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Endocrinology Vol. 140, No. 5 2117-2124
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential Effects of Leptin in Regulation of Tissue Glucose Utilization in Vivo1

Jin-lin Wang, Narumol Chinookoswong, Sheila Scully, Meiying Qi and Zhi-Qing Shi

Departments of Pharmacology and Pathology, Amgen, Inc., Thousand Oaks, California 91320

Address all correspondence and requests for reprints to: Zhi-Qing Shi, M.D., Ph.D., Department of Pharmacology, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320. E-mail: jshi{at}amgen.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown that leptin enhances systemic insulin sensitivity and whole body glucose utilization in the rat. This study examines our hypothesis that leptin has differential effects in regulating glucose utilization among the tissues, i.e. stimulating glucose utilization in brown adipose tissue (BAT) and skeletal muscle but suppressing glucose utilization in white adipose tissue (WAT) in normal male rats (275–350 g BW). The rats were treated with sc infusion of recombinant murine leptin (4 mg/kg·day) or vehicle (V) with Alzet osmotic pumps or with vehicle and pair-feeding (PF) for 7 days. Leptin significantly decreased food intake (leptin, 11.5 ± 0.4 g/day; V, 16.8 ± 1.5 g/day; P < 0.05) and body weight (maximum change, 5.0 ± 0.2%; P < 0.05 vs. V) and lowered plasma triglyceride, insulin, and glucose levels, but raised ß-hydroxybutyrate levels. Glucose utilization by individual tissues was determined with an iv bolus of [1-14C]2-deoxyglucose (2-DG) after a 90-min hyperinsulinemic (2 mU/kg·min) euglycemic clamp. With leptin treatment, the 2-DG-determined glucose utilization in interscapular BAT was almost 3-fold that in V-treated rats and 70% greater than that in PF rats. In contrast, in the epididymal WAT, glucose utilization was reduced by leptin treatment to only 34% that in V-treated rats and 45% that in PF rats. Leptin increased 2-DG uptake by extensor digitorum longus muscle and soleus muscle compared with that in the V and PF groups. With leptin treatment, the GLUT4 glucose transporter mRNA and protein levels were increased in BAT, but decreased in WAT (both P < 0.05). There was no significant change in GLUT4 mRNA and protein expression in extensor digitorum longus muscle and soleus muscle. Oxygen consumption was significantly increased (32.1 ± 7.4%) in BAT (139.0 ± 8.2 nmole O2/30 min·106 cells) of leptin-treated rats vs. that in V control rats (105.3 ± 6.7 nmole O2/30 min·106 cells). In conclusion, leptin has differential, tissue-specific effects on glucose and oxygen utilization, which contribute to the reduction in whole body adiposity by enhancing energy consumption in BAT and muscle while attenuating energy storage in WAT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, a 16-kDa protein hormone produced primarily from adipocytes (1, 2), inhibits food intake, reduces body weight, and stimulates energy expenditure (3, 4). Leptin has been reported to normalize hyperglycemia and hyperinsulinemia in ob/ob mice (5). Several studies, including our own work, have shown that leptin increases insulin sensitivity in vivo (6, 7, 8, 9). These studies open the possibility that leptin may be involved in the regulation of glucose transport. The importance of glucose transport as the rate-limiting step in whole body glucose utilization becomes more significant in metabolic disease states such as obesity and NIDDM, in which insulin-mediated glucose transport is impaired (10). Although leptin has been shown to increase whole body glucose utilization, the effect of leptin on glucose transport at the tissue level remains controversial. Leptin by itself stimulates glucose transport and glycogen synthesis in the C2 C12 myotube cell line independently of insulin (11). In isolated rat adipocytes, leptin impairs the metabolic action of insulin, which suggests that leptin per se acts to down-regulate glucose transport in adipose tissues (12). Another study has shown that in vitro exposure of skeletal muscle or adipocytes to leptin for 2 h did not alter glucose transport in the absence or presence of insulin (13). In isolated rat adipocytes and cultured 3T3-L1 adipocytes, leptin had no effect on basal and insulin-stimulated glucose transport (14). Therefore, the effect of leptin on tissue glucose utilization and its mechanism are still not well defined.

The objective of this study was to examine our hypothesis that leptin may have differential, tissue-specific effects on glucose utilization in vivo. This study focuses on the chronic effect of leptin on regulation of glucose transport in insulin-sensitive tissues such as muscle and adipose tissues. As the GLUT-4 glucose transporter is considered to be the primary and regulatable transporter species in these tissues (15), we have examined the effect of leptin on GLUT4 mRNA and protein expression in these tissues. Brown adipose tissue (BAT) has been identified as a major site of thermogenesis, and leptin treatment increases uncoupling protein expression and heat production (16). The effect of leptin on oxygen consumption in brown adipocytes was also studied.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant murine leptin (r-metMuLeptin) was provided by Amgen, Inc. (Thousand Oaks, CA). Alzet osmotic pumps (2ML1) were obtained from Alza Corp. (Palo Alto, CA). [1-14C]2-Deoxy-D-glucose ([14C]2-DG) was purchased from New England Nuclear (Boston, MA). Rabbit antiserum against cytoplasmic 12-amino acid C-terminal peptide of GLUT4 and the Western blot kit were purchased from Alphadiagnostic International Co. (San Antonio, TX). RNA STAT-60 was purchased from Tel-Test B, Inc. (Friendswood, TX). [{alpha}-32P]UTP was obtained from Amersham (Arlington Heights, IL). The Maxi Script SP6/T7 Transcription Kits and the RPA II ribonuclease protection assay kits were obtained from Ambion, Inc. (Austin, TX). D-Glucose, Somogyi reagents, and protein A-peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). Regular pork insulin was obtained from Eli Lilly & Co. (Indianapolis, IN).

Experimental animals
Male Sprague Dawley rats (275–350 g BW) were obtained from Harlan Sprague Dawley, Inc. (San Diego, CA). Animals were housed in separate cages in temperature-controlled rooms (22–24 C) with a 12-h light, 12-h dark cycle and were fed regular rat chow. Two experimental protocols were used. In the protocol for 2-DG uptake measurements, rats received chronic cannulation of the left carotid artery and right jugular vein as described previously (9). In the meantime, Alzet osmotic pumps were implanted sc which delivered r-Met-MuLeptin (4 mg/kg·day; n = 12) or phosphate buffer vehicle (V; n = 8) for 5 days. Another group of rats receiving vehicle infusion was pair-fed (PF; n = 9), with the amount of daily food consumption matching that of leptin-treated animals. Body weight and food intake were monitored every day. In another protocol for determination of glucose transporter mRNA and protein expression, three groups of rats (leptin, V, and PF) received corresponding osmotic pump implantation 5–7 days before the ribonuclease protection assay and Western blot analysis. Animals were killed by cervical dislocation after a 5-h fast, and all tissues were processed at the same time in a side by side fashion.

Measurement of glucose utilization in muscle and adipose tissues in vivo
The in vivo glucose uptake rate in individual tissues was determined and calculated from the measurement of 2-DG uptake under the conditions of a hyperinsulinemic euglycemic clamp (17, 18, 19, 20, 21, 22, 23). The experiments were carried out in unrestrained conscious rats. Animals were fasted 5–6 h before the experiments. Contents in the vascular cannulas were aspirated, and the cannulas were extended with PE-50 plastic tubings and flushed with fresh heparinized (10 U/ml) saline. Basal arterial blood samples were taken for biochemical and hormonal assays. The right jugular venous cannula was used for infusion of tracer, insulin, and glucose via serial Y needle connectors. A continuous infusion of regular porcine insulin (Eli Lilly & Co.) was given at 2 mU/kg·min and maintained throughout the experiment. An exogenous glucose infusion (30%) was given at variable rates to maintain plasma glucose at normal basal levels, according to instant plasma glucose measurements by a glucose oxidase method using a Beckman Coulter, Inc. Glucose Analyzer II (Beckman Coulter, Inc., Fullerton, CA). Arterial blood was sampled every 10 min throughout the experiment to ensure that euglycemia was maintained. A flash iv injection of 30 µCi [14C]2-DG was given at 90 min of the glucose clamp, and blood was sampled via the arterial catheter immediately before (0 min) and 3, 6, 10, 20, 30, and 40 min after the flash injection.

After the last blood sample, the rats were killed by cervical dislocation. Soleus muscle (SM), extensor digitorum longus muscle (EDL), white epididymal adipose tissue (WAT), interscapular BAT, and liver were excised and frozen in liquid nitrogen. The above tissues were digested by NaOH and deproteinated by either 6% HClO4 or the Somogyi procedure. The supernatant from the deproteinated samples were mixed with scintillation cocktail, and 14C radioactivity was quantitated (21, 22). After the plasma samples were deproteinated using the Somogyi procedure, an aliquot of the supernatant was evaporated overnight and then mixed with scintillation cocktail for determination of [14C]2-DG with a liquid scintillation counter. The rate of glucose uptake in individual tissues was calculated according to Kraegen’s method (23).

Western blot analysis
The isolated adipose tissues from both epididymal WAT and interscapular BAT were homogenized in 10 vol ice-cold buffer containing 25 mM HEPES, 250 mM sucrose, 4 mM EDTA, aprotinin (40 µg/ml), 25 mM benzamidine, 0.2 mM phenylmethylsulfonylfluoride, 1 µM leupeptin, and 1 µM pepstatin, pH 7.4. Homogenates from adipose tissue were centrifuged at 5,000 x g for 5 min at 4°C. The supernatant was then centrifuged at 150,000 xg for 2 h at 4°C to obtain the membrane fraction (24). The muscle tissues were homogenized in homogenization buffer (pH 7.4) containing 10 mM NaHCO3, 0.25 M sucrose, and 5 mM NaN3 at 4°C. Homogenates were centrifuged at 1,200 x g for 10 min at 4°C. The first and second supernatants were combined and centrifuged at 9,000 x g for 10 min at 4°C. The resultant supernatant was centrifuged at 200,000 x g for 90 min at 4°C. The pellet was resuspended and stored at -80°C before Western blot analysis (24, 25). Protein content in both muscle and adipose tissues was determined by a modification of the Lowry method, with BSA as a reference standard (26). Equivalent amounts of protein homogenates (30 µg) were separated in 10% SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane (polyvinylidene difluoride, Schleicher & Schuell, Inc., Keene, NH). The filters were blocked by incubation for 1 h in PBS with 5% nonfat milk. Blots were then washed in PBS-Tween and incubated with a rabbit polyclonal antiserum (1:1,000) raised against a peptide corresponding to the GLUT4 COOH-terminus (27). Blots were washed and incubated with a goat antirabbit secondary antiserum (1:2,000 dilution) conjugated to horseradish peroxidase (Sigma Chemical Co.). Detection of immune complex was accomplished using the enhanced chemiluminescent reagent (ECL, Amersham, Arlington Heights, IL). The filters were then exposed to Kodak XAR films (Eastman Kodak, Rochester, NY) and subjected to laser scanning densitometry (Molecular Dynamics, Inc., Sunnyvale, CA) to quantitate the results.

Ribonuclease protection assay
Animals were killed after 5 h of fasting on day 5 of leptin treatment. The STAT-60 reagent was used to extract total RNA from EDL, SM, interscapular BAT, and epididymal WAT. A segment of the rat GLUT4 sequence (Gb:D28561, nucleotides 1445–1629) was generated by RT-PCR from rat muscle RNA and cloned into the transcription vector pGEM-T (Promega Corp., Madison, WI). Radiolabeled antisense transcripts were synthesized from linearized plasmid templates using T7 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) and [32P]rat UTP (800 Ci/mol; Amersham). A 103-bp rat cyclophilin probe (Ambion, Inc. Austin, TX) was used as an internal control. The ribonuclease protection assay was performed using the RPA II kit (Ambion, Inc.) and 5 µg total RNA from each sample. Quantitation was performed with a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The ratio of the integrated volume of the GLUT4 band vs. the cyclophilin (housekeeping control) band was calculated, and the results were expressed as the mean ± SEM of the values in each group.

Measurement of oxygen consumption
After 5 h of fasting, animals were killed by cervical dislocation, and interscapular BAT was immediately excised from each group of rats. Brown adipocytes were isolated using the method described by Nedergaard et al. (28). The 1.4-ml cell suspension (2–3 x 106 cell/ml) in Kreb-Ringer buffer was added to a magnetically stirred chamber at 37 C. Oxygen consumption was measured polarographically using a Clark style oxygen electrode (4004 Clark oxygen probe, YSI, Inc., Yellow Springs, OH). These electrodes were connected to the biological oxygen monitor (YSI model 5300, YSI, Inc.), providing simultaneous recording of both the total O2 concentration and the rate of O2 consumption in the chamber (29, 30).

Calculation and statistical analysis
The rate of tissue glucose uptake (defined as the glucose metabolic index, Rg) was calculated as follows: Rg (µmol/100 g·min) = Cp Cm1(45)/{int}045 Cp1(t) dt, where Cp is the steady state plasma glucose concentration over the 45-min period of observation (millimoles per liter), Cm1 is the tissue accumulation of [14C]2-DG-6-phosphate per unit mass at 45 min (disintegrations per min/mg·wet wt), Cp1(t) is the plasma [14C]2-DG concentration (disintegrations per min/ml), and the tracer bolus is administered at time zero. All data are expressed as the mean ± SEM. Two-way ANOVA or unpaired t test was used to determine the statistical differences among the three groups or between two groups, respectively. Significance is assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of leptin on body weight and food intake
Leptin decreased body weight and food intake compared with V control and V PF control rats (Fig. 1Go). The baseline body weight for the experimental rats was about 275 g. There was a significant progressive reduction (5%) in the body weight of leptin-treated rats (P < 0.05 vs. V and PF), whereas the V control rats increased body weight (6%) during the study. A slight body weight loss was observed in PF rats on days 2 and 3, after which their body weight was slowly increased in a pattern similar to that of V rats. Leptin significantly reduced food intake (11.5 ± 0.4 g/day) compared with that of the vehicle control rats (16.8 ± 1.5 g/day; P < 0.05). The pair-fed animals received an amount of food equal to that consumed by the leptin-treated rats.



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  		Figure 1. The effects of leptin on body weight and food intake in the rats treated with chronic sc leptin infusion (4 mg/kg·day; n = 12), treated with V (n = 8), or PF (n = 9) for 7 days. Value are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01 (leptin vs. V). +, P < 0.05 (leptin vs. PF).

 
Effects of leptin on plasma hormones and metabolites
Fasting plasma hormones and metabolic substrates were measured at the end of the 7-day treatment. Blood samples were withdrawn from the tail vein, Blood was quickly centrifuged, and plasma was collected and stored at -70 C. As shown in Table 1Go, the plasma insulin level was decreased and the leptin level was increased in the leptin group compared with those in both V and PF control rats. The plasma T4 level was increased in the leptin-treated group compared with that in the V control group, but did not significantly differ from that in PF rats. Plasma glucagon and corticosterone levels did not change after leptin treatment. The plasma triglyceride concentration was significantly reduced in leptin-treated rats compared with that in both V and PF control rats. The plasma glucose level was also reduced in leptin-treated rats compared with that in the V control group, but did not differ from that in PF rats. Plasma ß-hydroxybutyrate was increased in the leptin-treated group compared with levels in the other two groups.


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  		Table 1. The effect of leptin on plasma hormone and metabolite concentrations in normal rats

 
Effect of leptin on the insulin-mediated increase in glucose infusion
Plasma glucose levels and exogenous glucose infusion rates during the hyperinsulinemic euglycemic clamp are shown in Fig. 2Go. The basal plasma glucose level was reduced in the leptin group (115.6 ± 3.8 mg/dl) compared with those in the V (126.7 ± 4.2 mg/dl) and PF (128.5 ± 2.9 mg/dl) groups. The plasma glucose levels were clamped at the respective baseline values during the glucose clamp. During the last 60 min, the glucose infusion rate required to maintain the clamp was 16.5 ± 0.3 mg/kg·min in the leptin group, 8.8 ± 0.6 mg/kg·min in the V group, and 13.0 ± 0.1 mg/kg·min in the PF group. There was a significant difference in the glucose infusion rate among the three groups of animals (P < 0.05).



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  		Figure 2. Summary of plasma glucose levels (upper panel) and exogenous glucose infusion rates (lower panel) during the hyperinsulinemic euglycemic glucose clamp. The glucose clamps were performed in rats treated with leptin (closed circle; n = 7) or V (open triangle; n = 5) or rats that were PF (open square; n = 6). The insulin infusion rate was 2 mU/kg·min. The glucose infusion rates are significantly different during the last 60 min of the clamp: **, P < 0.01 (leptin vs. V); 2+, P < 0.01 (leptin vs. PF).

 
Effect of leptin on glucose utilization in individual tissues
Glucose utilization rates in various tissues in the leptin, V, and PF groups are shown in Fig. 3Go. 2-DG uptake was examined in SM, EDL, interscapular BAT, epididymal WAT, and liver. The Rg (micromoles of glucose per 100 g tissue/min) is a glucose metabolic index calculated from tissue [14C]2-DG-6-phosphate accumulation and parameters derived from plasma glucose and [14C]2-DG data. A small, but significant, increase in the Rg value was observed in EDL and SM muscles in the leptin-treated group compared with those in V and PF groups. In BAT, 2-DG uptake in the leptin-treated group was significantly elevated and was 3-fold that in the V control group and 70% greater than that in the PF group. Intriguingly, 2-DG uptake in WAT was significantly reduced by leptin treatment, with a Rg value 50% less than those in the V and PF groups. No difference was found in 2-DG uptake by the liver among the three groups.



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  		Figure 3. Effect of leptin on glucose utilization in vivo in individual tissues of normal rats. Animals were received sc infusion of r-MetMuleptin (4 mg/kg·day) or PBS (in V or PF control group of rats) for 5 days. Euglycemic clamps were performed in chronically cannulated rats for 2.5 h at an insulin infusion rate of 2 mU/kg·min. An iv bolus of [14C]2-DG was administrated 45 min before the completion of the study, at which time various tissues were rapidly removed for subsequent analysis. Rg is the glucose metabolic index in each tissue. Values are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01 (leptin vs. V). +, P < 0.05 (leptin vs. PF).

 
Effect of leptin on GLUT4 glucose transporter protein expression
The GLUT4 glucose transporter plays a major role in regulation of tissue glucose transport in response to insulin. To examine the mechanism of leptin action on glucose transport, we determined GLUT4 mRNA and protein expression in these tissues in three groups of animals after 7 days of treatment. Figure 4Go shows the results from Western blot analysis. In BAT, GLUT4 protein levels were markedly increased in the leptin-treated group compared with those in the V and PF groups. No significant difference was observed in muscle tissues among the three groups. However, levels of GLUT4 protein were markedly reduced in WAT of leptin-treated rats compared with that of V and PF animals.



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  		Figure 4. Immunoblot analysis of GLUT4 protein expression in individual tissue of leptin (L)-treated, V-treated, and PF rats. The tissue samples were collected after 7 days of treatment, which included EDL, SM, interscapular BAT, and epididymal WAT. The membrane fractions of these tissues were prepared, and GLUT4 glucose transporter protein expression was determined by Western blot analysis. GLUT4 molecular weight is about 43 kDa, as indicated by the arrow (upper panel). Laser scanning densitometry was conducted to a quantitative difference within each group. Statistical significance was accepted at P < 0.05. *, P < 0.05 (leptin vs. V). +, P < 0.05 (leptin vs. PF).

 
Effect of leptin on GLUT4 mRNA
The effect of leptin on GLUT4 mRNA expression was also examined in individual tissues using the ribonuclease protection assay. The GLUT4 mRNA is expressed as the ratio of GLUT4/cyclophilin for quantitative measurements and comparisons. As shown in Fig. 5Go, leptin treatment significantly increased GLUT4 mRNA expression in BAT compared with that in both control groups. The ribonuclease protection assay result shows a very significant decrease in GLUT4 mRNA expression in WAT of leptin-treated animals, which is in agreement with the Western blot assay. No significant difference in GLUT4 mRNA expression in muscle tissues was observed between the leptin-treated and control (V and PF) groups.



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  		Figure 5. The effect of leptin on GLUT4 mRNA expression in muscle and adipose tissue. RPAs were performed to measure levels of GLUT4 mRNA in tissues from leptin-treated (L; n = 7), V-treated (n = 6), and PF control (n = 5) animals. A is a PhosphorImager printout of a representative experiment. Upper bands represent GLUT4-protected fragments (184 bp), and lower bands represent cyclophilin (internal control)-protected fragments (103 bp). In B, GLUT4 mRNA levels are expressed as GLUT4/cyclophilin. *, P < 0.05 (leptin vs. V); P < 0.05 (leptin vs. PF). **, P > 0.01 (leptin vs. V). 2+, P > 0.01 (leptin vs. PF).

 
Effect of leptin on oxygen consumption in isolated adipocytes
Figure 6Go shows the effect of leptin on oxygen consumption in isolated brown adipocytes. After 7 days of treatment with leptin or vehicle, oxygen consumption was measured in freshly isolated BAT during a 30-min incubation. The upper panel of Fig. 6Go shows the oxygen consumption rates (nanomoles of O2 per 106 cells) over the 30-min time course in the leptin- and vehicle-treated rats. The mean oxygen consumption rates per min are displayed in the lower panel. Oxygen consumption was significantly increased (32 ± 7%) in BAT (139 ± 8 nmol O2/30 min·106 cells) of leptin-treated rats vs. V control rats (105 ± 7 nmol/30 min·106 cells).



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  		Figure 6. The effects of chronic leptin and PBS infusion on cellular respiration in brown adipocytes are shown. The cells were isolated from interscapular BAT. The upper panel shows that oxygen consumption was measured for 30 min in adipocytes isolated from leptin-treated rats (closed circles; n = 10) or V-treated rats (open circles; n = 10). The lower panel shows the mean oxygen consumption rate measured in the control group (open bar; n = 10) or the leptin-treated group (black bar; n = 10). Values given are means from duplicate chambers in five separate experiments. **, P < 0.01 L (vs. V).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates tissue-specific effects of leptin in modulating glucose metabolism in various tissues in vivo. After chronic sc leptin treatment, glucose utilization in BAT and skeletal muscle is enhanced, whereas that in WAT is down-regulated. The differential effect of leptin on tissue glucose utilization is closely associated with a divergent effect of leptin on GLUT4 mRNA and protein expression in the various tissues in vivo. The current results demonstrate that GLUT4 mRNA and protein levels were significantly elevated in BAT, but reduced in WAT after leptin treatment. The differences in GLUT4 were consistent with corresponding differences in the rates of 2-DG uptake in these tissues. In the skeletal muscles, leptin treatment stimulated 2-DG uptake, even though GLUT4 mRNA and protein levels were not significantly affected in this study, suggesting an increased transport activity. Chronic leptin treatment also induced a significant increase in oxygen consumption in brown adipocytes.

Decreases in food intake and body weight in the leptin-treated rats have been common findings in many studies. The observation that the loss of body weight in the PF rats was much less than that in leptin-treated rats despite the same amount of daily food consumption suggests that leptin also induced an increase in energy expenditure, which is indeed evidenced by the current study.

BAT appears to play an important role in regulation of adiposity and energy expenditure, as brown fat deficiency in a transgenic mouse model produces severe leptin resistance (31). Our study demonstrated that chronic sc leptin treatment in the rat induced an increase in glucose uptake rate in BAT. Leptin also induced an increase in GLUT4 glucose transporter mRNA and protein expression in BAT. As GLUT4 mRNA and protein levels in BAT of PF animals were not affected, the effect of leptin must be independent of food restriction. The enhancement in GLUT4 mRNA and protein expression in BAT may directly contribute to the observed increase in glucose uptake in BAT of leptin-treated rats, because the GLUT4 glucose transporter is the main regulatable element in the glucose transport system in these insulin-sensitive tissues. It was recently shown that glucose uptake in BAT is increased 5 h after iv administration of leptin in the mouse (6). The same results were achieved after intracerebroventricular leptin administration, suggesting a central mechanism of leptin actions (6). Similar to the results found with acute iv injection, chronic iv administration of leptin for 4 days increased glucose utilization in BAT (32). However, in the same study, leptin given intracerebroventricularly had no effect (32). The researchers thus suggested a direct action mechanism of leptin in the BAT. It is thus unresolved whether such an increase in glucose uptake is mediated by leptin’s actions directly on BAT or indirectly, via an efferent pathway from the central nervous system. However, leptin given ip to intact mice was able to stimulate the release of norepinephrine from the sympathetic nerve endings in BAT (33). Leptin infusion to anesthetized rats increased BAT sympathetic nerve activity measured by direct recording from nerves innervating interscapular brown fat (34). These studies point to a possible mechanistic association that connects the leptin hormonal signal to the central nervous system and a sympathetic efferent output.

Thermogenesis in BAT may be an important mechanism by which leptin regulates body fat content. It has been shown that leptin increased uncoupling protein and lipoprotein lipase mRNA expression in BAT (35). If cellular respiration is directly coupled to the oxidative metabolism to a significant extent in BAT, then the rate of oxygen consumption in brown adipocytes would be expected to correlate with the rate of glucose uptake. This possibility is verified by our current data that demonstrated significant increases in oxygen consumption in brown adipocytes isolated from leptin-treated rats.

An intriguing finding in this study is that 2-DG uptake was significantly reduced in WAT of leptin-treated animals. This is in sharp contrast with the results in BAT. In addition, we found substantially reduced GLUT4 mRNA and protein levels in WAT in leptin-treated rats, and this effect of leptin again appears independent of food restriction. Our findings in WAT bear certain clinical importance in terms of energy storage. Fat cells require glucose to generate glycerol-3-phosphate, rather than using intracellular glycerol, for the synthesis of triglyceride, which is the major energy storage form. In this process, uptake of glucose from extracellular sources is an important step in lipogenesis in adipocytes. Therefore, a decrease in glucose transport into the WAT may lead to a reduced triglyceride store in white adipocytes. In our previous study, chronic leptin treatment also resulted in a significant diminution in hepatic triglyceride production (36). Assuming that therapeutic hyperleptinemia in humans could result in a similar reduction in glucose uptake in white adipocytes, the resultant suppression in lipogenesis could be an important part of the leptin action mechanism in the reduction of adiposity. Leptin has been shown to impair the effect of insulin on glucose transport in isolated white adipocytes in vitro (12). Our present study is the first report that leptin decreases glucose uptake and down-regulates GLUT4 mRNA and protein expression in WAT in vivo. It is apparent from our current results that glucose uptake in individual tissues is distinctly regulated and that leptin treatment produces a tissue-specific effect on glucose utilization and oxygen consumption.

The muscle tissue is the major site of insulin-mediated glucose disposal in vivo. Our data show that leptin treatment increased glucose uptake in the EDL (a glycolytic muscle type) and the SM (an oxidative muscle type) in leptin-treated animals. The increase in glucose uptake in muscle tissues may largely reflect the whole body glucose utilization, and these results are in agreement with those of a study in mice (6). However, the chronic leptin infusion did not appear to alter GLUT4 mRNA and protein expression in skeletal muscles tested in this study. Thus, the mechanism by which leptin affects muscle insulin sensitivity is still unclear. A recent glucose clamp study performed in anesthetized rats demonstrated that a 4-day intracerebroventricular leptin treatment resulted in significant increments in insulin-mediated systemic glucose disposal and 2-DG uptake in various muscle types (37). However, similar changes were induced by pair-feeding in the control rats, suggesting that negative energy balance plays a role in enhancing insulin-mediated glucose metabolism (37). Kamohara et al. studied the effect of intracerebroventricular leptin on glucose uptake in denervated and intact EDL and SM in the same animal and found that leptin-stimulated glucose uptake was lower in the denervated leg than that in the intact muscle (6). This suggests that leptin may affect muscle insulin sensitivity through the central nervous system. In our recent study in unrestrained conscious rats, intracerebroventricular infusion of leptin, given either as one overnight bolus or a 1-week infusion, resulted in significant increases in whole body glucose utilization (9). These results collectively argue for a central mechanism of action rather than a direct leptin action on peripheral tissues. However, it has been shown that leptin affected muscle lipid metabolism, which leads to a decrease in muscle triglyceride content (38). The reduction in muscle triglyceride levels appears to be associated with increased insulin sensitivity in muscle (39, 40). These data seem to indicate a direct effect of leptin on peripheral tissue. The mechanism by which leptin may enhance insulin sensitivity without altering glucose transporter mRNA and protein expression in skeletal muscle remains to be identified. It is possible that changes in glucose transport activities is mediated by altered translocation and/or intrinsic activity of the glucose transporter(s) (41).

The decreases in plasma insulin, triglyceride, and glucose levels after sc leptin infusion are in agreement with the findings of recent studies by us and others (7, 9, 42). Leptin has been shown to enhance systemic insulin action in stimulating whole body glucose disposal and reducing hepatic glucose production under glucose clamp conditions (7, 9, 42). The decreased basal plasma glucose levels in the study may be associated with both a reduced hepatic glucose production and an increased glucose utilization. Plasma ß-hydroxybutyrate was elevated after leptin treatment for 7 days, indicating an accelerated rate of FFA oxidation, which, again, contributes to the overall effect of leptin in reducing adiposity. The elevated T4 levels suggest a possible involvement of thyroid hormones in the systemic actions of leptin, especially relating to calorigenic metabolism.

In summary, chronic sc leptin treatment produced a tissue-specific effect on glucose utilization in individual tissues in normal rats. Glucose uptake increases in BAT and muscle, but decreases in WAT. GLUT4 glucose transporter mRNA and protein expression were up-regulated in BAT, but down-regulated in WAT, by leptin, without a significant change in muscle tissues. The differential effects of leptin on tissue glucose utilization appear to be independent of food restriction and circulating insulin levels. The above results suggest that a marked reduction in adiposity induced by leptin is associated with both enhanced energy consumption in BAT and muscle and an attenuation in energy storage in WAT.


   Acknowledgments
 
The authors are grateful to Larry Ross and Sylvia Copon for their assistance in conducting the biological and hormonal assay, to Dr. Margery Nicolson and co-workers for conducting the leptin enzyme-linked immunosorbent assay, and to Dr. David Y. Hsu for his input in the 2-DG uptake data analysis.


   Footnotes
 
1 This study is supported by Amgen, Inc. All work described in this manuscript was performed at Amgen. Back

Received September 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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