Leptin administration improves skeletal muscle insulin
responsiveness in diet-induced insulin-resistant rats
Ben B.
Yaspelkis III1,
James R.
Davis2,
Maziyar
Saberi1,
Toby L.
Smith1,
Reza
Jazayeri1,
Mohenish
Singh1,
Victoria
Fernandez1,
Beatriz
Trevino1,
Narumol
Chinookoswong3,
Jinlin
Wang3,
Zhi Qing
Shi3, and
Nancy
Levin2
1 Exercise Biochemistry Laboratory, Department of
Kinesiology, California State University Northridge, Northridge
91330-8287; and Departments of 2 Neuroscience and
3 Pharmacology, Amgen Incorporated, Thousand Oaks, California
91320-1799
 |
ABSTRACT |
In addition to
suppressing appetite, leptin may also modulate insulin secretion and
action. Leptin was administered here to insulin-resistant rats to
determine its effects on secretagogue-stimulated insulin release, whole
body glucose disposal, and insulin-stimulated skeletal muscle glucose
uptake and transport. Male Wistar rats were fed either a normal (Con)
or a high-fat (HF) diet for 3 or 6 mo. HF rats were then treated with
either vehicle (HF), leptin (HF-Lep, 10 mg · kg
1 · day
1 sc), or
food restriction (HF-FR) for 12-15 days. Glucose tolerance and
skeletal muscle glucose uptake and transport were significantly impaired in HF compared with Con. Whole body glucose tolerance and
rates of insulin-stimulated skeletal muscle glucose uptake and
transport in HF-Lep were similar to those of Con and greater than those
of HF and HF-FR. The insulin secretory response to either glucose or
tolbutamide (a pancreatic
-cell secretagogue) was not significantly
diminished in HF-Lep. Total and plasma membrane skeletal muscle GLUT-4
protein concentrations were similar in Con and HF-Lep and greater than
those in HF and HF-FR. The findings suggest that chronic leptin
administration reversed a high-fat diet-induced insulin-resistant
state, without compromising insulin secretion.
ob gene product; high-fat diet; glucose tolerance; glucose uptake and transport; GLUT-4 protein
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INTRODUCTION |
LEPTIN, THE PRODUCT of
the ob gene (62), has received a great deal of
attention since its discovery in 1994, due to the ability of this
16-kDa protein hormone to reduce visceral adipose deposition (21,
37). This biological activity is important from a public health
perspective, as increases in visceral fat have been associated with
"insulin resistance syndrome" or Syndrome X (39).
Attenuation of insulin resistance will decrease the incidence of
metabolic abnormalities such as hypertriglyceridemia, reduced
high-density lipoproteins, elevated apolipoprotein B levels, and
hypertension. Furthermore, reduced visceral fat deposition may also
prevent the development of non-insulin-dependent diabetes (17).
It is believed that leptin exerts its primary effect by acting on
receptors in the hypothalamus, possibly via inhibition of neuropeptide
Y release (47). However, leptin receptor isoforms are
expressed in tissues other than the hypothalamus (12, 29, 51), and insulin action (e.g., phosphatidylinositol 3-kinase activity, skeletal muscle glucose uptake and transport) is improved in
these tissues after leptin treatment (3, 56, 57, 60). Improvements in insulin-stimulated glucose disposal after chronic leptin administration were initially demonstrated by Barzilai et al.
(3) and Sivitz et al. (44). Barzilai et al.
(3) reported that 8 days of leptin treatment increased
whole body glucose uptake in Sprague-Dawley rats as assessed by the
euglycemic clamp technique. In an extension to these findings, we
(60) found that 14 days of leptin administration increased
skeletal muscle glucose uptake and
3-O-methyl-D-glucose (3-MG) transport in
hindlimbs of Sprague-Dawley rats. However, these observations (3,
44, 60) were made in non-insulin-resistant animals. Although
interventions that improve carbohydrate metabolism in normal
animals also tend to be effective in insulin-resistant animals, it
is unknown whether chronic leptin administration will improve an
insulin-resistant state. Moreover, ex vivo pancreatic perfusion data
suggest the possibility that leptin may have a detrimental effect on
pancreatic secretagogue responsiveness (38).
Therefore, the aims of the present investigation were to evaluate the
impact of chronic leptin administration in an insulin-resistant rodent
model by assessing 1) the insulin secretory response to glucose and tolbutamide; 2) whole body glucose clearance;
3) insulin-stimulated skeletal muscle glucose uptake and
3-MG transport; and 4) if improvements in leptin-treated
insulin-resistant skeletal muscle result from alterations in enzymatic
activity, glycogen concentration, and/or GLUT-4 protein concentration.
 |
METHODS |
Animals.
All experimental procedures were approved by the Institutional Animal
Care and Use Committees at Amgen (Thousand Oaks, CA) and California
State University Northridge and conformed to the guidelines for use of
laboratory animals published by the United States Department of Health
and Human Resources.
Eight-week-old male Wistar rats (Harlan Sprague-Dawley, San Diego, CA)
were housed two per cage in a temperature-, humidity-, and
light-controlled room (lights on at 0630, lights off at 1830). Rats
were provided water and one of two purified powdered diets ad libitum
for 3 (experimental protocols 1 and 3) or 6 (experimental protocol 2) mo. The two diets were of either
normal fat content (control diet, 17% fat-derived calories, no.
112386; Dyets, Bethlehem, PA) or high- fat content (high-fat diet, 59%
fat-derived calories, no. 112387; Dyets). Protein and carbohydrate
contents of the diets were, respectively, 20 and 63% for control and
15 and 26% for the high-fat diet. The diets were identical with
respect to vitamin and mineral content. A similar high-fat diet has
previously been shown to induce skeletal muscle insulin resistance in
rats (48, 49). Diets were stored at 4°C, and fresh diet
was provided to all rats two times a week.
At the end of the 3- or 6-mo dietary lead in, glucose tolerance of the
rats was assessed after an intraperitoneal glucose challenge
[intraperitoneal glucose tolerance test (IPGTT)]. Rats were fasted in
the morning (food out at 0900) for 4 h before testing. At the
onset of the IPGTT, rats (nonanesthetized) were introduced to plastic
restrainers and were bled by producing a small incision in the tail
vein and milking the tail (~0.5 ml blood). After this time
0 sample was collected, rats were injected with glucose (2 g/kg
ip), and blood was collected from the tail (~0.2 ml blood) at 30 and
90 min after injection. Blood glucose levels were determined on all
samples using a One-Touch Meter (Lifescan, San Carlos, CA).
Glucose-intolerant rats were defined as those that displayed a blood
glucose value at 90 min that was 80% or more of the value at 30 min.
With the use of this criterion, between 25 and 35% of the high fat-fed
rats were found to be glucose intolerant. Serum insulin levels in the
time 0 samples were determined by RIA (Linco Research, St.
Louis, MO). Serum leptin levels in the time 0 sample were
determined by enzyme-linked immunosorbent assay (59).
Glucose-intolerant high-fat diet-fed rats were then assigned to
one of three treatment groups. Treatments were given for 12-15 days, during which time rats continued to consume the high-fat diet.
Two treatment groups [high-fat-fed rats (HF) and high-fat-fed food-restricted rats (HF-FR)] received twice daily subcutaneous injections of vehicle (PBS). The third treatment group [high-fat-fed leptin-treated rats (HF-Lep)] was injected subcutaneously twice daily
with recombinant murine leptin (5 mg · kg
1 · injection
1, 10 mg · kg
1 · day
1 total dose;
Amgen). The HF and HF-Lep groups consumed the high-fat diet ad libitum,
whereas the food-restricted (HF-FR) group was provided 15 g/day of the
high-fat diet for treatment days 1-6 and 13 g/day of
high-fat diet from days 7-15 of the experimental period. These food intake values were based on observations of food
intake in the HF-Lep rats (Davis and Levin, unpublished observations). A fourth treatment group consisted of rats that had been fed the control diet (Con) and demonstrated normal glucose tolerance. Con rats
continued to consume the control diet ad libitum during the treatment
periods and were injected two times daily with PBS subcutaneously.
Injections were administered at 0800 and 1800 using a volume of ~0.25 ml.
Experimental protocol 1.
For this protocol, rats were maintained on either the control or the
high-fat diet for 3 mo before selection of glucose-intolerant rats.
Glucose-intolerant, high-fat-fed rats were divided into treatment
groups (n = 10-12/group) and received either
vehicle (HF) or leptin (HF-Lep) for 15 days. An additional group of
glucose-intolerant, high-fat-fed rats was treated with vehicle and were
food restricted (HF-FR) following the predetermined schedule described
above that mimicked the intake of the HF-Lep rats. A group of rats fed
the control (Con) diet (n = 10) and injected with
vehicle was also studied. Treatments were initiated immediately after
the 3-mo dietary lead in. On day 7 of the 15-day treatment
period, rats were anesthetized with pentobarbital sodium and implanted
with carotid artery and jugular vein cannulas. Cannulas were
exteriorized in the midscapular region, and patency was maintained by
daily flushing with a heparin-saline solution. The insulin secretory response to an intravenous glucose challenge (1 g/kg) was assessed over
a 60-min period on treatment day 12 in conscious rats. The insulin secretory response to an intravenous tolbutamide injection (0.1 g/kg) was assessed over a 60-min period on treatment day 14 or 15 in conscious rats. Tolbutamide was employed to assess pancreatic insulin secretory capacity without the confound of glucose-sensing ability. Tolbutamide binds to the sulfonylurea receptor
in the
-cells of the pancreas, blocking the ATP-dependent potassium
(KATP) channels and resulting in insulin secretion. For
these tests, rats were fasted in the morning (food removed at 0900) for
4 h before and during testing. Fifteen minutes before testing, the
cannulas were extended with additional tubing (PE-50) to allow for
dosing and sampling without disturbing the rat, and a prestudy blood
sample (~0.5 ml) was drawn from the arterial cannulas. At time
0, a small (<0.1 ml) blood sample was drawn from the arterial
cannulas, and then the test substance was administered through the
venous cannulas. Small (<0.1 ml) blood samples were collected from the
arterial cannulas after glucose or tolbutamide dosing at 1.5, 3, 4.5, 6, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, and 60 min. Serum from
these samples was separated by centrifugation and stored at
20°C
until subsequent determination of glucose (by the glucose oxidase
method) and insulin (by RIA) levels.
Experimental protocol 2.
For this protocol, rats were maintained on the high-fat diet for 6 mo
before selection of glucose-intolerant rats. Treatment groups included
HF-Lep and HF-FR, and treatments were initiated immediately after the
6-mo dietary lead in. Glucose-intolerant, high-fat-fed rats were
divided into treatment groups (n = 5-6/group) and
received either vehicle or leptin (HF-Lep) for 12 days. The vehicle-treated rats were food restricted (HF-FR) following the predetermined schedule described above that mimicked the intake of the
HF-Lep rats. As in experiment protocol 1, rats were
outfitted with carotid artery and jugular vein cannulas on day
7 of treatment, and cannula patency was ensured by daily flushing
with heparin-saline.
On treatment day 12, rats were fasted in the morning for
4 h before initiation of the hyperinsulinemic-euglycemic clamp
study, as previously described by Shi et al. (43). The
jugular vein cannulas were used for infusion of insulin and glucose via
serial T-shaped needle connectors. Constant insulin infusion (4 mU · kg
1 · min
1) began at 0 min, and an exogenous glucose infusion (30%) was given at variable
rates to achieve and maintain stable euglycemia. Carotid arterial
samples were taken at timed intervals, and plasma was collected in
ice-chilled microtubes containing EGTA/aprotinin. Plasma glucose levels
were determined using a Glucose Analyzer II (Beckman Instruments,
Fullerton, CA). The packed blood cells were resuspended in heparinized
saline and reinfused.
Experimental protocol 3.
For this protocol, rats were maintained on either the control or the
high-fat diet for 3 mo before selection of glucose-intolerant rats.
Approximately 2-3 wk after the dietary lead in, animals were
divided among two perfusion groups. Perfusion group 1 consisted Con (n = 7), HF (n = 7),
HF-FR (n = 7), and HF-Lep (n = 7)
animals. Perfusion group 2 consisted of Con (n = 8), HF (n = 8), and HF-Lep (n = 8)
animals. Animals were treated with either leptin or vehicle for 12 days. After the 12-day treatment period, animals were anesthetized with
an intraperitoneal injection of pentobarbital sodium (6.5 mg/100 g body
wt) and surgically prepared for hindlimb perfusion as described
previously by Ruderman et al. (41) and modified by Ivy et
al. (25). After the surgical preparation, the soleus (Sol), plantaris (Plant), and red (RG) and white (WG) portions of the
gastrocnemius and quadricep were excised from the left leg,
clamp-frozen in liquid nitrogen, and stored at
80°C until analysis.
Total GLUT-4 protein concentration, enzymatic analysis, and muscle
glycogen content were assessed in the Sol, Plant, RG, and WG from
perfusion group 1. Total GLUT-4 protein concentration and
intramuscular triglyceride content was assessed in the quadriceps of
perfusion group 2. The right iliac artery was then
catheterized to the tip of the femoral artery to limit perfusate flow
to the right hindlimb. Catheterization of the lower abdominal vena cava to the tip of the iliac vein permitted the collection of effluent perfusate. Immediately after catheterization of the vessels, rats were
killed via an intracardiac injection of pentobarbital sodium as the
hindlimbs were being washed out with 10 ml of Krebs-Heinseleit buffer
(KHB). The catheters were then placed in line with a nonrecirculating perfusion system, and the hindlimb was allowed to stabilize during a
5-min washout period. The perfusate was gassed continuously with a
mixture of 95% O2-5% CO2 and was warmed
to 37°C. Perfusate flow rate was set at 5 ml/min during the 5-min
stabilization and the subsequent perfusion, during which the rates of
muscle glucose uptake and glucose transport were determined.
Perfusions were carried out in the presence of a submaximal insulin
concentration (500 µU/ml) for all experimental groups. The basic
perfusate medium consisted of 30% washed time-expired human
erythrocytes (HemaCare, Van Nuys, CA), KHB (pH 7.4), 4% dialyzed BSA
(Fraction V; Fisher Scientific, Fair Lawn, NJ), and 0.2 mM pyruvate.
Over the first 20 min, 8 mM glucose was present in the perfusate, and
it was during this period that glucose uptake was measured across the
hindlimb. Subsequent to the determination of glucose uptake, the
hindlimb was washed out with glucose-free perfusate for 1 min in
preparation for the measurement of glucose transport. Glucose transport
was measured over an 8-min period using an 8 mM concentration of the
nonmetabolizable glucose analog 3-MG (32 µCi
3-[3H]MG/mmol) and 2 mM mannitol (60 µCi
D-[1-14C]mannitol/mmol). Immediately at the
end of the transport period, the Sol, Plant, RG, and WG were excised
from the right leg, blotted on gauze dampened in cold KHB, and
clamp-frozen in tongs cooled in liquid N2. The muscles were
stored at
80°C until analyzed. Sol, Plant, RG, and WG from
perfusion group 1 were used to determine rates of
insulin-stimulated 3-MG transport, whereas the quadriceps from
perfusion group 2 were used to assess insulin-stimulated plasma membrane GLUT-4 protein concentration.
Glucose uptake was determined over a 20-min nonrecirculating perfusion
by collecting arterial perfusate samples before perfusion and
collecting the total venous effluent. Well-mixed aliquots of the
arterial perfusate and venous effluent were analyzed for glucose
concentration by a glucose oxidase method on a model 2300 STAT Plus
glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH).
Muscle glucose uptake, expressed in micromoles per gram per hour, was
calculated from the arteriovenous difference, the perfusate flow rate,
and the weight of the muscle perfused. The weight of the perfused
muscle was determined by dissection of the rat hindlimb
(42).
Muscle samples were weighed, homogenized in 1 ml of 10% TCA at 4°C,
and centrifuged in a microcentrifuge (Fisher Scientific, Houston, TX)
for 10 min. Duplicate 300-µl samples of the supernatant were
transferred to 7-ml scintillation vials containing 6 ml of Bio-Safe II
scintillation counting cocktail (Research Products International, Mount
Prospect, IL) and vortexed. For determination of perfusate specific
activity, 200 µl of the arterial perfusate were added to 800 µl of
10% TCA and treated the same as the muscle homogenates. The samples
were counted for radioactivity in a LS 1801 liquid scintillation
spectrophotometer (Beckman Instruments, Fullerton, CA) set for
simultaneous counting of 14C/3H. The
accumulation of intracellular 3-[3H]MG, which is
indicative of muscle glucose transport, was calculated by subtracting
the concentration of 3-[3H]MG in the extracellular space
from the total muscle 3-[3H]MG concentration. The
3-[3H]MG in the extracellular space was quantified by
measuring the concentration of [14C]mannitol in the homogenate.
Total skeletal muscle GLUT-4 glucose transporter content was determined
by Western blotting as described previously (61). Briefly,
portions of the freeze-clamped muscles from the left hindlimb were
weighed frozen and then homogenized in Hepes-EDTA-sucrose (HES)
buffer. The protein concentration of the homogenate was determined by
the Bradford (6) method. A 100-µl sample of the tissue
homogenate was diluted 1:1 with Laemmli (31) sample
buffer. An aliquot of the diluted homogenate sample containing 75 µg
of protein was subjected to SDS-PAGE run under reducing conditions on a
12.5% resolving gel on a Mini-Protean II dual slab cell (Bio-Rad, Richmond, CA). Resolved proteins were transferred to polyvinylidene difluoride (PVDF) sheets (Bio-Rad, Richmond, CA) by the method of
Towbin et al. (53) using a Bio-Rad wet transfer unit. The membranes were incubated with an affinity-purified polyclonal GLUT-4
antibody (donated by Dr. Samuel W. Cushman, National Institute of
Diabetes and Digestive and Kidney Diseases, Bethesda, MD) followed by
incubation with horseradish peroxidase-labeled protein A (Amersham Life
Science, Arlington Heights, IL). Antibody binding was visualized using
enhanced chemiluminescence autoradiography in accordance with the
manufacturer's instructions (Amersham Life Science). Labeled bands
were quantified by capturing images of the autoradiographs in a
Macintosh G3 computer. The captured images of the autoradiographs were
produced by an image scanner (ScanJet 4C; Hewlett Packard, Boise, ID)
equipped with a transparency module. The captured images were digitized
and imported into the public domain NIH image program (developed at the
United States National Institutes of Health and available on the
Internet at http://rsb.info.nih.gov/nih-image/). The density of the
labeled bands was calculated, corrected for background activity, and
expressed as a percentage of a standard (30 µg of heart homogenate
protein) run on each gel.
Plasma membrane fractions were prepared from portions of the perfused
quadricep according to the procedure of Turcotte et al.
(54). Briefly, a portion of the quadriceps was minced,
diluted 1:7 in a 10 mM Tris-15% sucrose solution (pH 7.5) that
contained 0.1 mmol/l phenylmethylsulfonyl fluoride, 10 mmol/l EGTA, and 10 mg/ml trypsin inhibitor, and homogenized with a PT 2100 Polytron homogenizer (Kinematica, Littau/Luzern, Switzerland). The homogenate was filtered and centrifuged at 100,000 g for 1 h using
a Sorvall T-1250 rotor (Kendro Laboratory Products, Newton, CT). The
pellet was resuspended in 10 mM Tris-15% sucrose buffer, and a small aliquot from this resuspension was collected and retained for analysis
and will be referred to as the crude homogenate. The remaining crude
homogenate suspension was layered on continuous sucrose gradients
(35-70%) and centrifuged at 120,000 g for 2 h in
a Sorvall Surespin 630/36 rotor. The plasma membrane layer was
collected, washed in 10 mM Tris buffer, and centrifuged for 1 h at
100,000 g in a Sorvall T-1250 rotor. The final plasma
membrane pellet was resuspended in a small volume of 10 mM Tris buffer (200 µl/g of original tissue), frozen in liquid nitrogen, and stored
at
80°C until analyzed. To assess the purity of the plasma membrane
fractions, protein content of the plasma membrane (6) and
activity of the plasma membrane marker enzyme 5'-nucleotidase was
measured (52) and compared with activity in the crude
homogenate fraction. Aliquots of the plasma membrane (70 µg of
protein) were treated with Laemmli sample buffer and subjected to
SDS-PAGE run under reducing conditions on a 10% resolving gel.
Resolved proteins were transferred to PVDF by the method of Towbin et
al. (53) using a Bio-Rad semidry transfer unit, and GLUT-4
protein content of the plasma membrane was determined by Western
blotting as described above.
Aliquots of muscle samples that were homogenized 1:20 in HES buffer
were used for enzymatic analysis. Hexokinase, an enzyme required for
glucose metabolism, was measured as described by Uyeda and Racker
(55). Citrate synthase, a marker of the tricarboxylic acid
cycle, was measured spectrophotometrically (45) after
dilution of the initial homogenate 1:10 in 1 M Tris · HCl + 0.4% Triton X (pH 8.1). For determination of
-hydroxyacetyl-CoA
dehydrogenase (
-OAC), a marker of
-oxidation, homogenates from
the WG were diluted 1:5, whereas homogenates from the Sol, Plant, and
RG were diluted 1:20 in 167 mM triethanolamine-HCl buffer, pH 7.0.
-OAC activity was determined spectrophotometrically according to the procedure described by Bass et al. (4).
Muscle glycogen concentration was determined in the Sol, Plant, RG, and
WG using a modification of the Passonneau and Lauderdale (36) procedure. Approximately 50 mg of muscle were
dissolved in 1 ml of 1 N KOH at 70°C for 30 min. One hundred
microliters of the dissolved homogenate were removed and neutralized
with 250 µl of 0.3 M sodium acetate buffer (pH 4.8) and 10 µl of
50% glacial acetic acid. The homogenate was then incubated for 2 h at 100°C after the addition of 200 µl of 2 N HCl. After the
incubation, the reaction mixture was neutralized with 2 N NaOH. Samples
were then analyzed by measuring glucosyl units by the Trinder reaction (Sigma, St. Louis, MO). A portion of the quadricep was homogenized 1:20
in 25 mM KF/20 mM EDTA buffer, pH 7.0, and subjected to lipid extraction as described by Burton et al. (9). Total
triglyceride content of the lipid extract was determined using a
commercially available kit (Infinity Triglycerides; Sigma-Aldrich, St.
Louis, MO).
Statistical analysis.
A one-way ANOVA was used on all variables in experimental
protocols 1 and 3 to determine whether significant
differences existed between the Con, HF-Lep, HF, and HF-FR groups. When
a significant F-ratio was obtained, a Fisher's protected
least significant difference post hoc test was employed to identify
statistically significant differences (P < 0.05) among
the means. Glucose infusion data in experimental protocol 2 were analyzed using a two-way ANOVA (treatment × time) for
repeated measures.
 |
RESULTS |
Effects of high-fat diet consumption.
Rats that had consumed the high-fat diet ad libitum for 12 wk
(experimental protocols 1 and 3) gained
significantly more weight and had higher fasting serum leptin
concentrations than did rats that consumed the control diet for the
same duration (Table 1). At the end of
the 12 wk of dietary manipulation, an IPGTT was performed to determine
which rats had developed glucose intolerance as a consequence of
consumption of the high-fat diet. Results from the IPGTT of rats used
in experimental protocol 3 are depicted in Fig.
1. Similar findings were observed in the
two independent cohorts of rats used in experimental protocols
1 and 2 (data not shown). In one of the three cohorts
(Fig. 1), fasting blood glucose levels immediately before the
intraperitoneal glucose injection were slightly but significantly
greater in the high-fat-fed rats compared with the controls.

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Fig. 1.
Blood glucose levels during intraperitoneal glucose tolerance test
(IPGTT) in rats fed either a control diet (Con, n = 8)
or a high-fat diet (HF, n = 24) for 12 wk before
testing. Values are means ± SE. * Significantly different from
Con (P < 0.05).
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Experimental protocol 1.
On treatment day 12, immediately before intravenous glucose
tolerance testing, a blood sample was drawn to determine fasting hormone and clinical chemistry of the rats. As expected, serum leptin
levels (sampled 4-6 h after the morning injection of leptin) were
significantly increased in the HF-Lep group compared with all other
groups (Table 2). Although HF-Lep and
HF-FR rats had consumed significantly less food than HF rats over the
12-day treatment period, the body weights of these animals were not
significantly less than the HF rats fed ad libitum (Table 2). Serum
thyroxine levels were also significantly and similarly reduced
in the HF-Lep and HF-FR groups relative to the HF group. Fasting
-hydroxybutyrate levels in the HF-Lep group were significantly
higher than all other groups. The significant reduction in
nonesterified fatty acid levels in the HF group, relative to the Con
group, was not observed in HF-FR or HF-Lep rats.
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Table 2.
Body mass, food intake, serum hormones, and clinical chemistry
after 12 days of treatment/feeding regimen
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Intravenous glucose tolerance tests were performed on day 12 of the treatment period. The areas under the curve (AUCs) for glucose,
calculated from 0 to 25 min (intravenous glucose administration at 0 min), are shown in Fig. 2A.
The glucose AUC in HF rats was significantly increased when compared
with Con rats (P < 0.05). This increase was reversed
by leptin treatment in the HF-Lep rats to an AUC similar to that
observed in the Con rats and significantly lower than the HF rats
(P < 0.01). The glucose AUC for HF-FR rats was not
different from that observed for HF rats.

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Fig. 2.
Serum glucose and insulin areas under the curve (AUCs)
after intravenous injection of glucose (A and B;
0-25 min) or tolbutamide (C; 0-60 min) in Con
(n = 10), HF (n = 11), high-fat diet
and leptin-treated (HF-Lep, n = 12), or high-fat diet
and food-restricted (HF-FR, n = 10) rats. Values are
means ± SE. * Significantly different from Con (P < 0.05). Significantly different from HF (P < 0.05).
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AUCs for insulin, calculated from 0 to 25 min (intravenous glucose
administration at 0 min), are shown in Fig. 2B. The insulin secretory response to the intravenous glucose challenge was not significantly different across the four treatments groups. There was a
tendency for the insulin AUC to be lowest in the HF-Lep group.
Importantly, no worsening of glucose tolerance was observed in the
HF-Lep rats, as they displayed glucose AUCs significantly lower than
those observed in HF rats (Fig. 2A). Leptin treatment therefore did not significantly affect insulin secretory output and was
accompanied by improved glucose tolerance in the insulin-resistant model. Further observations of the effects of chronic leptin
administration on glucose homeostasis were pursued in
experimental protocols 2 and 3.
To better characterize the insulin secretory capacity after chronic
leptin administration, rats from each of the treatment groups described
above underwent an intravenous tolbutamide tolerance test on
treatment day 14 or 15. The AUCs for insulin,
calculated from 0 to 60 min (intravenous tolbutamide administration at
0 min), are shown in Fig. 2C. The insulin secretory response
to the intravenous tolbutamide challenge was similar across the four treatments groups. Again, there was a tendency for the insulin AUC to
be lower in the HF-Lep group and in the case of tolbutamide treatment
was also slightly reduced in the HF-FR group.
Experimental protocol 2.
The glucose infusion rates necessary to maintain euglycemia (111 ± 2 mg/dl for HF-FR, 115 ± 2 mg/dl for HF-Lep) during
hyperinsulinemia are shown in Fig. 3.
There was a significant increase in the glucose infusion rate for the
HF-Lep rats compared with HF-FR rats, indicating that chronic leptin
treatment of glucose-intolerant rats resulted in an increase in the
insulin-mediated glucose clearance rate.

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Fig. 3.
Glucose infusion rates during hyperinsulinemic (4 mU · kg 1 · min 1 begun at
time 0)-euglycemic clamp of HF-FR (n = 6) or
HF-Lep (n = 5) rats. Values are means ± SE. There
was a significant effect of time (F = 89.560, P < 0.0001) and a significant interaction of time and treatment
(F = 1.808, P < 0.05), whereas the main effect
for treatment approached significance (F = 3.885, P = 0.08).
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Experimental protocol 3.
At day 0 of the experimental treatment period, body mass of
the HF-Lep, HF, and HF-FR groups were similar, but all high-fat diet
animals were significantly heavier than the Con animals (Table 3). After the 12-day treatment period,
body mass of the HF-Lep and HF-FR animals was reduced compared with
day 0. However, at day 12, the body mass of the
HF-Lep, HF, and HF-FR animals remained greater than that of the Con
group.
Sol mass was similar among the high-fat diet groups and among the Con,
HF, and HF-FR groups (Table 3), although the mass of the Sol in the
HF-Lep animals was heavier compared with Con animals. Plant mass was
similar between the HF-Lep and HF animals, but Plant muscles of both
groups were significantly heavier compared with Con animals (Table 3).
HF animals were also found to have Plant muscles that were heavier than
the HF-FR animals. Epididymal fat pad mass was similar among the
HF-Lep, HF, and HF-FR groups (Table 3). Con animals had significantly
lighter epididymal fat pads compared with the HF-Lep, HF, and HF-FR groups.
The rates of submaximal insulin-stimulated skeletal muscle glucose
uptake were reduced by ~48 and 25% in the HF and HF-FR animals,
respectively, when compared with the Con group (Fig. 4). In contrast, rates of
insulin-stimulated glucose uptake were not different between the Con
and HF-Lep animals. Glucose uptake in the hindlimbs of the HF-Lep
animals was greater than that of the HF-FR animals, and HF-FR animals
had rates of glucose uptake that were greater than the HF animals.

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Fig. 4.
Glucose uptake in hindlimbs of Con, HF-Lep, HF, and HF-FR rats
during perfusion with 8 mM glucose and a submaximal (500 µU/ml)
insulin concentration; n = 7/group. Values are
means ± SE. * Significantly different from Con (P < 0.05). Significantly different from HF-Lep (P < 0.05). # Significantly different from HF (P < 0.05).
|
|
Rates of glucose transport were determined in the Sol, Plant, WG, and
RG under submaximal insulin-stimulated conditions using the glucose
analog 3-MG (Fig. 5). 3-MG is carried by
the glucose transporter but is not phosphorylated, which results in its
intracellular accumulation representing the glucose transport process
independent of intracellular disposal. The rates of 3-MG transport in
the Sol, Plant, and RG were significantly reduced in the HF animals compared with both the Con and HF-Lep animals. Similarly, rates of 3-MG
transport in the Sol, Plant, and RG of the HF-FR group were reduced
compared with Con and HF-Lep animals. However, no difference in rates
of 3-MG transport in the Sol, Plant, and RG existed between the HF and
HF-FR groups. Rates of 3-MG transport in the WG were similar among all
experimental groups.

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Fig. 5.
3-O-methylglucose (3-MG) transport in the presence
of 500 µU/ml insulin in hindlimb muscles of Con, HF-Lep, HF, and
HF-FR rats; n = 7/group. Sol, soleus; Plant, plantaris;
WG, white gastrocnemius; RG, red gastrocnemius. Values are means ± SE. * Significantly different from Con (P < 0.05). Significantly different from HF-Lep (P < 0.05).
|
|
Total GLUT-4 protein concentrations in the Sol, Plant, and RG (Fig.
6) and quadricep (Fig.
7) were significantly greater in the
Con and HF-Lep animals compared with either the HF or HF-FR groups. No
difference in GLUT-4 protein concentration existed in the Sol, Plant,
and RG between Con and HF-Lep or HF and HF-FR animals (Fig. 6). GLUT-4
protein concentration in the WG was similar among all experimental
groups (Fig. 6).

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Fig. 6.
GLUT-4 protein concentration, expressed as a percentage of a heart
standard, in skeletal muscles from rats that were divided among 1 of 4 groups: Con, HF-Lep, HF, and HF-FR; n = 7/group. Values
are means ± SE. * Significantly different from Con
(P < 0.05). Significantly different from HF-Lep
(P < 0.05).
|
|

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Fig. 7.
Total and insulin-stimulated plasma membrane (PM) GLUT-4 protein
concentration, expressed as a percentage of a heart standard, in
quadricep muscles from rats that were divided among 1 of 3 groups (Con,
HF-Lep, and HF) and subjected to hindlimb perfusion; n = 8/group. Values are means ± SE. * Significantly different
from Con (P < 0.05). Significantly different
from HF-Lep (P < 0.05).
|
|
Insulin-stimulated plasma membrane GLUT-4 protein concentration was
similar in the Con and HF-Lep animals and was significantly greater
compared with the HF group (Fig. 7). Protein concentration (mg/g wet
wt) of the plasma membrane fractions was similar across the three
treatment groups (Con: 0.57 ± 0.03; HF-Lep: 0.56 ± 0.09; HF: 0.55 ± 0.05). Assessment of 5'-nucleotidase activity
(µmol · min
1 · mg
protein
1) indicated that the plasma membrane fractions
were purified compared with the crude homogenate (Con: 52.6 ± 1.4 vs. 147.2 ± 9.8; HF-Lep: 42.0 ± 3.9 vs. 172.9 ± 6.6;
HF: 37.4 ± 1.4 vs. 167.7 ± 9.1).
After the 12-day treatment period, muscle glycogen concentration was
similar in the Plant, RG, and WG among all experimental animals in
perfusion group 1 (Table 4).
In contrast, glycogen concentration in the Sol of the Con group was
significantly greater compared with HF-Lep, HF, and HF-FR animals. The
glycogen concentration in the Sol of the HF-FR animals was also greater
compared with the HF-Lep animals. Intramuscular triglyceride (IMTG)
content was assessed in the quadriceps of animals in perfusion
group 2 (Table 4). A 3-mo high-fat diet significantly increased
intramuscular triglyceride content as evidenced in the HF group
compared with Con. Of interest, a 12-day leptin treatment period
significantly reduced intramuscular triglyceride levels such that the
intramuscular triglyceride of the Con and HF-Lep animals was similar.
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Table 4.
Glycogen and intramuscular triglyceride concentration in selected
muscles from male Wistar rats after the 12-day treatment period
|
|
Skeletal muscle enzyme activity data are shown in Table
5. Hexokinase activity was similar among
all experimental groups and muscles except in the Sol. Hexokinase
activity in the Sol of the Con group was significantly greater compared
with HF and HF-FR animals. Citrate synthase activity was similar across
all experimental groups and skeletal muscles assessed (Table 5).
-OAC activity was similar in the Sol and RG of all experimental groups (Table 5). However,
-OAC activity was significantly elevated in the Plant and WG of the HF-Lep, HF, and HF-FR groups compared with
Con animals.
 |
DISCUSSION |
Chronic leptin administration has been reported to increase whole
body glucose clearance, insulin-stimulated skeletal muscle glucose
uptake, and 3-MG transport in non-insulin-resistant rats (3, 44,
60). However, little information has been presented to date that
has evaluated the effect of chronic leptin administration in
glucose-intolerant rodents. In this investigation, we provided male
Wistar rats a high-fat diet before leptin treatment as this feeding
regimen has previously been shown to induce skeletal muscle insulin
resistance in rats (48, 49). This diet was effective in
inducing an insulin-resistant state as it was observed that glucose
tolerance was significantly impaired in high-fat-fed animals (Fig. 1).
Our initial inquiry was to determine if chronic leptin administration
improved whole body glucose tolerance in the high-fat-diet-induced insulin-resistant rats (Fig. 2). To assess these effects, we subjected animals to an intravenous glucose tolerance test. Glucose AUC in the
HF-Lep animals were similar to those of the Con animals and were
significantly reduced compared with the HF animals. Of particular
interest, the decreased glucose AUC in the HF-Lep animals occurred with
insulin AUC being slightly lowered. It has been reported that chronic
leptin administration inhibits insulin secretion from isolated islets
(35) and perfused rat pancreas (18). To
further characterize the effects of chronic leptin administration on
pancreatic function, we subjected animals to an intravenous tolbutamide
tolerance test. Tolbutamide binds to the sulfonylurea receptor in the
-cells of the pancreas, which blocks the KATP channels,
leading to an increased intracellular Ca2+ concentration
and increased insulin secretion (32). Unlike the isolated
islet or perfused pancreas studies, we did not find a difference in
tolbutamide-stimulated insulin secretion across treatment groups. These
observations suggest that improvements in whole body glucose tolerance
after chronic leptin treatment result from changes in insulin action,
as opposed to alterations in pancreatic insulin secretion. This
contention is further supported by our observation that
insulin-mediated glucose clearance was enhanced in leptin-treated
animals during a hyperinsulinemic-euglycemic clamp (Fig. 3). Although
these observations have not been reported previously in
insulin-resistant animals, they are consistent with those
investigations that have administered leptin to non-insulin-resistant animals and subsequently found improvements in whole body insulin sensitivity and glucose clearance rates (2, 3, 43).
As over 80% of a glucose load is disposed of by skeletal muscle
(14), it was determined if alterations in skeletal muscle might account for the suppressed whole body glucose disposal rates in
response to a high-fat diet. Rates of insulin-stimulated skeletal muscle glucose uptake (Fig. 4) and 3-MG transport (Fig. 5) were reduced
significantly in the HF and HF-FR animals. This observation is
consistent with Wilkes et al. (58), Hansen et al.
(22), and Buettner et al. (8) who reported
that a high-fat diet reduces insulin-stimulated 3-MG transport
(22, 58) and 2-deoxyglucose uptake (8, 22) in
rodent skeletal muscle. The key observation in the present
investigation was that 12 days of leptin administration completely
reversed high-fat-diet-induced skeletal muscle insulin resistance,
resulting in similar rates of insulin-stimulated skeletal muscle
glucose uptake and 3-MG transport in Con and HF-Lep animals.
The high-fat diet appeared to induce skeletal muscle insulin resistance
by suppressing the expression of the glucose transporter GLUT-4 (Fig.
6). This observation is similar to that of Kahn and Pedersen
(26), who reported total GLUT-4 protein concentration is
reduced in quadricep muscle of Sprague-Dawley rats subjected to a
high-fat diet. Hansen et al. (22) also investigated the effects of a high-fat diet on GLUT-4 protein in rodent skeletal muscle.
Although these investigators, did not evaluate total skeletal muscle
GLUT-4 protein concentration, they did find that a high-fat diet
reduced insulin-stimulated cell surface GLUT-4 protein concentration. In agreement with Hansen et al., we observed insulin-stimulated translocation of glucose transporters to the plasma membrane to be
reduced in the HF animals (Fig. 7).
Most striking, we found that 12 days of leptin administration
completely normalized total skeletal muscle (Figs. 6 and 7) and
insulin-stimulated plasma membrane GLUT-4 protein concentrations (Fig.
7) in animals fed a high-fat diet. When rates of 3-MG transport (Fig.
5) and skeletal muscle GLUT-4 protein concentration (Fig. 6) were
compared, the alterations in total skeletal muscle GLUT-4 protein
concentration could virtually account for either the reduction or
elevation in rates of insulin-stimulated 3-MG transport. This observation is consistent with previous reports that have shown insulin-stimulated skeletal muscle 3-MG glucose transport rates to be
related to the total GLUT-4 protein concentration (1, 7, 23,
28). Furthermore, several investigations have demonstrated that
the total pool of skeletal muscle GLUT-4 protein is associated with the
amount of GLUT-4 translocated to the plasma membrane in response to
insulin (15, 40). Therefore, it is reasonable to suggest
that insulin-stimulated skeletal muscle 3-MG transport was improved in
the HF-Lep animals due to leptin treatment normalizing the total GLUT-4
protein concentration, which in turn resulted in a greater number of
glucose transporters being translocated to the plasma membrane in
response to insulin. However, chronic leptin administration did not
appear to affect 3-MG transport or GLUT-4 protein concentration in the
WG (a type IIb fiber), which suggests that leptin does not affect the
glucose transport pathway in glycolytic muscle fibers.
While plausible to suggest that chronic leptin administration improves
insulin-stimulated glucose uptake and transport due to reductions in
visceral fat concentration, it has been reported recently that the
effect of leptin administration on peripheral insulin action cannot be
explained solely by decreases in visceral fat deposition
(2). Therefore, the possibility exists that secondary
effects in response to leptin treatment may have accounted for these
effects. Leptin administration alters skeletal muscle metabolism by
shifting the muscle from lipid storage to fat oxidation (33). Because a reduction in skeletal muscle triglyceride
levels improves whole body glucose tolerance (30), leptin
may improve insulin-stimulated glucose uptake and transport, in part,
by altering the intramuscular triglyceride concentration
(8). This possibility is consistent with our finding that
leptin administration for 12 days reduced intramuscular triglyceride
levels in animals that had been subjected to 3 mo of a high-fat diet
(Table 4). Chronic leptin treatment may also improve an
insulin-resistant state by attenuating the effects of an elevated blood
tumor necrosis factor-
(TNF-
) level. TNF-
is secreted from
highly active adipocytes in the abdominal region, and serum TNF-
levels have been reported to be elevated in obese, type II diabetics
(27). An excess of TNF-
attenuates in vitro expression
of GLUT-4 (46), inhibits insulin receptor tyrosine kinase
activity (24), and impairs insulin-stimulated glucose
uptake in C2C12 muscle cells (16). Of particular relevance, exogenous leptin administration has been reported to attenuate the effects of elevated TNF-
levels
(50).
Alternatively, the improvements in insulin-stimulated glucose uptake
and transport may be due to leptin exerting a primary effect in the
skeletal muscle. Skeletal muscle expresses both long and short isoforms
of the leptin receptor (20, 51). The leptin receptor is a
member of the gp130 family of cytokine receptors, which stimulate gene
transcription via activation of cytosolic STAT proteins
(5). Both leptin receptor isoforms have signal transduction capabilities (20, 34). Although the
activation and effect of these signals in skeletal muscle in response
to chronic leptin administration are unknown, it is possible that chronic leptin treatment may improve insulin-stimulated glucose transport in skeletal muscle by initiating increases in GLUT-4 protein concentration.
Rates of skeletal muscle glucose uptake and transport can be elevated
in response to increased whole body energy expenditure (21,
37) and/or decreased caloric intake (11, 60), both of which could result in a reduced muscle glycogen concentration. A
reduction in muscle glycogen will facilitate insulin-stimulated glucose
uptake and transport (19). However, muscle glycogen concentration was similar across all treatment groups, with the exception of the Sol (Table 4). Therefore, muscle glycogen levels cannot completely account for differences in rates of glucose uptake
and transport that were observed among the experimental groups (Figs. 4
and 5). This finding is in agreement with Dean et al. (13)
who reported that caloric restriction does not reduce skeletal muscle
glycogen levels or directly influence rates of insulin-stimulated
glucose transport. Cartee and associates (10, 13) have
reported that caloric intake can influence insulin action and membrane
permeability to glucose in skeletal muscle. Although 3-MG transport may
have been elevated in the HF-Lep animals due to caloric restriction,
the HF-Lep and HF-FR animals consumed a similar amount of food
throughout the experimental period. Thus any differences in
insulin-stimulated glucose uptake and transport that existed between
the HF-Lep and HF-FR animals can presumably be attributed to the
effects of chronic leptin treatment.
In summary, we found that a high-fat diet reduced glucose tolerance and
insulin-stimulated skeletal muscle glucose disposal, glucose uptake,
and 3-MG transport. Insulin resistance in the skeletal muscles of the
animals subjected to the high-fat diet appeared to be due to a reduced
GLUT-4 protein concentration. Animals subjected to a high-fat diet and
subsequently treated with leptin exhibited rates of insulin-stimulated
glucose uptake and 3-MG transport that were identical to Con animals.
It appeared that this improvement was due to leptin treatment reversing
a high-fat diet-induced skeletal muscle insulin resistance, at least in
part, by normalizing the total skeletal muscle GLUT-4 protein concentration.
 |
ACKNOWLEDGEMENTS |
We thank Lily Ansari and Ric O'Conner for excellent technical assistance.
 |
FOOTNOTES |
Work done at California State University Northridge was supported in
part by National Institute of General Medical Sciences Grant GM-48680,
the California State University-Northridge Probationary Faculty Support
Program, and by Amgen, Inc.
Current address for N. Levin: Trega Biosciences Inc., 9880 Campus Point
Dr., San Diego, CA 92121 (E-mail: nlevin{at}trega.com).
Address for correspondence and reprint requests: B. B. Yaspelkis III, Dept. of Kinesiology, California State University
Northridge, 1811 Nordhoff St., Northridge, CA 91330-8287 (E-mail:
ben.yaspelkis{at}csun.edu).
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.
Received 10 April 2000; accepted in final form 20 September 2000.
 |
REFERENCES |
1.
Banks, EA,
Brozinick JT, Jr,
Yaspelkis BB, III,
Kang HY,
and
Ivy JL.
Muscle glucose transport, GLUT4 content and degree of exercise training in obese Zucker rats.
Am J Physiol Endocrinol Metab
263:
E1010-E1015,
1992[Abstract/Free Full Text].
2.
Barzilai, N,
She L,
Liu L,
Wang J,
Hu M,
Vuguin P,
and
Rossetti L.
Decreased visceral adiposity accounts for leptin effect on hepatic but not peripheral insulin action.
Am J Physiol Endocrinol Metab
277:
E291-E298,
1999[Abstract/Free Full Text].
3.
Barzilai, N,
Wang J,
Massilon D,
Vuguin P,
Hawkins M,
and
Rossetti L.
Leptin selectively decreases visceral adiposity and enhances insulin action.
J Clin Invest
100:
3105-3110,
1997[Abstract/Free Full Text].
4.
Bass, A,
Brdiczka D,
Eyer P,
Hofer S,
and
Pette D.
Metabolic differentiation of distinct muscle types at the level of enzymatic organization.
Eur J Biochem
10:
198-206,
1969[ISI][Medline].
5.
Baumann, H,
Morella KK,
White DW,
Dembski M,
Bailon PS,
Kim H,
Lai CF,
and
Tartaglia LA.
The full-length leptin receptor has signaling capabilities of interleukin 6-type receptors.
Proc Natl Acad Sci USA
93:
8374-8378,
1996[Abstract/Free Full Text].
6.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
7.
Brozinick Jr, JT,
Etgen GJ, Jr,
Yaspelkis BB, III,
Kang HY,
and
Ivy JL.
Effects of exercise training on muscle GLUT4 protein content and translocation in obese Zucker rats.
Am J Physiol Endocrinol Metab
265:
E419-E427,
1993[Abstract/Free Full Text].
8.
Buettner, R,
Newgard CB,
Rhodes CJ,
and
O'Doherty RM.
Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia.
Am J Physiol Endocrinol Metab
278:
E563-E569,
2000[Abstract/Free Full Text].
9.
Burton, GW,
Webb A,
and
Ingold KU.
Methods: a mild, rapid and effecient method of lipd extraction for use in determining vitamin E/lipid ratios.
Lipids
20:
29-39,
1985[ISI][Medline].
10.
Cartee, G,
Kietzke E,
and
Briggs-Tung C.
Adaptation of muscle glucose transport with caloric restriction in adult, middle-aged, and old rats.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R1443-R1447,
1994[Abstract/Free Full Text].
11.
Chen, G,
Koyama K,
Yuan X,
Lee Y,
Zhou Y-T,
O'Doherty R,
Newgard CB,
and
Unger RH.
Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy.
Proc Natl Acad Sci USA
93:
14795-14799,
1996[Abstract/Free Full Text].
12.
Cioffi, JA,
Shafer AW,
Zupancic TJ,
Smith-Gbur J,
Mikhail A,
Platika D,
and
Snodgrass HR.
Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction.
Nat Med
2:
585-589,
1996[ISI][Medline].
13.
Dean, JJ,
Brozinick JT, Jr,
Cushman SW,
and
Cartee GD.
Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle.
Am J Physiol Endocrinol Metab
275:
E957-E964,
1998[Abstract/Free Full Text].
14.
Defronzo, RA,
Jacot E,
Jequier E,
Maeder E,
Wahren J,
and
Felber JP.
The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization.
Diabetes
30:
1000-1007,
1981[ISI][Medline].
15.
Dela, F,
Mikines KJ,
Linstow M,
Seccher NH,
and
Galbo H.
Effect of training on insulin-mediated glucose uptake in human muscle.
Am J Physiol Endocrinol Metab
263:
E1134-E1143,
1992.
16.
Del Aguila, LF,
Claffey KP,
and
Kirwan JP.
TNF-
impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells.
Am J Physiol Endocrinol Metab
276:
E849-E855,
1999[Abstract/Free Full Text].
17.
Després, JP.
Abdominal obesity as an important component of insulin resistance syndrome.
Nutrition
9:
452-459,
1993[ISI][Medline].
18.
Fehmann, HC,
Peiser C,
Bode HP, SM,
Staats P,
Hedetoft C,
Lang RE,
and
Goke B.
Leptin: a potent inhibitor of insulin secretion.
Peptides
18:
1267-1273,
1997[ISI][Medline].
19.
Fell, RD,
Terblanche SE,
Ivy JL,
Young JC,
and
Holloszy JO.
Effect of muscle glycogen content on glucose uptake following exercise.
J Appl Physiol
52:
434-437,
1982[Abstract/Free Full Text].
20.
Ghilardi, N,
Ziegler S,
Wiestner A,
Stoffel R,
Heim M,
and
Radek SC.
Defective STAT signaling by the leptin receptor in diabetic mice.
Proc Natl Acad Sci USA
93:
6231-6235,
1996[Abstract/Free Full Text].
21.
Halaas, JL,
Gajiwala KS,
Maffei M,
Cohen SL,
Chait BT,
Rabinowitz D,
Lallone RL,
Burley SK,
and
Friedman JF.
Weight-reducing effects of the plasma protein encoded by the obese gene.
Science
269:
543-546,
1995[ISI][Medline].
22.
Hansen, PA,
Han DH,
Marshall BA,
Nolte LA,
Chen MM,
Mueckler M,
and
Holloszy JO.
A high fat diet impairs stimulation of glucose transport in muscle. Functional evaluation of potential mechanisms.
J Biol Chem
273:
26157-26163,
1998[Abstract/Free Full Text].
23.
Henriksen, EJ,
Bourey RE,
Rodnick KJ,
Koranyi L,
Permutt MA,
and
Holloszy JO.
Glucose transporter protein content and glucose transport capacity in rat skeletal muscles.
Am J Physiol Endocrinol Metab
259:
E593-E598,
1990[Abstract/Free Full Text].
24.
Hotamisligil, GS,
and
Spiegelman BM.
Tumor necrosis factor
: a key component of the obesity-diabetes link.
Diabetes
43:
1271-1278,
1994[Abstract].
25.
Ivy, JL,
Brozinick JT, Jr.,
Torgan CE,
and
Kastello GM.
Skeletal muscle glucose transport in obese Zucker rats after exercise training.
J Appl Physiol
66:
2635-2641,
1989[Abstract/Free Full Text].
26.
Kahn, BB,
and
Pedersen O.
Suppression of GLUT4 expression in skeletal muscle of rats that are obese from high fat feeding but not from high carbohydrate feeding or genetic obesity.
Endocrinology
132:
13-22,
1993[Abstract].
27.
Katsuki, A,
Sumida Y,
Murashima S,
Murata K,
Takarada Y,
Ito K,
Fujii M,
Tsuchihashi K,
Goto H,
Nakatani K,
and
Yano Y.
Serum levels of tumor necrosis factor-alpha are increased in obese patients with non-insulin dependent diabetes mellitus.
J Clin Endocrinol Metab
83:
859-862,
1998[Abstract/Free Full Text].
28.
Kern, M,
Wells JA,
Stevens JM,
Elton CW,
Friedman JE,
Tapscott EB,
Pekala PH,
and
Dohm GL.
Insulin responsiveness in skeletal muscle is determined by glucose transporter (GLUT4) protein level.
Biochem J
270:
397-400,
1990[ISI][Medline].
29.
Kieffer, TJ,
Heller RS,
and
Habner JF.
Leptin receptors expressed on pancreatic beta-cells.
Biochem Biophys Res Commun
224:
522-527,
1996[ISI][Medline].
30.
Koyama, K,
Chen G,
Lee Y,
and
Unger RH.
Tissue triglycerides, insulin resistance, and insulin production: implications for hyperinsulinemia of obesity.
Am J Physiol Endocrinol Metab
273:
E708-E713,
1997[ISI][Medline].
31.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophase T4.
Nature
227:
680-685,
1970[ISI][Medline].
32.
Mariot, P,
Gilon P,
Nenquin M,
and
Henquin JC.
Tolbutamide and diazoxide influence insulin secretion by changing the concentration but not the action of cytoplasmic Ca2+ in
-cells.
Diabetes
47:
365-373,
1998[Abstract].
33.
Muoio, DM,
Dohm GL,
Fiedorek FT,
Tapscott EB,
and
Coleman RA.
Leptin directly alters lipid partitioning in skeletal muscle.
Diabetes
46:
1360-1363,
1997[Abstract].
34.
Murakami, T,
Yamashita T,
Iida M,
Kuwajima M,
and
Shima K.
A short form of leptin receptor performs signal transduction.
Biochem Biophys Res Commun
231:
26-29,
1997[ISI][Medline].
35.
Pallett, AL,
Morton NM,
Cawthorne MA,
and
Emilsson V.
Leptin inhibits insulin secretion and reduces insulin mRNA levels in rat isolated pancreatic islets.
Biochem Biophys Res Commun
238:
267-270,
1997[ISI][Medline].
36.
Passonneau, JV,
and
Lauderdale VR.
A comparison of three methods of glycogen measurement in tissues.
Anal Biochem
60:
405-412,
1974[ISI][Medline].
37.
Pellymounter, MA,
Cullen MJ,
Baker MB,
Hecht R,
Winters D,
Boone T,
and
Collins F.
Effects of the obese gene product on body weight regulation in ob/ob mice.
Science
269:
540-543,
1995[ISI][Medline].
38.
Poitout, V,
Rouault C,
Guerre-Millo M,
and
Reach G.
Does leptin regulate insulin secretion?
Diabetes Metab
24:
321-326,
1998[ISI][Medline].
39.
Reaven, GM.
Pathophysiology of insulin resistance in human disease.
Physiol Rev
76:
473-486,
1995.
40.
Reynolds, TH,
Brozinick JT,
Rogers MA,
and
Cushman SW.
Effects of exercise training on glucose transport and cell-surface GLUT-4 in isolated rat epitrochlearis muscle.
Am J Physiol Endocrinol Metab
274:
E773-E778,
1997[ISI].
41.
Ruderman, NB,
Houghton CRS,
and
Helms R.
Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism.
Biochem J
124:
639-651,
1971[ISI][Medline].
42.
Sherman, WM,
Katz AL,
Cutler CL,
Withers RT,
and
Ivy JL.
Glucose transport: locus of muscle insulin resistance in obese Zucker rats.
Am J Physiol Endocrinol Metab
255:
E374-E382,
1988[Abstract/Free Full Text].
43.
Shi, ZQ,
Nelson A,
Whitcomb L,
Wang J,
and
Cohen AM.
Intracerebroventricular administration of leptin markedly enhances insulin sensitivity and systemic glucose utilization in conscious rats.
Metabolism
47:
1274-80,
1998[ISI][Medline].
44.
Sivitz, WI,
Walsh SA,
Morgan DA,
Thomas MJ,
and
Haynes WG.
Effects of leptin on insulin sensitivity in normal rats.
Endocrinology
138:
3395-3401,
1997[Abstract/Free Full Text].
45.
Srere, PA.
Citrate synthase.
In: Methods in Enzymology. New York: Academic, 1969, p. 3-5.
46.
Stephens, JM,
and
Pekala PH.
Transcriptional repression of the GLUT4 and C/EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-alpha.
J Biol Chem
266:
21839-21845,
1991[Abstract/Free Full Text].
47.
Stephens, TW,
Basinski M,
Bristow PK,
Bue-Valleskey JM,
Burgett SG,
Craft L,
Hale J,
Hoffman J,
Hsiung HM,
Kriauciunas A,
MacKellar W,
Rosteck PR,
Schoner B,
Smith D,
Tinsely FC,
Zhang X-Y,
and
Heiman M.
The role of neuropeptide Y in the antiobesity action of the obese gene product.
Nature
377:
530-532,
1995[ISI][Medline].
48.
Storlien, LH,
James DE,
Burleigh KM,
Chisolm DJ,
and
Kraegen EW.
Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats.
Am J Physiol Endocrinol Metab
251:
E576-E583,
1986[Abstract/Free Full Text].
49.
Storlien, LH,
Jenkins AB,
Chisolm DJ,
Pascoe WS,
Khouri S,
and
Kraegen EW.
Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid.
Diabetes
40:
280-289,
1991[Abstract].
50.
Takahashi, N,
Waelput W,
and
Guisez Y.
Leptin is an endogenous protective protein against the toxicity exerted by tumor necrosis factor.
J Exp Med
189:
207-212,
1999[Abstract/Free Full Text].
51.
Tartaglia, L,
Dembski M,
Weng X,
Deng G,
Culpepper J,
Devos R,
Richards GJ,
Campfield LA,
Clark FT,
Deeds J,
Muir C,
Sanker S,
Moriarty A,
Moore KJ,
Smutko JS,
Mays GG,
Woolf EA,
Monrow CA,
and
Tepper RI.
Identification and expression cloning of a leptin receptor, OB-R.
Cell
83:
1263-1271,
1995[ISI][Medline].
52.
Touster, O,
Aronson NN,
Dulaney JT,
and
Hendrickson H.
Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes.
J Cell Biol
47:
604-618,
1970[Abstract/Free Full Text].
53.
Towbin, H,
Staehelin T,
and
Gordon J.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354,
1979[Abstract].
54.
Turcotte, LP,
Swenberger JR,
Tucker MZ,
and
Yee AJ.
Training-induced elevation in FABPpm is associated with increased palmitate use in contracting muscle.
J Appl Physiol
87:
285-293,
1999[Abstract/Free Full Text].
55.
Uyeda, K,
and
Racker E.
Regulatory mechanisms in carbohydrate metabolism. VII. Hexokinase and phosphofructokinase.
J Biol Chem
240:
4682-4688,
1965[Free Full Text].
56.
Wang, JL,
Chinookoswong N,
Scully S,
Qi M,
and
Shi ZQ.
Differential effects of leptin in regulation of tissue glucose utilization in vivo.
Endocrinology
140:
2117-2124,
1999[Abstract/Free Full Text].
57.
Wang, MY,
Zhou YT,
Newgard CB,
and
Unger RH.
A novel leptin receptor isoform in rat.
FEBS Lett
392:
87-90,
1996[ISI][Medline].
58.
Wilkes, JJ,
Bonen A,
and
Bell RC.
A modified high-fat diet induces insulin resistance in rat skeletal muscle but not adipocytes.
Am J Physiol Endocrinol Metab
275:
E679-E686,
1998[Abstract/Free Full Text].
59.
Wu-Peng, XS,
Chua J, SC,
Okada N,
Liu S-M,
Nicolson M,
and
Leibel RL.
Phenotype of the obese Koletsky (f) rat due to Tyr763stop mutation in the extracellular domain of the leptin receptor (Lepr).
Diabetes
46:
513-518,
1997[Abstract].
60.
Yaspelkis, BB, III,
Ansari L,
Ramey EA,
and
Loy SF.
Chronic leptin administration increases insulin-stimulated skeletal muscle glucose uptake and transport.
Metabolism
48:
671-676,
1999[ISI][Medline].
61.
Yaspelkis, BB, III,
Castle AL,
Farrar RP,
and
Ivy JL.
Contraction-induced intracellular signals and their relationship to muscle GLUT-4 concentration.
Am J Physiol Endocrinol Metab
272:
E118-E125,
1997[Abstract/Free Full Text].
62.
Zhang, Y,
Proenca R,
Maffei M,
Barone M,
Leopold L,
and
Friedman JM.
Positional cloning of the mouse obese gene and its human homologue.
Nature
372:
425-432,
1994[ISI][Medline].
Am J Physiol Endocrinol Metab 280(1):E130-E142
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