Decreased visceral adiposity accounts for leptin effect on hepatic but not peripheral insulin action

Nir Barzilai1,2, Li She2, Lisen Liu2, Jiali Wang2, Meizu Hu2, Patricia Vuguin3, and Luciano Rossetti2

Divisions of 1 Geriatrics and 3 Pediatric Endocrinology, Department of Medicine, and the 2 Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, New York 10461


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Leptin decreases visceral fat (VF) and increases peripheral and hepatic insulin action. Here, we generated similar decreases in VF using leptin (Lep), beta 3-adrenoreceptor agonism (beta 3), or food restriction (FR) and asked whether insulin action would be equally improved. For 8 days before the in vivo study, Sprague-Dawley rats (n = 24) were either fed ad libitum [control (Con)], treated with Lep or beta 3 (CL-316,243) by implanted osmotic mini-pumps, or treated with FR. Total VF was similarly decreased in the latter three groups (Lep, 3.11 ± 0.96 g; beta 3, 2.87 ± 0.48 g; and FR, 3.54 ± 0.77 g compared with 6.91 ± 1.41 g in Con; P < 0.001) independent of total fat mass (by 3H2O) and food intake. Insulin (3 mU · kg-1 · min-1) clamp studies were performed to assess hepatic and peripheral insulin sensitivity. Decreased VF resulted in similar and marked improvements in insulin action on glucose production (GP) (Lep, 1.19 ± 0.51; beta 3, 1.46 ± 0.68; FR, 2.27 ±0.71 compared with 6.06 ± 0.70 mg · kg-1 · min-1 in Con; P < 0.001). By contrast, reduction in VF by beta 3 and FR failed to reproduce the stimulation of insulin-mediated glucose uptake (~60%), glycogen synthesis (~80%), and glycolysis (~25%) observed with Lep. We conclude that 1) a moderate decrease in VF uniformly leads to a marked increase in hepatic insulin action, but 2) the effects of leptin on peripheral insulin action are not due to the associated changes in VF or beta 3 activation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A MAJOR ROLE of a centripetal distribution of adiposity in the pathophysiology of insulin resistance has been suggested by numerous epidemiological studies (9, 25). However, the covariance of central adiposity and insulin resistance may also be due to tightly associated hormonal or metabolic parameters. We have recently reported that the administration of the "anorectic" fat-derived hormone, leptin, to moderately obese rats leads to a selective decrease in intra-abdominal adiposity and to marked improvements in both hepatic and peripheral insulin action (7). This suggests that some of the chronic metabolic effects of leptin might be secondary to the decrease in visceral fat (VF).

Leptin suppresses appetite and augments energy expenditure mainly via its interaction with hypothalamic receptors (41, 42), and it has been postulated that some of the downstream effects of leptin are mediated by the beta 3-adrenoreceptor system (17). A mutation in this receptor is associated with insulin resistance (44), morbid obesity (10), and increased visceral adiposity (40). Administration of beta 3-adrenoreceptor agonists increases thermogenesis through their action on uncoupling protein 1. Although this action occurs mainly in brown fat, beta 3-adrenoreceptors are present in a variety of white fat tissue in humans (26). Because the administration of selective beta 3-adrenoreceptor agonists to rodents affects visceral more than subcutaneous fat (20), it is possible that the selective effect of leptin on VF is due, in part, to the activation of this neuronal pathway (12). Furthermore, important metabolic actions of leptin on energy expenditure, substrate partitioning, insulin action, and storage of body fat have also emerged (19, 32, 34).

In fact, whereas chronic administration of leptin improves insulin action in animal models (7, 19, 34), recent studies have also shown acute modulation of insulin action by leptin in vivo (22, 39, 43) and in vitro (11). Thus insulin action may be improved before and independently of the leptin-induced decrease in VF.

To delineate the contribution of the leptin-induced changes in VF to the potent effects of leptin on in vivo insulin action, in the current study we generated similar decreases in VF by alternative means and compared their impact on hepatic and peripheral insulin action. We utilized the beta 3-adrenoreceptor agonist CL-316,243, which caused decreased VF (by ~60%) with no changes in food intake and modest decline in total fat mass (~10%) (20), and caloric restriction designed to achieve a similar decrease in VF.

We hypothesize that if decreased VF is solely responsible for the leptin-induced improvement in insulin action, the latter will be independent of the modality by which VF is decreased. Alternatively, leptin may play a direct role in the modulation of peripheral or hepatic insulin action.


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

Experimental animals. Four groups of male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) received the following treatment by osmotic minipumps for 8 days: 1) Con (n = 6), saline; 2) Lep (n = 6), recombinant mouse leptin at the rate of ~0.5 mg · kg-1 · day-1 (Amgen, Thousand Oaks, CA; >95% pure by SDS-PAGE); 3) beta 3 (n = 6), a beta 3-adrenoreceptor agonist, at the rate of ~0.1 mg · kg-1 · day-1 (CL-316,243 provided by Wyeth-Ayrest Research); and 4) FR (n = 6), saline and food restriction at the physiological level of 17 kcal/day. Data obtained from four of the six Lep rats were included in a previous publication (7) and are reported here solely to facilitate comparison with beta 3 and FR. These rats were selected on the basis of their VF to match that obtained with the alternative interventions. Food intake and body weights were measured every 24 h during the 8-day infusion period. Rats were housed in individual cages and subjected to a standard light (6 AM to 6 PM)-dark (6 PM to 6 AM) cycle. Eight days before the in vivo study, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), the osmotic minipumps were placed in the subcutaneous interscapular area, and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery (4, 5, 7, 38, 39). The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch.

Body composition. Body composition was assessed as in Refs. 3, 5, and 7. Briefly, rats received an intra-arterial bolus injection of 20 µCi of tritiated-labeled water (3H2O; New England Nuclear, Boston, MA), and plasma samples were obtained at 30-min intervals for 3 h. Steady-state conditions for plasma 3H2O specific activity were achieved within 45 min in all studies. Five plasma samples obtained between 1 and 3 h were used in the calculation of the whole body distribution space of water. VF (i.e., epididymal, perinephric, and mesenteric fat depots) was dissected and weighed at the end of each experiment.

Measurements of in vivo glucose kinetics. Measurements were performed as in Refs. 7 and 39. Briefly, a primed-continuous infusion of HPLC-purified [3-3H]glucose (New England Nuclear; 40 µCi bolus, 0.4 µCi/min) was administered for the duration of the study. Two hours after the basal period, a primed-continuous infusion of somatostatin (1.5 µg · kg-1 · min-1) and regular insulin (3 mU · kg-1 · min-1) were administered, and a variable infusion of a 25% glucose solution was started at time 0 and periodically adjusted to clamp the plasma glucose concentration at ~7.5 mM for the rest of the studies. Samples for determination of [3H]glucose specific activity were obtained every 10 min, and plasma samples for determination of plasma insulin, glycerol, and free fatty acid (FFA) concentrations were obtained every 30 min during the study. At the end of the infusions, rats were anesthetized (pentobarbital, 60 mg/kg body wt iv), the abdomen was quickly opened, portal vein blood was obtained, and muscle and liver were freeze-clamped in situ with aluminum tongs precooled in liquid nitrogen.

Rates of glycolysis and glycogen synthesis were estimated as in Refs. 7 and 37. Rates of hepatic glucose fluxes were determined as in Refs. 7, 36, and 38. Gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) by RT-PCR were determined as in Ref. 29.

Analytic procedures. Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Palo Alto, CA). Plasma corticosterone and insulin (with rat and porcine insulin standards) were measured by radioimmunoassay. Plasma glucagon and leptin (RIA kit, Linco Research, St. Charles, MO) concentrations were measured by radioimmunoassay. The plasma concentration of FFA was determined by an automated kit according to the manufacturer's specifications (Waco Pure Chemical Industries, Osaka, Japan). Plasma [3H]glucose radioactivity was measured in duplicates in the supernatants of Ba(OH)2 and ZnSO4 precipitates of plasma samples (20 µl) after evaporation to dryness to eliminate tritiated water. UDP-glucose and PEP concentrations and specific activities in the liver were obtained through two sequential chromatographic separations, as previously reported (7, 36, 38).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Caloric intake, body weight, and fat distribution. Because our design required matching VF by various experimental means, rats had to be preselected for assignment to each study group according to their body weights. Marked decreases in body weight were anticipated after 8 days of Lep and FR; thus rats were weighed before initiation of treatment. Con and beta 3 rats weighed 303 ± 19 and 288 ± 18 g, whereas Lep and FR rats weighed 351 ± 3 and 338 ± 6 g. As expected, administration of exogenous leptin decreased food intake by ~50%, and administration of CL-316,243 (beta 3) resulted in similar food intake as Con (Table 1). Because we had previously shown that pair-feeding to Lep was not sufficient to reproduce the effect of Lep on total abdominal fat (7), in this study FR rats received approximately one-half the caloric consumption of Lep. After these protocols, similar body weight and lean body mass (LBM) were achieved in all groups (Table 1, Fig. 1A), and epididymal, perinephric, and mesenteric fat depots were similarly decreased by all interventions (Table 1, Fig. 1C). Thus the remaining differences in body composition among the groups were due to variations in the amount of total fat mass (Fig. 1B). However, the latter was significantly lower in Lep (34 ± 8 g), beta 3 (28 ± 9 g), and FR (17 ± 8 g) compared with Con (54 ± 4 g).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Caloric intake and body composition



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Body composition. Lean body mass (LBM, A), total fat mass (B), and total epididymal, perinephric, and mesenteric visceral fat (VF, C) obtained at end of 8-day infusion of interventions from rats treated with saline (Con), leptin (Lep), beta 3-adrenoreceptor agonist CL-316,243 (beta 3), or food restriction (FR). Fat mass was calculated from whole body volume of distribution of water, estimated by 3H20 bolus injection in each experimental rat. See detail in MATERIALS AND METHODS. Total VF was similar for all intervention groups vs. Con. * P < 0.001 vs. Con; ** P < 0.001 vs. Con and leptin.

Decreasing VF per se markedly enhances hepatic insulin sensitivity. Plasma leptin levels were markedly increased in Lep (39 ± 8 ng/ml) compared with beta 3, FR, and Con (2 ± 1, 3 ± 1, and 4 ± 1 ng/ml, respectively). During the insulin clamp studies, the plasma glucagon (116 ± 11, 96 ± 18, 125 ± 12, and 102 ± 9 pg/ml) and corticosterone (154 ± 22, 126 ± 28, 168 ± 21, and 186 ± 25 ng/ml in Con, Lep, beta 3, and FR, respectively) concentrations were similar in all groups. Table 2 displays the basal biochemical parameters in all experimental groups. Postabsorptive (6 h of fasting) plasma glucose concentrations were similar in all groups. However, plasma insulin levels were markedly decreased by interventions and were significantly lower in Lep compared with all other groups. Basal plasma FFA and glycerol levels were similar at basal in all groups. At basal, glucose production (GP, Fig. 2A) was similar in all groups (11.2 ± 0.9, 12.2 ± 1.0, 11.5 ± 0.9, and 12.4 ± 1.3 mg · kg-1 · min-1 in Con, Lep, beta 3, and FR, respectively). During the insulin clamp, plasma insulin levels increased to similar levels, and plasma FFA and glycerol levels decreased similarly in all groups (Table 2). Although GP (Fig. 2B) was decreased from basal in all groups, it was about threefold lower (and similar) in all intervention groups (6.1 ± 0.7, 1.2 ± 0.5, 1.5 ± 0.7, and 2.3 ± 0.7 mg · kg-1 · min-1 in Con, Lep, beta 3, and FR, respectively).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Metabolic characteristics



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Hepatic insulin sensitivity. Basal glucose production (GP, A), GP during insulin infusion (3 mU · kg-1 · min-1, B), and percent (%) contribution of gluconeogenesis to GP in Con, Lep, beta 3, and FR. Although basal GP was similar in all groups, insulin inhibited GP to a greater extent in the 3 intervention groups. Gluconeogenesis increased more in Lep than in FR and beta 3. * P < 0.001 vs. all others.

Effect of decreasing VF with Lep, beta 3, or FR on gluconeogenesis and glycogenolysis. The direct contribution of plasma glucose to the hepatic glucose 6-phophate (G-6-P) pool was calculated from the specific activities of UDP-glucose and plasma glucose (Table 3), and it was similar in all groups. Thus decreased VF resulted in similar decreases (to <30% of Con) in the rates of GP, flux through G-6-Pase or total glucose output (TGO), and glucose cycling (GC) in response to physiological hyperinsulinemia. Lep and FR increased the percentage of hepatic G-6-P pool that is derived from PEP-gluconeogenesis (GN), but the rate of GN was similar in all intervention groups (Table 4). In a net sense, the major contribution to the decreased GP in all intervention groups was due to a marked decrease in glycogenolysis (to <20% of Con).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Hepatic glucose fluxes during insulin clamp


                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Hepatic glucose fluxes during insulin clamp

Effect of decreasing VF with Lep, beta 3, or FR on PEPCK and G-6-Pase gene expression. Multiple densitometric scanning of PCR products (examples shown in Fig. 3A) shows that when the hepatic G-6-Pase and PEPCK mRNA levels were compared with those of Con, they were increased by approximately twofold in Lep and more modestly in FR, but not in beta 3.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Gene expression of hepatic glucose-6-phosphatase (Glc-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Individual livers from each of the rats were rapidly obtained, clamp-frozen with liquid nitrogen, and stored in -80°C for subsequent analysis. RT-PCR analysis for Glc-6-Pase, PEPCK, and beta -actin is described in text. A: example of RT-PCR analysis from Con, Lep, beta 3, and FR. B: analysis of all RT-PCR data obtained from all rats, corrected for intensity of beta -actin, and presented in arbitrary units. * P < 0.01 vs. Con and beta 3.

Leptin, but not decreased VF per se, augments insulin action on glucose uptake, glycogen synthesis, and glycolysis. During the insulin clamp studies, glucose uptake (Rd, Fig. 4A) was increased by 63% (P < 0.001) in Lep (17.5 ± 1.1, 28.6 ± 1.3, 19.2 ± 1.3, and 20.7 ± 1.3 mg · kg-1 · min-1 in Con, Lep, beta 3, and FR; respectively). This improvement in peripheral insulin action was accounted for by a twofold increase in the rate of glycogen synthesis (Fig. 4B; 5.6 ± 0.9, 11.6 ± 1.2, 8.7 ± 1.3, and 9.7 ± 1.1 mg · kg-1 · min-1 in Con, Lep, beta 3, and FR; P < 0.001) and by a 26% increase in glycolysis (11.9 ± 1.6, 15.0 ± 1.2, 10.0 ± 1.2, and 11.2 ± 0.3 mg · kg-1 · min-1 in Con, Lep, beta 3, and FR; P < 0.01).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Peripheral insulin sensitivity. Glucose uptake (Rd, A), glycogen synthesis (GS, B), and glycolysis (C) during insulin infusion (3 mU · kg-1 · min-1). Glycolysis was determined by conversion rate of [3-3H]glucose to 3H2O, and GS was determined as the difference between Rd and glycolysis. Rd was increased by contribution from both GS and glycolysis. * P < 0.001 vs. all others; ** P < 0.01 vs. all others.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we attempted to delineate whether the potent effects of leptin on in vivo insulin action are secondary to the associated changes in body composition. Decreasing VF led to a striking improvement in hepatic insulin sensitivity that was independent of total fat mass (FM) and of the modalities used to achieve the "target" VF. This observation provides support for a cause-effect relationship between intra-abdominal deposition of fat and hepatic insulin resistance. Conversely, the marked stimulation of insulin-mediated glucose uptake, glycolysis, and glycogen synthesis induced by leptin treatment could not be reproduced by decreasing VF by alternative means. The latter finding indicates that, at least within the time frame of the present study, the effects of leptin on peripheral insulin action are not likely to be solely mediated via decreased VF and/or activation of the beta 3-adrenoreceptor system. Furthermore, rapid changes in VF modulate hepatic much more than peripheral insulin action.

"Manipulating" body composition. It is well established that weight loss is commonly associated with decreased plasma insulin concentrations and increased insulin sensitivity (8, 13, 15, 16, 27). Early studies in obese mice reported marked improvements in glucose tolerance after leptin treatment (19, 34). However, whereas some reports suggested that the improvement in glucose tolerance may precede the decline in body weight and total fat mass (34, 41), it has been difficult to discern the relative contribution of the associated changes in body composition to the improved glucose tolerance observed with leptin treatment (19, 34). Furthermore, although pair-feeding vehicle-treated rats to the level of leptin-treated rats resulted in similar decreases in body weight and FM, leptin caused a selective and marked decrease in visceral adiposity (7). The latter observation further complicates the interpretation of potential effects of leptin treatment on in vivo insulin action.

Administration of beta 3-adrenoreceptor agonists causes marked decreases in circulating leptin concentrations (18, 28, 30); however, consistent with previous reports (20), in the present study food intake was not decreased compared with Con (Table 1). Despite similar caloric intake, beta 3 rats gained less weight and their FM was significantly lower than Con rats. This may be due to increased energy expenditure and thermogenesis in this group (20).

To generate similar VF with FR it was necessary to further decrease the caloric intake by ~50%; this intervention resulted in much lower FM than in the other groups. This finding is a dramatic confirmation of the selective effects of Lep and beta 3 on intra-abdominal adiposity. This model is also different from the administration of leptin and beta 3-adrenoreceptor agonists, because energy expenditure is expected to be markedly decreased. Thus similar declines and final mass of VF were obtained in the three intervention groups despite differences in food consumption, weight gain, energy expenditure, food intake, and whole body adiposity.

VF and hepatic insulin sensitivity. All interventions that decreased VF resulted in similar fasting plasma glucose levels despite lower plasma insulin levels compared with Con rats, suggesting an improvement in postabsorptive hepatic insulin sensitivity. To directly test whether hepatic insulin sensitivity was improved by decreasing VF, we performed low-dose insulin clamp studies in combination with somatostatin infusions. The plasma glucose, FFA, glycerol, and insulin concentrations during the insulin clamp studies were similar in all groups (Table 2). This procedure also erased the portal-venous insulin gradient, matching peripheral and hepatic insulin levels in all groups. Decreasing VF resulted in a marked decrease in GP during the insulin infusion, indicating heightened hepatic insulin sensitivity (Fig. 2B). This improvement in insulin action was independent of the modalities by which decreased VF was achieved, and it is consistent with other animal models, such as the calorie-restricted "old" rats (2) and rats with surgical removal of VF (6). Although the mechanism(s) whereby VF regulates insulin sensitivity remain to be delineated, it is evident that the impacts of changes in VF on hepatic glucose fluxes are remarkable. It has been suggested that the unique metabolic characteristics of the intra-abdominal fat depots that concern the turnover of glycerol, FFA, and lactate play a role through a "portal effect" (9), i.e., the hepatic load of FFA, lactate, and glycerol can modulate liver glucose metabolism (31, 35). However, it should be noted that, in this experimental model, the peripheral concentrations of these substrates were unchanged during the basal and insulin clamp periods. Although potential effects of long-term differences in plasma FFA, lactate, or glycerol levels on hepatic enzymes cannot be excluded, alternative hypotheses should also be considered for the "cross-talk" between intra-abdominal fat depots and the liver. For example, a fat-derived and secreted peptide, tumor necrosis factor-alpha (TNF-alpha ), causes peripheral and hepatic insulin resistance via its antagonism of early insulin signaling (14, 21).

Consistent with the lower GP, the rates of TGO and GC were also markedly decreased in parallel with the changes in VF. This suggests a marked decrease in the in vivo flux through G-6-Pase. In a net sense, the decreased GP in the intervention groups was mainly the result of a marked suppression of hepatic glycogenolysis, which was most pronounced in the group treated with leptin (Table 2). Overall, whereas hepatic insulin sensitivity improved similarly with all interventions designed to decrease VF, there were some changes in the intraheptic distribution of hepatic glucose flux and in the gene expression of key hepatic enzymes that appear to be treatment specific. For example, the percent contribution of GN to TGO was increased by Lep right-arrow FR right-arrow beta 3 (Fig. 2C). This is supported by the increased expression of hepatic PEPCK in Lep and FR. Although leptin has similar effects on PEPCK mRNA when administered acutely via a peripheral vein (39) or in a cerebral ventricle (29), it should be pointed out that the decline in plasma insulin concentrations might also contribute to the upregulation of this gene. By contrast, it is noteworthy that acute and chronic stimulation of the beta -adrenergic systems has frequently divergent effects. We have previously shown that the acute (6-h) administration of the same beta 3-adrenoreceptor agonist increased the gene expression of G-6-Pase and PEPCK, perhaps via activation of hypothalamic efferent pathway(s) (29). The waning of this effect after more prolonged exposure to the agonist may be due to either the associated marked decline in leptin levels and/or central or peripheral downregulation of the beta -adrenoreceptor system. This also suggests that the stimulation of the beta 3-adrenoreceptor system is not likely to mediate the effects of chronic leptin administration on hepatic gene expression.

Unique effects of leptin on peripheral insulin sensitivity. A major effect of insulin in vivo is to stimulate the disposal of glucose into peripheral tissues (mostly in skeletal muscle). During physiological hyperinsulinemia, the rate of tissue glucose uptake in Lep was improved by >60%, whereas only a mild increase in peripheral glucose uptake was noted in the other intervention groups. This improvement in peripheral insulin action was accounted for by about a twofold increase in the rate of glycogen synthesis and by an ~25% increase in the rate of whole body glycolysis. On the basis of epidemiological evidence correlating insulin resistance and hyperinsulinemia with intra-abdominal adiposity (9, 25), it has been suggested that decreasing VF should lead to a marked improvement in the action of insulin on peripheral glucose disposal. Indeed, modest increases in the rates of insulin-mediated glucose uptake and glycogen synthesis were detected when VF was markedly decreased using caloric restriction or beta 3-adrenergic agonism. However, this improvement could only account for a small fraction (up to 30%) of the effects of leptin on glucose uptake. Thus 8-day leptin administration exerts potent effects on peripheral insulin action, which are largely independent of the associated decrease in VF. Several mechanism(s) may be invoked to account for the enhanced muscle insulin sensitivity in rats treated with leptin. Leptin has been shown to increase skeletal muscle glucose uptake quite rapidly in some rodent studies (22, 43), and activation of early insulin signaling by leptin has been demonstrated in a muscle cell line (23) and in a preliminary report in rats (24). However, acute exposure of skeletal muscle and adipose cells to leptin, with and without insulin, failed to alter the glucose transport system in some studies (46). Thus leptin may augment muscle insulin signaling via a direct action on local receptors or via hypothalamic efferent pathways. An additional explanation may be found in the "lipopenic" effects of leptin (32) and in the close correlation between intramyocellular lipid levels and insulin sensitivity (33). In fact, leptin enhances lipid oxidation and depletes triglyceride stores in preadipocytes, pancreatic beta -cells, and muscle (1, 32, 45). The latter effects may be mediated in part via decreased gene expression of acetyl-CoA carboxylase.

Taken together with the hepatic actions of leptin, the above data suggest that a prolonged elevation in circulating leptin favors the storage of energy into glycogen rather than into lipid stores. The latter metabolic adaptation may represent a response to signals generated by leptin in the hypothalamic "lipostat" and/or the results of peripheral actions of the hormone.

In conclusion, decreasing intra-abdominal adiposity by ~60% via three different means results in a dramatic increase in hepatic insulin sensitivity. Conversely, the potent effect of leptin administration on peripheral insulin action cannot be solely explained on the basis of the associated decrease in VF mass. Understanding the biochemical mechanism(s) that are responsible for the specific action of leptin on skeletal muscle glucose disposal should help to clarify the link between nutrient excess, weight gain, and insulin resistance.


    ACKNOWLEDGEMENTS

We thank Jie Wu, Robin Squeglia, and Rong Liu for expert technical assistance, and Drs. Michael McCaleb and Nancy Levin (Amgen, Thousand Oaks, CA) for providing recombinant mouse leptin.


    FOOTNOTES

This work was supported by grants from the National Institutes of Health (KO8-AG-00639 and R29-AG-15003 to N. Barzilai, R01-DK-45024 and ROI-DK-48321 to L. Rossetti), the American Diabetes Association, and the Core Laboratories of the Albert Einstein Diabetes Research and Training Center (DK-20541). Dr. Barzilai is a recipient of the Paul Beeson Physician Faculty Scholar in Aging Award. Dr. Rossetti is the recipient of a Career Scientist Award from the Irma T. Hirschl Trust.

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: N. Barzilai, Divisions of Geriatrics and Endocrinology, Dept. of Medicine, Belfer Bld. #701, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: barzilai{at}aecom.yu.edu).

Received 20 January 1999; accepted in final form 8 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bai, Y., S. Zhang, K.-S. Kim, J.-K. Lee, and K.-H. Kim. Leptin inhibits acetyl-CoA carboxylase in 3T3 pre-adipocytes. J. Biol. Chem. 271: 13939-13942, 1996[Abstract/Free Full Text].

2.   Barzilai, N., S. Banerjee, M. Hawkins, W. Chen, and L. Rossetti. Caloric restriction reverses hepatic insulin resistance in aging rats by decreasing visceral fat. J. Clin. Invest. 101: 1353-1361, 1998[Abstract/Free Full Text].

3.   Barzilai, N., D. Massillon, and L. Rossetti. Effects of fasting on hepatic and peripheral glucose metabolism in conscious rats with near-total fat depletion. Biochem. J. 310: 819-826, 1995[Medline].

4.   Barzilai, N., and L. Rossetti. Role of glucokinase and glucose-6-phosphatase in the acute and chronic regulation of hepatic glucose fluxes by insulin. J. Biol. Chem. 268: 25019-25025, 1993[Abstract/Free Full Text].

5.   Barzilai, N., and L. Rossetti. Age-related changes in body composition are associated with hepatic insulin resistance in conscious rats. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E930-E936, 1996[Medline].

6.   Barzilai, N., L. She, B.-Q. Liu, M. Hu, and L. Rossetti. Surgical removal of visceral fat (VF) in rats reverses hepatic insulin resistance. Diabetes 48: 341-345, 1999.

7.   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].

8.   Bessard, T., Y. Schutz, and E. Jequier. Energy expenditure and postprandial thermogenesis in obese women before and after weight loss. Am. J. Clin. Nutr. 38: 680-693, 1983[Abstract].

9.   Bjorntorp, P. Metabolic implications of body fat distribution. Diabetes Care 14: 1132-1143, 1991[Abstract].

10.   Clement, K., C. Vaisse, B. S. Manning, A. Basdevant, B. Guy-Grand, J. Ruiz, K. D. Silver, A. R. Shuldiner, P. Froguel, and A. D. Strosberg. Genetic variation in the beta 3-adrenergic receptor and an increased capacity to gain weight in patients with morbid obesity. N. Engl. J. Med. 333: 352-354, 1995[Abstract/Free Full Text].

11.   Cohen, B., D. Novick, and M. Rubinstein. Modulation of insulin activities by leptin. Science 274: 1185-1188, 1996[Abstract/Free Full Text].

12.   Cusin, I., K. E. Zakrzewska, O. Boss, P. Muzzin, J.-P. Giacobino, D. Ricquier, B. Jeanrenaud, and F. Rhohner-Jeanrenaud. Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins. Diabetes 47: 1014-1019, 1998[Abstract].

13.   Diaz, E. O., A. M. Prentice, G. R. Goldberg, P. R. Murgatroyd, and W. A. Coward. Metabolic response to experimental overfeeding in overweight healthy volunteers. Am. J. Clin. Nutr. 56: 641-655, 1992[Abstract].

14.   Feinstein, R., H. Kanety, M. S. Papa, B. Lunenfeld, and A. Karasic. Tumor necrosis factor-alpha supresses insulin-induced tyrosine phosphorylation of insulin receptor and its substrates. J. Biol. Chem. 268: 26055-26057, 1993[Abstract/Free Full Text].

15.   Felber, J. P., E. Ferrannini, A. Golay, H. U. Meyer, D. Thiebaud, and R. A. Defronzo. Role of lipid oxidation in pathogenesis of insulin resistance of obesity and type II diabetes. Diabetes 36: 1341-1350, 1987[Abstract].

16.   Flatt, J. P. Dietary fat, carbohydrate balance, and weight maintenance: effect of exercise. Am. J. Clin. Nutr. 45: 296-306, 1987[Medline].

17.   Flier, J. S. Leptin expression and action: new experimental paradigms. Proc. Natl. Acad. Sci. USA 94: 4242-4245, 1997[Free Full Text].

18.   Gettys, T. W., P. J. Harkness, and P. M. Watson. The beta 3-adrenergic receptor inhibits insulin-stimulated leptin secretion from isolated rat adipocytes. Endocrinology 137: 4054-4057, 1996[Abstract].

19.   Halaas, J. L., K. S. Gajiwala, M. Maffei, S. L. Cohen, B. T. Chait, D. Rabinowitz, R. L. Lallone, S. K. Burley, and J. M. Friedman. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269: 543-546, 1995[Medline].

20.   Himms-Hagen, J., J. Cui, E. Danforth, Jr., D. J. Taatjes, S. S. Lang, B. L. Waters, and T. H. Claus. Effect of CL-316,243, a thermogenic beta 3-agonist, on energy balance and brown and white adipose tissues in rats. Am. J. Physiol. 266 (Regulatory Integrative Comp. Physiol. 35): R1371-R1382, 1994[Abstract/Free Full Text].

21.   Hotamisligil, G. S., N. S. Shargill, and B. M. Spiegelman. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 259: 87-91, 1993[Medline].

22.   Kamohara, S., R. Burcelin, J. L. Halaas, J. M. Friedman, and M. J. Charron. Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389: 374-377, 1997[Medline].

23.   Kellerer, M., M. Koch, E. Metzinger, J. Mushack, E. Capp, and H. U. Haring. Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways. Diabetologia 40: 1358-1362, 1997[Medline].

24.   Kim, Y.-B., S. Uotani, J. S. Flier, and B. B. Kahn. In vivo administration of leptin rapidly activates PI3-kinase in insulin sensitive tissues. Diabetes 47, Suppl. 1: 1226, 1998.

25.   Kissebah, A. H. Insulin resistance in visceral obesity. Int. J. Obes. 15, Suppl. 2: 109-115, 1991[Medline].

26.   Krief, S., F. Lonnqvist, S. Raimbault, B. Baude, A. Van Spronsen, P. Arner, A. D. Strosberg, D. Ricquier, and L. J. Emorine. Tissue distribution of beta 3-adrenergic receptor mRNA in man. J. Clin. Invest. 91: 344-349, 1993[Medline].

27.   Larson, B. Regional obesity as health hazard in men---prospective studies. Acta Med. Scand. 723: 45-51, 1992.

28.   Li, H., M. Matheny, and P. J. Scarpace. beta 3-Adrenergic-mediated suppression of leptin gene expression in rats. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E1031-E1036, 1997[Abstract/Free Full Text].

29.   Liu, L., G. Karkanias, J. Morales, M. Hawkins, N. Barzilai, J. Wang, and L. Rossetti. Intracerebroventricular (ICV) leptin regulates hepatic but not peripheral glucose fluxes. J. Biol. Chem. 273: 31160-31167, 1998[Abstract/Free Full Text].

30.   Mantzoros, C. S., D. Qu, R. C. Frederich, V. S. Susulic, B. B. Lowell, E. Maratos-Flier, and J. S. Flier. Activation of beta(3) adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes 45: 909-914, 1996[Abstract].

31.   Massillon, D., N. Barzilai, M. Hawkins, D. Prus-Wertheimer, and L. Rossetti. Induction of hepatic glucose-6-phosphatase gene expression by lipid infusion. Diabetes 46: 153-157, 1997[Abstract].

32.   Muoio, D. M., G. L. Dohn, F. T. Fiedorek, E. B. Tapscott, and R. A. Coleman. Leptin directly alters lipid partitioning in skeletal muscle. Diabetes 46: 1360-1363, 1997[Abstract].

33.   Pan, D. A., S. Lillioja, A. D. Kriketos, M. R. Milner, L. A. Baur, C. Bogardus, A. B. Jenkins, and L. H. Storlien. Skeletal muscle triglyceride levels are inversely related to insulin. Diabetes 46: 983-988, 1997[Abstract].

34.   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[Medline].

35.   Rebrin, K., G. M. Steil, S. D. Mittelman, and R. N. Bergman. Causal linkage between insulin suppression of lipolysis and suppression of liver glucose output in dogs. J. Clin. Invest. 98: 741-749, 1996[Abstract/Free Full Text].

36.   Rossetti, L., N. Barzilai, W. Chen, T. Harris, D. Yang, and C. E. Rogler. Hepatic overexpression of insulin-like growth factor-II in adulthood increases basal and insulin-stimulated glucose disposal in conscious mice. J. Biol. Chem. 271: 203-208, 1996[Abstract/Free Full Text].

37.   Rossetti, L., and A. Giaccari. Relative contribution of glycogen synthesis and glycolysis to insulin-mediated glucose uptake. A dose-response euglycemic clamp study in normal and diabetic rats. J. Clin. Invest. 85: 1785-1792, 1990[Medline].

38.   Rossetti, L., A. Giaccari, N. Barzilai, K. Howard, G. Sebel, and M. Hu. Mechanism by which hyperglycemia inhibits hepatic glucose production in conscious rats. Implications for the pathophysiology of fasting hyperglycemia in diabetes. J. Clin. Invest. 92: 1126-1134, 1993[Medline].

39.   Rossetti, L., D. Massillon, N. Barzilai, P. Vuguin, J. Wu, R. Liu, and J. Wang. Short-term effects of leptin on hepatic gluconeogenesis and in vivo insulin action. J. Biol. Chem. 272: 27758-27763, 1997[Abstract/Free Full Text].

40.   Sakane, N., T. Yoshida, T. Umekawa, M. Kondo, Y. Sakai, and T. Takahashi. Beta 3-adrenergic-receptor polymorphism: a genetic marker for visceral fat obesity and the insulin resistance syndrome. Diabetologia 40: 200-204, 1997[Medline].

41.   Schwartz, M. W., D. G. Baskin, T. R. Bukowski, J. L. Kuijper, D. Foster, G. Lasser, D. E. Prunkard, D. J. Porte, S. C. Woods, R. Seeley, and D. 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].

42.   Schwartz, M. W., R. J. Seeley, L. A. Campfield, P. Burn, and D. G. Baskin. Identification of targets of leptin action in rat hypothalamus. J. Clin. Invest. 98: 1101-1106, 1996[Abstract/Free Full Text].

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

44.   Weigle, D. S., T. R. Bukowski, D. C. Foster, S. Holderman, J. M. Kramer, G. Lasser, C. E. Lofton-Day, D. E. Prunkard, C. Raymond, and J. L. Kuijper. Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J. Clin. Invest. 96: 2065-2070, 1995[Medline].

45.   Zhou, Y. T., M. Shimabukuro, Y. Lee, K. Koyama, M. Higa, T. Ferguson, and R. H. Unger. Enhanced de novo lipogenesis in the leptin-unresponsive pancreatic islets of prediabetic Zucker diabetic fatty rats: role in the pathogenesis of lipotoxic diabetes. Diabetes 47: 1904-1909, 1998[Abstract].

46.   Zierath, J. R., E. U. Frevert, J. W. Ryder, P.-O. Berggren, and B. B. Kahn. Evidence against a direct effect of leptin on glucose transport in skeletal muscle and adipocytes. Diabetes 47: 1-4, 1998[Abstract].


Am J Physiol Endocrinol Metab 277(2):E291-E298
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society