wt ip) to hand-held conscious animals.
Thereafter, time points for glucose measurements were made at 60,
120, and 180 min and insulin at 60 and 180 min.
Surgery and clamp procedure. Rats were inserted with cannulae on
day 16 of the protocol in the same manner as we have described
previously (22). Euglycemic hyperinsulinemic clamp studies were
performed 7 to 9 days after cannulation in fasted rats, as before, but
with one minor technical modification. Instead of arterial blood
sampling, blood was collected via the tail vein to be consistent with
the sampling technique used in our GTT procedure. Basal blood
samples were drawn at ⫺60 and 0 min. A priming dose (5 ␮Ci) of
D-[3-3H]glucose tracer (New England Nuclear, Boston, MA) was
administered by bolus injection. Then (at ⫺60 min) tracer was
administered by constant infusion (0.167 ␮Ci/min) for 1 h to further
assist in the equilibration of tritium with the glucose pool. At 0 min,
a solution of cold glucose (50% dextrose; Abbott Labs, Chicago, IL)
was infused at a variable rate along with the infusion of tritium plus
insulin (25 mU 䡠 kg⫺1 䡠 min⫺1 Novlin R; Novo Nordisk, Copenhagen,
Denmark) which was infused at a fixed rate (16.7 ␮l/min). Variable
and fixed infusions of glucose and tritium plus insulin solutions were
started simultaneously and maintained throughout the duration of the
clamp. Glucose pumps were adjusted as needed to correct for changes
in blood glucose levels during insulin stimulation. This was achieved
by manually adjusting pump rates every 10 min until euglycemia was
reached. Blood samples were taken at the completion of the 2-h clamp
for determination of glucose, insulin, free fatty acids (FFA), adiponectin, and lactate. All blood samples were immediately centrifuged, and
plasma was stored at ⫺80°C. A terminal dose of Nembutal (100
mg/kg iv) was administered after clamping to dissect liver, as well as
extensor digitorum longus (EDL) muscle, from euthanized rats.
Analysis of plasma hormones and metabolites. Plasma insulin and
plasma adiponectin were measured via double-antibody radioimmunoassay techniques specific for insulin and adiponectin, respectively
(LINCO Research, St. Charles, MO). Plasma FFA was measured
enzymatically using a commercially available kit (NEFA C; Wako
Chemicals USA, Richmond, VA). Plasma lactate was determined with
a YSI 1500 SPORT plasma analyzer fitted with YSI 2329 membranes
suitable for L-lactate analysis (Yellow Springs Instrument, Yellow
Springs, OH).
Analysis of the oligomeric distribution of serum adiponectin.
Plasma samples (2 ␮l) were fractionated by PAGE on a 4 –15% gel
under nonreducing conditions, transferred onto PVDF membrane
(Immobilon-P, Millipore), and blotted with a rat adiponectin antibody
(Affinity BioReagents, Golden, CO). Membranes were then incubated
with horseradish peroxidase-conjugated secondary antibody before
chemiluminescence detection (Pierce, Rockford, IL). Band intensities
for LMW and HMW adiponectin were quantified by densitometry
using a Macintosh computer connected to an ARCUS scanner by way
of NIH Image 1.6 software.
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identical body weights, and both were significantly (P ⬍ 0.05)
heavier (15–20%) than regular chow-fed rats. Thus we performed GTTs in moderately obese rats as well as in normal
chow-fed animals, and the results, as shown in Fig. 1, indicate
that TPM treatment prevented the HFD-induced glucose intolerance (Fig. 1C) and also lowered basal and postprandial
insulin levels (Fig. 1D).
Euglycemic clamp studies. To confirm the results of the
GTTs showing that TPM treatments improve whole body
glucose homeostasis in HFD Wistar rats, we subjected all three
groups to euglycemic hyperinsulinemic clamp studies. Table 2
shows some general characteristics of Wistar rats carrying
indwelling catheters and receiving treatments of TPM or placebo in the postsurgical state. As seen in Table 2, chronically
catheterized Wistars on HFD and undergoing treatments with
or without TPM were equal in body size at the time of
hyperinsulinemic euglycemic testing and significantly heavier
than normal rats (P ⬍ 0.05). All groups of cannulated rats on
HFD displayed modest but significant increases (P ⬍ 0.05) in
basal blood glucose (⫹1.5 -2 mM) and basal plasma insulin
(⫹1–1.5 ng/ml) concentrations compared with normal chowfed controls. Clamped glucose and clamped lactate levels were
identical among all groups of rats (HFD, HFD ⫹ TPM, chow).
Basal plasma FFAs were equal among HFD, HFD ⫹ TPM, and
chow groups. Plasma FFAs are normally suppressed during hyperinsulinemic euglycemic clamp studies, and an overall suppression of serum FFAs occurred in all three groups of rats (P ⬍ 0.05
for glucose clamped vs. basal). TPM-treated rats showed a greater
suppression of serum FFAs than HFD controls (P ⬍ 0.05) and
demonstrated significantly lower serum FFA levels during glucose clamps compared with HFD controls (P ⬍ 0.05).
Figure 2 shows the glucose infusion rates required to maintain steady-state blood glucose concentrations in the clamped
rats from the experimental groups. As seen in Fig. 2, HFD led
to a 25% decrease in the glucose infusion rate (GINF), whereas
the GINF was increased by 25– 40% (P ⬍ 0.05) in HFD animals
on TPM compared with HFD rats treated with placebo. The
GINF in TPM-treated rats (40 – 45 mg䡠kg⫺1 䡠min⫺1) was similar to the GINF of chow-fed rats (40 – 45 mg䡠kg⫺1 䡠 min⫺1) and
significantly higher (P ⬍ 0.05) than the GINF in HFD control
rats (30 –35 mg䡠kg⫺1 䡠min⫺1).
Next, we assessed the effects of TPM treatment on muscle
and liver insulin action by calculating [3H]glucose tracerderived GDR and HGP. As seen in Fig. 3A, HFD led to insulin
resistance resulting in the expected decrease in GDR, whereas
TPM treatment led to an increase in GDR in the HFD rats.
Because the majority of GDR occurs in skeletal muscle, these
Fig. 3. TPM treatment ameliorates effect of diet in muscle without correcting
insulin resistance in liver. Glucose disposal rates [GDR and insulin-stimulated
(IS)-GDR], are shown in A and C, respectively. Hepatic glucose production
(HGP) is shown in B. Metabolic effects of insulin were determined by
hyperinsulinemic clamp in combination with [3H]glucose infusion and analysis
of tracer in dehydrated plasma. A: TPM treatment increased GDR in HFD-fed
rats; ¶¶P ⬍ 0.01 vs. HFD-fed controls; *P ⬍ 0.05 vs. chow-fed. B: HGP
suppression by insulin was impaired by HFD, and insulin-stimulated HGP was
impaired in HFD ⫹ TPM-treated rats; *P ⬍ 0.05 vs. chow. C: net effect of
insulin on in vivo glucose uptake (IS-GDR). IS-GDR was reduced in HFD rats;
*P ⬍ 0.05 vs. chow. Also, HFD ⫹ TPM-treated rats had increased IS-GDR;
¶P ⬍ 0.05 vs. HFD-fed controls, and IS-GDR in TPM animals was similar to
that in normal animals. Single filled bars represent means ⫾ SE of GDR and
IS-GDR. Double bars are for basal (filled) and insulin-stimulated (open) levels
of HGP (n ⫽ 6).
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results indicate a marked enhancement of in vivo muscle
glucose disposal in TPM rats compared with the HFD group
and the chow-fed rats. With respect to liver, there was no
improvement in hepatic insulin sensitivity resulting from
TPM treatment (Fig. 3B). Figure 3B shows that hepatic insulin resistance developed in both treated and untreated
HFD animals, as shown by a decreased effect of insulin to
suppress HGP.
To more accurately quantitate the effect of TPM treatment
on muscle insulin sensitivity, we calculated insulin-stimulated
GDR (IS-GDR). IS-GDR is typically used to illustrate the net
effect of insulin on in vivo muscle glucose uptake (IS-GDR ⫽
GDR ⫺ basal HGP). As shown in Fig. 3C, the HFD-induced
decrease in IS-GDR was completely prevented by TPM
treatment.
Circulating adiponectin levels. Adiponectin is an adipocytesecreted peptide with an ability to augment insulin sensitivity,
and previous studies have shown that circulating adiponectin
levels are low in insulin-resistant states, such as HFD, and are
upregulated when insulin sensitivity is improved by treatment
with thiazolidinediones (1). To assess the effects of TPM
treatment on adiponectin secretion, total adiponectin was measured in the experimental groups. As seen in Fig. 4A, HFD led
to a decrease in total adiponectin levels, whereas TPM treatment led to a marked increase.
AMPK phosphorylation and IMTG content. Pharmacological studies (2, 19) have shown that EDL skeletal muscles and
primary hepatocytes respond directly to adiponectin. Therefore, EDL and liver sections were harvested from clamped
animals and prepared, as described in MATERIALS AND METHODS,
for quantification of total AMPK␣ and phospho-AMPK levels.
Figure 5 shows that AMPK protein levels and AMPK
phosphorylation in liver tissue were the same among all study
groups. In Fig. 6A, it can be seen that the level of phosphoAMPK in EDL skeletal muscle was decreased by HFD (P ⬍
0.05) and that this was reversed by TPM treatment, resulting in
an increase to a level above that of normal animals (P ⬍ 0.05).
The HFD also led to an approximately twofold increase in
IMTG, which was completely prevented when the HFD-fed
animals were treated with TPM (Fig. 6B). This is fully consistent with the TPM-enhanced increase in muscle AMPK
activity and insulin sensitivity.
DISCUSSION
Insulin resistance is a central feature of type 2 diabetes as
well as a number of other common human insulin-resistant
states; therefore, identification of a new insulin-sensitizing
therapeutic would be of great importance. Topiramate is a
currently marketed antiepileptic agent that has been shown to
cause weight loss and improve insulin sensitivity in both
humans (4, 13) and animals (12, 14). Our own studies have
shown that Topiramate treatment in insulin-resistant female
ZDF rats can lead to amelioration of insulin resistance independently of weight loss. Although overall in vivo insulin
sensitivity was improved in these animals, when tissues were
studied ex vivo adipose tissue retained its insulin-sensitive
state, whereas the effects in skeletal muscle were lost after
removal from the in vivo environment. This led us to conclude
that Topiramate leads to insulin sensitization, exerting its
effects predominantly in adipose tissue.
AJP-Endocrinol Metab • VOL
Fig. 4. Circulating levels of adiponectin (Acrp30) are increased in TPMtreated rats. Rats were subjected to a 2-h hyperinsulinemic clamp as described
in MATERIALS AND METHODS. Plasma was collected afterward, stored at ⫺80°C,
and, upon completion of all studies, analyzed for total Acrp30 protein by RIA.
TPM-treated rats displayed significantly increased plasma levels of total
Acrp30 compared with placebo-treated (¶P ⬍ 0.01) and chow-fed controls.
Bars represent means ⫾ SE (n ⫽ 6). C, chow; HF, HFD; HMW and LMW,
high- and low-molecular-weight Acrp30, respectively. *P ⬍ 0.05.
Adiponectin is an adipocyte-secreted factor that can cause
insulin sensitization in a variety of conditions (6, 26). This
adipokine circulates in HMW and LMW components, and
recent evidence suggests that it is the HMW fraction of
adiponectin that is primarily responsible for the insulin sensi-
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Our experiments also reveal potential mechanisms to
explain these Topiramate effects. Thus we measured adiponectin in all of the experimental groups. As seen in Fig. 4,
HFD causes a decrease and Topiramate treatment leads to an
increase in total circulating adiponectin levels compared
with chow-fed controls.
Adiponectin is known to stimulate AMPK activity, and this
could result in the enhanced fat oxidation (19, 25) and all of the
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Fig. 5. AMP-activated protein kinase (AMPK) phosphorylation (P-AMPK) in
liver in TPM-treated rats. TPM-treated rats displayed normal AMPK phosphorylation levels compared with HFD-fed and regular (chow) controls.
tizing effects, presumably by binding to its G protein-coupled
receptor-like cell surface receptor and activating AMPK in
target tissues. We have recently found (15) that ectopic expression of adiponectin, using adenoviral mediated gene transfer,
leads to increased insulin sensitivity and complete protection
from the effects of HFD to cause insulin resistance. Taken
together, these results led us to speculate that Topiramate may
exert its insulin-sensitizing effects through an adiponectinrelated mechanism.
In the current study, we treated HFD-fed male Wistar rats
with Topiramate or placebo, followed by measurement of
glucose tolerance, insulin sensitivity, adiponectin levels, and
tissue AMPK activity. The major findings from this study are
that Topiramate treatment prevents HFD-induced glucose intolerance and hyperinsulinemia as measured during GTTs.
Hyperinsulinemic euglycemic clamp studies showed that HFD
led to insulin resistance, as shown by a 40% decrease in the
IS-GDR. Because the great majority of overall GDR is into
skeletal muscle, this result shows HFD-induced skeletal muscle insulin resistance. This HFD-induced decrease in IS-GDR
was completely prevented by Topiramate, and, in fact, IS-GDR
values in HFD ⫹ TPM animals were somewhat higher than in
chow-fed controls. To the extent that suppression of FFA
levels during the clamp procedure reflect insulin’s antilypolytic
effects, the HFD impaired, whereas Topiramate treatment enhanced, adipose tissue insulin sensitivity. Interestingly, HFD
also led to hepatic insulin resistance as manifested by an
impaired ability of insulin to suppress HGP, and this was not
affected by Topiramate treatment.
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Fig. 6. A: P-AMPK is upregulated in muscle in TPM-treated rats. EDL
muscles harvested at the end of the euglycemic clamp studies were analyzed
for AMPK␣ and P-AMPK (Thr172). TPM-treated rats displayed significantly
increased in vivo levels of AMPK phosphorylation; ¶¶P ⬍ 0.01 vs. HFD-fed
controls; *P ⬍ 0.05 vs. regular fed controls. B: intramuscular triglyceride
(IMTG) levels were increased by HFD (*P ⬍ 0.05 vs. chow) and decreased by
TPM treatment (¶P ⬍ 0.05 vs. HFD alone). Bars represent means ⫾ SE (n ⫽
5– 6).
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AJP-Endocrinol Metab • VOL
is comparable to the pattern reported in humans, raising the
possibility that Topiramate may work in man similarly to rats.
In conclusion, these studies show that Topiramate treatment
prevents HFD-induced insulin resistance in male Wistar rats.
This is accompanied by a drug-induced increase in adipose
tissue-derived circulating HMW adiponectin, with increased
skeletal muscle AMPK activity. Taken together, these results
suggest that Topiramate treatment can enhance muscle insulin
sensitivity, and this may reflect a direct effect on adipocytes to
increase adiponectin secretion.
ACKNOWLEDGMENTS
Betsy Hansen assisted in the preparation of the manuscript.
GRANTS
This work was supported by research grants from the National Institute of
Diabetes and Digestive and Kidney Diseases (DK-33651) and Johnson and
Johnson Pharmaceutical Research & Development.
REFERENCES
1. Arner P. The adipocyte in insulin resistance: key molecules and the
impact of the thiazolidinediones. Trends Endocrinol Metab 14: 137–145,
2003.
2. Berg AH, Combs TP, Du X, Brownlee M, and Scherer PE. The
adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat
Med 7: 947–953, 2001.
3. Ceddia RB, Somwar R, Maida A, Fang X, Bikopoulos G, and Sweeney
G. Globular adiponectin increases GLUT-4 translocation and glucose
uptake but reduces glycogen synthesis in rat skeletal muscle cells. Diabetologia 48: 132–139, 2005.
4. Chengappa KNR, Rathore D, Levine J, Atzert R, Solai L, Parepally H,
Levin H, Moffa N, Delaney J, and Brar JS. Topiramate as add-on
treatment for patients with bipolar mania. Bipolar Disord 1: 42–53, 1999.
5. Combs TP, Berg AH, Obici S, Scherer PE, and Rossetti L. Endogenous
glucose production is inhibited by the adipose-derived protein Acrp30.
J Clin Invest 108: 1875–1881, 2001.
6. Combs TP, Pajvani UB, Berg AH, Lin Y, Jelicks LA, Laplante M,
Nawrocki AR, Rajala MW, Parlow AF, Cheeseboro L, Ding YY,
Russell RG, Lindemann D, Hartley A, Baker GR, Obici S, Deshaies Y,
ludgate M, Rossetti L, and Scherer PE. A transgenic mouse with a
deletion in the collagenous domain of adiponectin displays elevated
circulating adiponectin and improved insulin sensitivity. Endocrinology
145: 367–383, 2004.
7. Frayn KN and Maycock PF. Skeletal muscle triacylglycerol in the rat
method for sampling and measurement and studies of biological variability. J Lipid Res 21: 139 –144, 1980.
8. Haluzik M, Colombo C, Gavrilova O, Chua S, Wolf N, Chen M,
Stannard B, Dietz KR, LeRoith D, and Reitman ML. Genetic background (C57BL/6J versus FVB/N) strongly influences the severity of
diabetes and insulin resistance in ob/ob mice. Endocrinology 145: 3258 –
3264, 2004.
9. Hotta K, Funahashi T, Bodkin NL, Ortmeyer HK, Arita Y, Hansen
BC, and Matsuzawa Y. Circulating concentrations of the adipocyte
protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys.
Diabetes 50: 1126 –1133, 2001.
10. Pajvani UB, Du X, Combs TP, Berg AH, Rajala MW, Schulthess T,
Engel J, Brownlee M, and Scherer PE. Structure-function studies of the
adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity. J Biol Chem 278: 9073–9085, 2003.
11. Pajvani UB, Hawkins M, Combs TP, Rajala MW, Doebber T, Berger
JP, Wagner JA, Wu M, Knopps A, Xiang AH, Utzschneider KM,
Kahn SE, Olefsky JM, Buchanan TA, and Scherer PE. Complex
distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity. J Biol Chem 279:
12152–12162, 2004.
12. Picard F, Deshaies Y, Lalonde J, Samson P, and Richard D. Topiramate reduces energy and fat gains in lean (Fa/?) and obese (fa/fa) Zucker
rats. Obes Res 8: 656 – 663, 2000.
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other insulin-sensitizing effects of adiponectin that have been
described. We measured AMPK activity in muscle and liver
tissue from these animals and found enhanced AMPK phosphorylation in muscle, but not in liver, in the HFD ⫹ TPM
animals, consistent with the in vivo effects of Topiramate on
IS-GDR but not HGP. Additionally, plasma FFAs were found
to be lower after insulin stimulation in glucose-clamped
HFD ⫹ TPM animals compared with controls, which could
have also affected muscle insulin sensitivity. Interestingly, the
HFD-induced accumulation of IMTG was also prevented by
Topiramate treatment.
Topiramate has been reported to cause decreased food intake
and weight loss, which are largely transient in rodents. Indeed,
there was a modest difference in body weights of Topiramatetreated and untreated non-pair-fed HFD animals. However, this
was not a factor in our studies, since all comparisons were
made between HFD ⫹ TPM-treated animals and pair-fed
HFD ⫹ placebo groups, and food intake and body weights
were identical between these two groups (Table 1 and Fig. 1A).
Indeed, over the last 24 h of the study, food intake was only ⬃1
g/day less in the Topiramate-treated and pair-fed placebo
groups compared with the ad libitum-fed HFD animals.
The literature contains mixed results as to the tissue site of
adiponectin’s insulin-sensitizing effects. Some studies have
shown effects predominantly in liver (2, 5) while others have
clearly shown direct adiponectin actions on skeletal muscle
(25, 3). Studies, for example, with 5-h-fasted mice given
recombinant adiponectin (20 ng䡠g body wt⫺1 䡠 min⫺1) to
acutely elevate plasma adiponectin concentrations showed that
adiponectin improves hepatic insulin sensitivity without improving peripheral insulin sensitivity. These hepatic effects
were associated with reductions in glucose flux through the
glucose-6-phosphatase metabolic pathway, with no effect on
glycolysis or glycogen synthesis. Indeed, others have also
reported on adiponectin’s liver selective actions. Berg et al. (2)
showed that the full-length adiponectin protein has glucoselowering effects in ob/ob and nonobese diabetic mice, and also
demonstrated direct effects of full-length adiponectin on isolated primary hepatocytes. In contrast, globular adiponectin has
direct effects in muscle to activate AMPK, stimulate glucose
uptake and FFA oxidation (25), and cause GLUT4 translocation in L6 cells (3). In our own earlier studies (15), using
adenovirus gene transfer to achieve ectopic transgenic expression of adiponectin in Wistar rats on HFD, we observed that
the predominant insulin-sensitizing effects of the expressed
adiponectin were exerted in skeletal muscle, as demonstrated
by increased in vivo IS-GDR and enhanced muscle AMPK
activity. This is similar to the current studies, where we find
that Topiramate treatment leads to enhanced muscle, but not
liver, insulin sensitivity. Although the reason for the differences between various animal studies is unknown, it is possible
that they may be partially related to differences in adiponectin
receptor expression. For example, Yamauchi et al. (24) identified two adiponectin receptors, Adipo-R1 and Adipo-R2. In
mice, Adipo-R1 is expressed predominantly in muscle, with
Adipo-R2 highly expressed in liver, and exogenous adiponectin exerts potent effects to enhance hepatic insulin sensitivity in
mice. In rats, both Adipo-R1 and -R2 are expressed more
highly in muscle, possibly explaining adiponectin-induced
muscle insulin-sensitizing effects in rats. Interesting, the pattern of adiponectin receptor expression that we observed in rats
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TOPIRAMATE IS INSULIN SENSITIZING IN HF-FED WISTAR RATS
AJP-Endocrinol Metab • VOL
21. Tsao TS, Murrey HE, Hug C, Lee DH, and Lodish HF. Oligomerization state-dependent activation of NF-kappa B signaling pathway by
adipocyte complement-related protein of 30 kDa (Acrp30). J Biol Chem
277: 29359 –29362, 2002.
22. Wilkes JJ, Hevener A, and Olefsky JM. Chronic ET-1 treatment leads
to insulin resistance in vivo. Diabetes 52: 1904 –1909, 2003.
23. Wilkes JJ, Nelson E, Osborne E, Demarest KT, and Olefsky JM.
Topiramate is an insulin-sensitizing compound in vivo with direct effects
on adipocytes in female ZDF rats. Am J Physiol Endocrinol Metab 288:
E617–E624, 2005.
24. Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S,
Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki
T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y,
Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai
R, and Kadowaki T. Cloning of adiponectin receptors that mediate
antidiabetic metabolic effects. Nature 423: 762–769, 2003.
25. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S,
Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P,
Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, and
Kadowaki T. Adiponectin stimulates glucose utilization and fatty-acid
oxidation by activating AMP-activated kinase. Nat Med 8: 1288 –1295,
2002.
26. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori
Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma
Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K,
Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel
P, and Kadowaki T. The fat-derived hormone adiponectin reverses
insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:
941–946, 2001.
289 • DECEMBER 2005 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.6 on July 28, 2017
13. Reife R, Pledger G, and Wu SC. Topiramate as add-on therapy: pooled
analysis of randomized controlled trials in adults. Epilepsia 41: S66 –S71,
2000.
14. Richard D, Ferland J, Lalonde J, Samson P, and Deshaies Y. Influences of Topiramate in the regulation of energy balance. Nutrition 16:
961–966, 2000.
15. Satoh H, Nguyen MT, Trujillo M, Imamura T, Usui I, Scherer PE,
and Olefsky JM. Adenovirus-mediated adiponectin expression augments
skeletal muscle insulin sensitivity in male Wistar rats. Diabetes 54:
1304 –1313, 2005.
16. Scherer PE, Williams S, Fogliano M, Baldini G, and Lodish HF. A
novel serum protein similar to C1q, produced exclusively in adipocytes.
J Biol Chem 270: 26746 –26749, 1995.
17. Statnick MA, Beavers LS, Conner LJ, Corominola H, Johnson D,
Hammond CD, Rafaeloff-Phail R, Seng T, Suter TM, Sluka JP,
Ravussin E, Gadski RA, and Caro JF. Decreased expression of apM1 in
omental and subcutaneous adipose tissue of humans with type 2 diabetes.
Int J Exp Diabetes Res 1: 81– 88, 2000.
18. Steele R. Influence of glucose loading and injected insulin on hepatic
glucose output. Ann NY Acad Sci 82: 420 – 430, 1959.
19. Tomas E, Tsao TS, Saha AK, Murrey HE, Zhang C, Samar I, Lodish
HF, and Ruderman NB. Enhanced muscle fat oxidation and glucose
transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition
and AMP-activated protein kinase activation. Proc Natl Acad Sci USA 99:
16309 –16313, 2002.
20. Tonelli J, Li W, Kishore P, Pajvani UB, Kwon E, Weaver C, Scherer
PE, and Hawkins M. Mechanisms of early insulin-sensitizing effects of
thiazolidinediones in type 2 diabetes. Diabetes 53: 1621–1629, 2004.