Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia

Roland Buettner1,2, Christopher B. Newgard1,2,3, Christopher J. Rhodes1,2,4, and Robert M. O'Doherty1,2

1 Gifford Laboratories for Diabetes Research, and Departments of 2 Internal Medicine, 3 Biochemistry, and 4 Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human obesity and high fat feeding in rats are associated with the development of insulin resistance and perturbed carbohydrate and lipid metabolism. It has been proposed that these metabolic abnormalities may be reversible by interventions that increase plasma leptin. Up to now, studies in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy with mixed results. Our study sought to determine whether a sustained, moderate increase in plasma leptin, achieved by administration of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin) would be effective in reversing the metabolic abnormalities of the obese phenotype. Wistar rats fed a high-fat diet (HF) were heavier (P < 0.05), had increased fat mass and intramuscular triglycerides (mTG), and had elevated plasma glucose, insulin, triglyceride, and free fatty acids compared with standard chow-fed (SC) control animals (all P < 0.01). HF rats also had impaired glucose tolerance and were markedly insulin resistant, as demonstrated by a 40% reduction in insulin-stimulated muscle glucose uptake (P < 0.001). Increasing plasma leptin levels to 29.0 ± 1.5 ng/ml (from 7.0 ± 1.4 ng/ml, P < 0.001) for a period of 6 days decreased adipose mass by 40% and normalized plasma glucose and insulin levels. In addition, insulin-stimulated skeletal muscle glucose uptake was normalized in hyperleptinemic rats, an effect that correlated closely with a 60% (P < 0.001) decrease in mTG. Importantly, HF rats that received a control adenovirus containing the beta -galactosidase cDNA and were calorically matched to AdCMV-leptin-treated animals remained hyperglycemic, hyperinsulinemic, insulin resistant, and maintained elevated mTG. We conclude that a gene-therapeutic intervention that elevates plasma leptin moderately for a sustained period reverses diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance, and that these improvements are tightly linked to leptin-induced reductions in mTG.

obesity; tissue triglycerides; leptin action; insulin action


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OBESITY IS A METABOLIC DISORDER characterized by weight gain, increased adiposity, insulin resistance, perturbed carbohydrate and lipid metabolism, and in many cases the development of non-insulin-dependent diabetes mellitus (2, 3, 7). Administration of leptin corrects the metabolic abnormalities of the ob/ob mouse, which lacks the adipocyte hormone (17, 21, 24), whereas hyperleptinemia in the normal rat reduces plasma and tissue lipids and increases insulin sensitivity (1, 5, 26). On the basis of these observations it has been proposed that leptin therapy may be effective in the treatment of human obesity.

To date, studies on the efficacy of leptin therapy in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy, with mixed results. Thus in high fat fed (HF) animals, a widely used model of obesity, intraperitoneal delivery of recombinant leptin reduces food intake and weight gain (4, 8). However, these studies did not address the ability of leptin therapy to reverse the metabolic abnormalities associated with high fat feeding. Human trials have reported weight loss in response to delivery of leptin by injection (7), but the losses have generally been minor compared with the weight of those individuals before the initiation of leptin therapy. Leptin injection therapy may be complicated by the short half-life of the peptide in the circulation (16, 31). Thus a more effective therapy may require a sustained, steady-state increase in leptin levels. Indeed, a recent study has demonstrated the greater effectiveness of sustained leptin increases over intraperitoneal leptin delivery in decreasing food intake and weight gain in the ob/ob mouse (16).

We have demonstrated previously that sustained, moderate hyperleptinemia can be achieved in normal rats by use of a recombinant adenovirus gene delivery system (5, 19). In the present study we have used this system to induce hyperleptinemia in the HF rat and tested the effects of this gene-therapeutic intervention on the metabolic abnormalities associated with obesity. The results demonstrate that hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance are corrected by a sustained, moderate increase in plasma leptin. The reversal of skeletal muscle insulin resistance is strongly correlated to a decrease in muscle triglyceride levels.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care and maintenance. Male Wistar rats were purchased from Charles River, at a weight of 150-175 g. After arrival, rats were caged individually with free access to water and either a high-fat diet (HF diet, 45% of calories from fat, Harlan Teklad, Madison WI., TD 96001) or standard rat chow diet (SC diet, 11% of calories from fat, Harlan Teklad), except where indicated below, and food intake and body weight were recorded on a daily basis. Animals were held on a 12:12-h light-dark cycle.

Experimental design. After 6 wk of a HF or SC diet, animals were anesthetized with pentobarbital sodium (50-100 mg/100 g body wt ip) between 9 and 10 AM. Samples of the medial part of the right gastrocnemius muscle were rapidly excised and frozen in liquid nitrogen and used to gain a representative measure of hindlimb muscle triglyceride levels. Blood samples were collected into EDTA-rinsed vials for analysis of plasma variables. Solei muscles were excised, and basal and insulin-stimulated 2-deoxyglucose uptake was measured as described below. Mesenteric, perirenal, and epididymal fat pads were excised, patted dry of excess fluid, and weighed. Additional groups of 6-wk HF- or SC-fed animals received recombinant adenovirus containing either the rat leptin cDNA (AdCMV-leptin) (5) or the Escherichia coli beta -galactosidase gene (AdCMV-beta Gal) (10). Where indicated, food intake in control groups was matched to animals receiving AdCMV-leptin. HF-fed or SC-fed animals were infused with 1 × 1012 viral particles in 200-250 µl PBS into a tail vein via a 22-gauge catheter as previously described (28). To reduce immunologic responses to the infused viruses, animals were treated with 15 mg · kg-1 · day-1 cyclosporin A (Calbiochem, La Jolla, CA) intraperitoneally one day before the virus infusion, on the infusion day, and on the following day. Rats were also treated with 1.5 mg · kg-1 · day-1 methylprednisone (Pharmacia Upjohn) intramuscularly on the same days. Four to five hours before the virus infusion, an extra injection of 3 mg/kg prednisone was given intramuscularly. No immunosuppression occurred for the five remaining days before an experiment. Six days after administration of the recombinant adenoviruses, blood samples were taken, solei and right medial gastrocnemius muscles were excised to measure 2-deoxyglucose uptake and triglyceride levels, respectively, and fat pads were isolated.

Oral glucose tolerance tests and measurement of insulin-stimulated skeletal muscle glucose uptake. Oral glucose tolerance tests were begun at 9:00 AM after an overnight fast. The rats were weighed, and a whole blood sample was taken from the capillary bed of the tail tip. Two grams of glucose/kg body wt were administered orally by gavage in a 40% glucose solution. Whole blood samples were taken 30, 60, 90, and 120 min after the gavage. Glucose values were measured in the samples with a HemoCue glucometer (HemoCue AB, Angelholm, Sweden). 2-Deoxyglucose uptake was measured in whole rat soleus muscles by use of a modification of previously described methods (9, 29). Briefly, muscles were quickly excised with animals under pentobarbital anesthesia, weighed, mounted onto stainless steel clips, and subjected to a series of incubations in modified Krebs-Ringer-Henseleit (KRH) buffer (118.4 mM NaCl, 1.19 mM KH2PO4, 4.76 mM KCl, 1.19 mM MgSO4, 24.9 mM NaHCO3, 1.2 mM CaCl2, 0.1% fat-free BSA) with additions, as described below, at 29°C in a shaking metabolic waterbath and under 95% O2-5% CO2. The muscles were first incubated in KRH buffer containing 8 mM glucose and 32 mM mannitol for 30 min and then in KRH buffer containing 40 mM mannitol for 10 min. They were transferred to KRH buffer containing 39 mM [14C]mannitol (NEN, 51.5 Ci/mmol) and 1 mM 2-deoxy[3H]glucose (NEN, 25.5 Ci/mmol) for 30 min in the presence or absence of insulin at a final concentration of 1 mU/ml. The muscles were then hydrolyzed at 70°C for 1 h in 1 N NaOH. A 200-µl aliquot was assayed for radioactivity in scintillation cocktail, and 2-deoxyglucose uptake was calculated as nanomoles of 2-deoxyglucose per microliters intracellular water per hour.

Skeletal muscle triglycerides. Muscle triglycerides were determined as described previously (28) with slight modifications. Briefly, frozen muscle samples were first powdered under liquid nitrogen. Twenty to fifty milligrams of frozen muscle powder were then weighed into 1 ml of a chloroform-methanol mix (2:1) and incubated for 1 h at room temperature with occasional shaking to extract the lipid. After addition of 200 µl H2O, vortexing, and centrifugation for 5 min at 3,000 g, the lower lipid phase was collected and dried at room temperature. The lipid pellet was redissolved in 60 µl tert-butanol and 40 µl of a Triton X-114-methanol (2:1) mix, and triglycerides were measured by means of the GPO-triglyceride kit (Sigma, St. Louis, MO) with Lintrol lipids as standards (Sigma).

Plasma measurements. Plasma triglycerides and free fatty acids were measured with kits from Sigma and Boehringer Mannheim, respectively. Plasma glucose was measured with a HemoCue glucose analyzer (HemoCue AB). Plasma leptin and insulin were measured using rat-specific RIA kits (Linco Research, St. Charles, MO).

Statistical methods. All results are expressed as means of 4-10 independent experiments ± SE. Statistical significance was determined by an unpaired Student's t-test by use of the statistics module of Microsoft Excel, Version 5.0 (Microsoft, Seattle, WA). Statistical significance was assumed at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fasting plasma variables, fat depots, and skeletal muscle triglyceride levels in the high fat fed rat. After 6 wk, marked alterations in carbohydrate and lipid metabolism were observed in HF rats compared with SC control animals (Table 1). Fasting plasma insulin, glucose, and leptin levels were increased in HF animals by 6.5-, 1.4-, and 7.0-fold, respectively. In addition, skeletal muscle triglycerides were increased 3.6-fold, and there were marked increases in plasma triglycerides (2.0-fold), free fatty acids (1.9-fold), and visceral fat mass (2.7-fold). Finally, HF rats were significantly heavier then SC controls. Taken together, these data demonstrate that the HF rats display a metabolic profile consistent with obesity, insulin resistance, and a perturbed state of lipid and carbohydrate homeostasis.

                              
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Table 1.   Basal variables in SC and HF rats

Confirmation of insulin resistance induced by high fat feeding. To confirm diet-induced insulin resistance, SC and HF animals were fasted overnight and then subjected to an oral glucose tolerance test and analysis of insulin-stimulated glucose uptake in skeletal muscle (Figs. 1 and 2). Administration of a glucose bolus to HF animals resulted in whole blood glucose levels that were significantly greater than those of SC animals at all time points tested (Fig. 1). In addition, at 120 min postglucose bolus, when blood glucose had returned to baseline in the SC group, blood glucose in the HF animals had decreased only marginally from the maximum value at 60 min postglucose bolus (Fig. 1), demonstrating severe whole body glucose intolerance and implying the presence of insulin resistance. Because a major site of glucose disposal is skeletal muscle, basal and insulin-stimulated glucose uptakes were measured in the isolated soleus muscle of HF and SC animals. Basal glucose uptake was unchanged by high fat feeding; however, insulin-stimulated glucose uptake was reduced by 40% compared with that of SC animals (Fig. 2). These data demonstrate insulin resistance in skeletal muscle of HF rats.


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Fig. 1.   Male Wistar rats were fed a high fat (HF) or a standard chow (SC) diet for 6 wk; n = 4 animals for each condition. At end of period all animals were fasted overnight (18 h) and then received 2.0 g/kg glucose by gavage. Tail vein blood samples were taken 0, 30, 60, 90, and 120 min after gavage, and blood glucose levels were measured. *, ** Significant difference between HF and SC animals at P < 0.05 and P < 0.01, respectively.



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Fig. 2.   Male Wistar rats were fed HF or SC diet for 6 wk. At end of period all animals were fasted overnight (18 h), soleus muscles were excised, and 2-deoxyglucose (2-DG) uptake was measured in absence (basal) or presence (+ insulin) of insulin. *** Significant difference between HF and SC groups (P < 0.001). There were 9 independent measurements for each condition.

Leptin action in the high fat fed rat. We next determined the effects of a sustained, moderate increase in leptin on the obese phenotype. Recombinant adenovirus containing the leptin cDNA was administered to both SC and HF animals, and a number of variables of leptin action were monitored (Fig. 3 and Table 2). A control HF group received an adenovirus expressing beta -galactosidase, and food intake was subsequently matched to food intake in hyperleptinemic HF animals. Hyperleptinemia decreased caloric intake in both HF and SC animals (Fig. 3A), but the decrease was significantly greater in SC animals (Table 2). Cumulative weight loss induced by hyperleptinemia was significant in SC, but not in HF animals (Table 2); however, when expressed as grams lost per day, HF rats lost significant body wt from day 5 to day 6 of hyperleptinemia (Fig. 3B). Visceral fat mass was markedly decreased by hyperleptinemia in HF rats (Table 2), and the loss was substantially greater in absolute terms compared with hyperleptinemic SC and calorically matched, AdCMV-beta Gal-treated HF rats.


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Fig. 3.   Male Wistar rats were fed HF or SC diet for 6 wk. At end of period animals received a recombinant adenovirus encoding leptin cDNA (HF-Lep, n = 9 and SC-Lep, n = 7). Food intake (A) and weight (B) were monitored for the next 6 days. Control HF group received a recombinant adenovirus expressing beta -galactosidase and were maintained on same caloric intake as HF-Lep (HF-beta Gal-CM, n = 5). * Significant difference between HF-Lep and HF-beta Gal-CM (P < 0.05).


                              
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Table 2.   Effects of 6 days of hyperleptinemia or caloric matching on weight, food intake, and visceral fat mass in HF and SC animals

Effects of hyperleptinemia on plasma metabolic variables and skeletal muscle insulin sensitivity. We next tested the hypothesis that sustained hyperleptinemia would correct the metabolic abnormalities and skeletal muscle insulin resistance associated with diet-induced obesity. First, hyperleptinemia resulted in a reduction in plasma glucose and insulin in HF animals to levels comparable with those in SC animals (Table 3). Importantly, these effects were specific, because HF animals that received AdCMV-beta Gal and were calorically matched to the hyperleptinemic HF rats remained hyperglycemic and hyperinsulinemic compared with SC and hyperleptinemic HF rats (Table 3). As previously reported (5), hyperleptinemia in SC rats also reduced plasma glucose and insulin (Table 3), an effect that was not observed in SC calorically matched animals (data not shown). These data suggest that insulin sensitivity is improved by hyperleptinemia. To evaluate this directly, we measured insulin-stimulated glucose uptake in skeletal muscle from hyperleptinemic HF and control animals. Skeletal muscle insulin sensitivity was restored in hyperleptinemic HF rats to levels that were similar to those in SC animals (Fig. 4). It is noteworthy that treatment of HF rats with AdCMV-beta Gal and subsequent caloric matching to hyperleptinemic HF rats did not correct skeletal muscle insulin resistance. Finally, plasma triglycerides and intramuscular triglycerides, but not free fatty acids, were decreased in hyperleptinemic HF rats compared with HF controls (Table 3 and Fig. 5). Caloric matching of AdCMV-beta Gal-treated HF rats to hyperleptinemic rats also reduced plasma triglycerides and free fatty acids; however, skeletal muscle triglycerides in calorically matched animals remained at levels similar to those in HF animals fed ad libitum. Because insulin resistance was not corrected in calorically matched animals, these data suggest that the leptin-mediated reduction in skeletal muscle triglyceride stores underlies improvements in insulin action. Indeed, a comparison of tissue triglyceride levels to skeletal muscle insulin sensitivity in individual animals demonstrated a negative correlation of r -0.7 (Fig. 6), lending further support to this possibility.

                              
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Table 3.   Effects of 6 days of hyperleptinemia or caloric matching on plasma variables in HF and SC rats



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Fig. 4.   Male Wistar rats were treated as detailed in legends of Figs. 2 and 3. After overnight fast, 2-DG uptake was measured in absence (basal) or presence (+ insulin) of insulin in soleus muscle preparations from SC (n = 9), HF (n = 9), SC-Lep (n = 5), hyperleptinemic HF (HF-Lep, n = 9), and HF-beta Gal-CM (n = 5). *** Significant difference between indicated and corresponding HF groups (P < 0.001).



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Fig. 5.   Male Wistar rats were treated as detailed in legends of Figs. 2 and 3. After overnight fast, skeletal muscle triglyceride levels were measured in SC (n = 9), HF (n = 9), SC-Lep (n = 5), HF-Lep (n = 9), and HF-beta Gal-CM (n = 5). *** Significant difference between indicated group and HF group (P < 0.001).



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Fig. 6.   Relationship between skeletal muscle triglyceride levels and insulin-stimulated 2-DG uptake in HF, SC, hyperleptinemic HF (HF-Lep), hyperleptinemic SC (SC-Lep), and HF rats that received adenovirus containing beta -galactosidase cDNA (AdCMV-beta Gal) and were subsequently calorically matched to HF-HL (HF-beta Gal-CM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The ability of leptin administration to reverse metabolic abnormalities in the ob/ob mouse (17, 21, 24) and improve insulin action in normal animals (1, 5, 26) has led to the proposal that leptin may serve as an effective therapy for human obesity. However, a number of questions remain unanswered in this regard. First, it is not clear that increasing plasma leptin levels will be sufficient to correct the metabolic abnormalities associated with obesity, principally insulin resistance and perturbed lipid and carbohydrate metabolism. Second, leptin therapy involving multiple injections has had mixed results both in animal models of obesity and in human trials, and this suggests that alternative strategies such as sustained increases in plasma leptin should be considered. The current study addressed these questions in the HF rat, a model of obesity that displays a metabolic phenotype similar to that in human obesity.

Previous studies in the HF obese model demonstrated that leptin administration can alter food intake and weight gain (4, 8) but did not address the capacity of leptin to correct the obese phenotype. This is an important question, because correction of the metabolic abnormalities of obesity with leptin has been demonstrated only in the ob/ob mouse, a model that lacks endogenous leptin. Thus the current study extends previous observations of leptin action in obesity by demonstrating in a nongenetic model that hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance are corrected by a sustained, moderate increase in plasma leptin mediated by recombinant adenovirus administration. It is important that we do not observe a similar correction of insulin action or plasma variables in HF animals that received a control adenovirus expressing beta -galactosidase and were calorically matched to the HF hyperleptinemic animals.

The increased skeletal muscle insulin sensitivity induced by hyperleptinemia in HF rats is associated with decreases in muscle triglyceride levels. It is noteworthy that muscle triglycerides remained elevated and insulin resistance was maintained in HF animals calorically matched to HF hyperleptinemic animals, whereas plasma variables of lipid metabolism (free fatty acids and triglycerides) were similar in the two groups. A comparison of muscle triglycieride levels with muscle insulin-stimulated glucose uptake demonstrated a strong negative correlation (r = -0.7). Of interest in this regard is the clustering of insulin-"sensitive" groups (SC, hyperleptinemic SC and hyperleptinemic HF) and insulin-"resistant" groups (HF and AdCMV-beta Gal-treated, calorically matched HF). Although these observations do not demonstrate a mechanistic link between tissue triglycerides and insulin sensitivity, a number of other studies have demonstrated a close relationship between perturbed lipid metabolism and insulin resistance (see Ref. 15 for review). Moreover, recent studies in rat and humans (12, 18, 20, 22) have demonstrated a similar relationship between insulin sensitivity and skeletal muscle triglyceride depots. In the Zucker diabetic fatty rat, increases in tissue triglycerides occur in parallel with the development of the insulin resistance characteristic of this model (13), whereas insulin resistance does not develop when pharmaceutical interventions are used that reduce or prevent muscle triglyceride accumulation during a high fat diet or in genetic models of obesity (14, 27). Although the mechanistic link between triglyceride accumulation and insulin resistance remains to be defined, lipids have been implicated in altering elements of the insulin signaling pathway (11, 33) and are known to regulate fuel disposal (23, 25, 32). Additional studies are required to determine the mechanisms underlying leptin-mediated increases in muscle insulin sensitivity.

Given that a subset of obese patients will likely be amenable to leptin therapy, much as some insulin-resistant type II diabetics respond to insulin therapy, an important consideration will be the method of leptin delivery. Injection therapy and more sustained increases in plasma leptin have been proposed as treatment methods. In humans, weight loss after injection therapy has been minor compared with the starting weight of the subjects (7). In animal models of obesity, a diminished (8) or absent (30) response to peripherally injected leptin has been reported. These effects may be explained by increases in plasma leptin levels that are transitory, thus compromising the efficiency of leptin action. Indeed, a recent study (16) in the ob/ob mouse demonstrated that intraperitoneally injected leptin was undetectable in plasma only 3 h after injection, having reached a peak value of 335 ng · ml-1 · 1 h-1 after injection. This study also demonstrated that intraperitoneal leptin injection was less effective at promoting decreases in food intake and weight gain compared with the effects of a sustained increase in plasma leptin. Concern about temporary increases in leptin levels was circumvented in the current study by use of a gene-therapeutic intervention that resulted in a sustained moderate increase in leptin. This strategy is clearly effective, because a quite modest increase in leptin from 7 to 29 ng/ml had a substantial impact on fuel homeostasis. However, additional studies are required to determine whether therapeutic strategies that maintain constant elevated leptin levels will be an effective therapy in the treatment of human obesity.

This study and others (4, 6, 8, 30) have demonstrated that diet-induced obesity is associated with elevated plasma leptin levels, but with accelerated weight gain and normal or increased caloric intake, leading to speculation that animals on high fat diets are leptin resistant. The data in the present study demonstrate that, if present, leptin resistance can be overcome by an intervention that sustains a moderate increase in plasma leptin levels above the levels present in the HF rat. Thus food intake and visceral adiposity were decreased substantially by hyperleptinemia in both SC and HF rats. Indeed, visceral fat loss in HF hyperleptinemic rats was greater in absolute terms than in hyperleptinemic SC and HF calorically matched animals. This suggests that leptin was more effective at mobilizing lipid stores in the HF animals. Caloric intake in HF hyperleptinemic animals was reduced but remained ~15% above that of SC hyperleptinemic rats. Because caloric intake before hyperleptinemia and the level of hyperleptinemia achieved were similar in the two groups, these data may indicate that HF rats are partially resistant to leptin effects on food intake. Although weight loss occurred in SC hyperleptinemic rats, it did not reach significance in the HF hyperleptinemic animals. This may be due to the higher caloric intake, the greater energy reserves in the form of adipose tissue, or a partial resistance to weight loss in HF hyperleptinemic animals. Given that leptin resistance has been proposed to play a role in the pathogenesis of obesity, it is important that the nature and extent of leptin resistance in the HF rat be defined.


    ACKNOWLEDGEMENTS

These studies were supported by National Institutes of Health Grant P50H2598801 (to C. B. Newgard) and Novo Nordisk.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. M. O'Doherty, Univ. of Pittsburgh Medical Center, E1112 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: odohertyr{at}msx.dept-med.pitt.edu).

Received 11 March 1999; accepted in final form 13 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barzilai, N., J. Wang, D. Massilon, P. Vuguin, M. Hawkins, and L. Rossetti. Leptin selectively decreases visceral adiposity and enhances insulin action. J. Clin. Invest. 100: 3105-3110, 1997[Abstract/Free Full Text].

2.   Bjorntorp, P., and B. N. Brodoff (Editors). Obesity. Philadelphia: Lippincott, 1992.

3.  Bray, G. (Editor). Obesity. Endocrinol. Metab. Clin. North Am. 25, 1996.

4.   Campfield, L. A., F. J. Smith, Y. Guisez, R. Devos, and P. Burn. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269: 546-549, 1995[ISI][Medline].

5.   Chen, G., K. Koyama, X. Yuan, Y. Lee, Y. T. Zhou, R. M. O'Doherty, C. B. Newgard, and R. H. Unger. 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].

6.   Frederich, R. C., A. Hamann, S. Anderson, B. Lollman, B. B. Lowell, and J. S. Flier. Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nature Med. 1: 1311-1314, 1995[ISI][Medline].

7.   Friedman, J. M., and J. L. Halaas. Leptin and the regulation of body weight in mammals. Nature 395: 763-770, 1998[ISI][Medline].

8.   Halaas, J. L., C. Boozer, J. Blair-West, N. Fidahusein, D. A. Denton, and J. M. Friedman. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc. Natl. Acad. Sci. USA 94: 8878-8883, 1997[Abstract/Free Full Text].

9.   Henriksen, E. J., and M. E. Tischler. Glucose uptake in rat soleus: effect of acute unloading and subsequent reloading. J. Appl. Physiol. 64: 1428-1432, 1988[Abstract/Free Full Text].

10.   Herz, J., and R. D. Gerard. Adenovirus-mediated transfer of low density lipoprotein lipase receptor gene acutely accelerates cholesterol clearance in normal mice. Proc. Natl. Acad. Sci. USA 90: 2812-2816, 1993[Abstract].

11.   Heydrick, S. J., D. Jullien, N. Gautier, J. F. Tanti, S. Giorgetti, E. van Obberghen, and Y. Le Marchand-Brustel. Defect in skeletal muscle phosphatidylinositol-3-kinase in obese insulin resistant mice. J. Clin. Invest. 91: 1358-1366, 1993[ISI][Medline].

12.   Koyama, K., G. Chen, Y. Lee, and R. H. Unger. Tissue triglycerides, insulin resistance, and insulin production: implications for hyperinsulinaemia of obesity. Am. J. Physiol. Endocrinol. Metab. 273: E708-E713, 1997[ISI][Medline].

13.   Lee, Y., M. Hirose, J. H. Ohneda, J. H. Johnson, J. D. McGarry, and R. H. Unger. beta -Cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment of adipocyte-beta -cell relationships. Proc. Natl. Acad. Sci. USA 91: 10878-10882, 1994[Abstract/Free Full Text].

14.   Matsui, H., K. Okumura, K. Kawakami, M. Hibnio, Y. Toki, and T. Ito. Improved insulin sensitivity by bezafibrate in rats. Relationship to fatty acid composition of skeletal muscle triglycerides. Diabetes 46: 348-353, 1997[Abstract].

15.   McGarry, J. D. Disordered metabolism in diabetes: have we underemphasized the fat cell component? J. Cell. Biochem. 555: 29-38, 1994.

16.   Morsy, M. A., M. C. Gu, J. Z. Zhao, D. J. Holder, I. T. Rogers, W. J. Pouch, S. L. Motzel, H. J. Klein, S. K. Gupta, X. Liang, M. R. Tota, C. I. Rosenblum, and C. T. Caskey. Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse. Gene Ther. 5: 8-18, 1997[ISI].

17.   Murphy, J. E., Z. Shangzen, K. Giese, L. T. Williams, J. A. Escobedo, and J. Dwarki. Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin. Proc. Natl. Acad. Sci. USA 94: 13921-13926, 1997[Abstract/Free Full Text].

18.   Oakes, N. D., G. J. Cooney, S. Camilleri, D. J. Chisholm, and E. W. Kraegen. Mechanisms of liver and muscle insulin resistance induced by chronic high-fat feeding. Diabetes 46: 1768-1774, 1997[Abstract].

19.   O'Doherty, R. M., P. R. Anderson, A. Z. Zhao, K. E. Bornfeldt, and C. B. Newgard. Sparing effect of leptin on liver glycogen stores during the fed-to-fasted transition. Am. J. Physiol. Endocrinol. Metab. 277: E544-E550, 1999[Abstract/Free Full Text].

20.   Pan, D. A., A. D. Lillioja, M. R. Kriketos, L. A. Baur, A. B. Bogardus, A. B. Jenkins, and L. H. Storlein. Skeletal muscle triglyceride levels are inversely related to insulin action. J. Clin. Invest. 46: 983-987, 1997.

21.   Pelleymounter, M. A., M. J. Cullen, M. B. Baker, R. Hecht, D. Winters, T. Boone, and F. Collins. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540-543, 1995[ISI][Medline].

22.   Phillips, D. I. W., S. Caddy, V. Ilic, K. N. Fielding, A. C. Borthwick, and R. Taylor. Intramuscular triglyceride and muscle insulin sensitivity: evidence for a relationship in nondiabetic subjects. Metabolism 45: 947-950, 1996[ISI][Medline].

23.  Randle, P. J., P. B. Garland, C. N. Hales, and E. A. Newsholme. The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet i: 785-794, 1963.

24.   Schwartz, M. W., D. G. Baskin, T. R. Bukoski, J. L. Kuijber, D. Foster, G. Lasser, D. E. Prunkard, D. Porte, Jr., S. C. Woods, R. J. Seely, and D. S. Weigle. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 45: 531-535, 1996[Abstract].

25.   Shrago, E., G. Woldegiorgis, A. E. Ruoho, and C. C. DiRusso. Fatty acyl-CoA esters as regulators of cell metabolism. Prostaglandins Leukotrienes Essent. Fatty Acids 52: 163-166, 1995[ISI][Medline].

26.   Sivitz, W. I., S. A. Walsh, D. A. Morgan, M. J. Thomas, and W. G. Haynes. Effects of leptin on insulin sensitivity in normal rats. Endocrinology 138: 3395-3401, 1997[Abstract/Free Full Text].

27.   Storlein, L. H., N. D. Oakes, D. A. Pan, M. Kusunoki, and A. B. Jenkins. Syndromes of insulin resistance in the rat. Inducement by diet and amelioration with benfluorex. Diabetes 42: 457-462, 1993[Abstract].

28.   Trinh, K., R. M. O'Doherty, P. R. Anderson, A. J. Lange, and C. B. Newgard. Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats. J. Biol. Chem. 273: 31615-31620, 1998[Abstract/Free Full Text].

29.   Turinsky, J., W. Nagel, J. S. Elmendorf, A. Damrau-Abney, and T. Smith. Sphingomyelinase stimulates 2-deoxyglucose uptake by skeletal muscle. Biochem. J. 313: 215-222, 1996[ISI][Medline].

30.   Van Heek, M., D. S. Compton, C. F. France, R. P. Tedesco, A. B. Fawzi, M. P. Graziano, E. J. Sybertz, C. D. Strader, and H. R. Davis, Jr. Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J. Clin. Invest. 99: 385-390, 1997[Abstract/Free Full Text].

31.   Van Heek, M., D. E. Mullins, M. A. Wirth, M. P. Graziano, A. B. Fawzi, D. S. Compton, C. F. France, L. M. Hoos, R. L. Casale, E. J. Sybertz, C. D. Strader, and H. R. Davis, Jr. The relationship of tissue localization, distribution and turnover to feeding after intraperitoneal 125I-leptin administration to ob/ob and db/db mice. Horm. Metab. Res. 28: 653-658, 1996[ISI][Medline].

32.   Wititsuwannakul, D., and K. Kim. Mechanism of palmitoyl coenzyme A inhibition of liver glycogen synthase. J. Biol. Chem. 252: 7812-7817, 1977[Abstract].

33.   Zierath, J. R., K. L. Houseknecht, L. Gnudi, and B. B. Kahn. High fat feeding impairs insulin-stimulated GLUT4 recruitment via an early insulin-signaling defect. Diabetes 46: 215-223, 1997[Abstract].


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