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Originally published In Press as doi:10.1074/jbc.M408665200 on September 10, 2004

J. Biol. Chem., Vol. 279, Issue 48, 49704-49715, November 26, 2004
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Melanocortin-independent Effects of Leptin on Hepatic Glucose Fluxes*

Roger Gutiérrez-Juárez{ddagger}, Silvana Obici, and Luciano Rossetti§

From the Departments of Medicine & Molecular Pharmacology, Diabetes Research & Training Center, Albert Einstein College of Medicine, Bronx, New York, 10461

Received for publication, July 30, 2004 , and in revised form, September 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin and insulin share some hypothalamic signaling molecules, but their central administration induces different effects on hepatic glucose fluxes. Acute insulin infusion in the third cerebral ventricle inhibits endogenous glucose production (GP), whereas acute leptin infusion stimulates gluconeogenesis but does not alter GP because of a compensatory decrease in glycogenolysis. Because melanocortin agonists also stimulate hepatic gluconeogenesis, here we examined whether central melanocortin blockade modifies the acute effects of leptin on GP, on gluconeogenesis, on glycogenolysis, and/or on the hepatic expression of the gluconeogenic enzymes glucose-6-phosphatase (Glc-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Systemic or central administration of leptin alone did not alter GP, despite increasing both the rate of gluconeogenesis and the expression of Glc-6-Pase and PEPCK. When activation of the central melanocortin pathway was prevented, the effects of leptin on gluconeogenesis, Glc-6-Pase, and PEPCK were abolished, and a marked suppression of glycogenolysis resulted in decreased GP. We conclude that leptin regulates hepatic glucose fluxes through a melanocortin-dependent pathway leading to stimulation of gluconeogenesis and a melanocortin-independent pathway causing inhibition of GP and glycogenolysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin, the 167-amino acid polypeptide product of the ob gene, is secreted by the adipose tissue and acts mainly on the central nervous system to regulate energy balance (1). Leptin regulates food intake and body adiposity partly via activation of melanocortin receptors in the hypothalamus and in other areas within the central nervous system (2, 3). It is now well recognized that leptin also plays an important role in the regulation of insulin action and intermediate metabolism (4–9). Indeed, prolonged administration of recombinant leptin to non-diabetic and diabetic rats (10–13) as well as to lipodystrophic humans and mice (4, 14) markedly improves in vivo insulin action. Similarly, bi-directional modulation of the activity of the central melanocortin pathway leads to significant changes in hepatic and peripheral insulin action (15). On the other hand, the prolonged administration of either leptin or melanocortin agonists or antagonists also impacts on the distribution of body adiposity and on lipid homeostasis (10, 15–18). These effects are in turn likely to secondarily influence the in vivo action of insulin on metabolic parameters (15). Thus, it is important to delineate the acute effects of leptin and melanocortins on metabolic fluxes prior to the onset of changes in energy balance, body composition, and lipid storage.

In this regard, acute administration of leptin to postabsorptive rats causes a marked redistribution of intrahepatic glucose fluxes, with a marked increase in the relative contribution of gluconeogenesis and a parallel decrease in the contribution of glycogenolysis to hepatic glucose output (5, 6). This effect is seen following either systemic or central administration of the hormone (5, 6). Which are the mechanism(s) by which leptin stimulates gluconeogenesis and inhibits glycogenolysis? Leptin appears to exert its pleiotropic behavioral, metabolic, and neuroendocrine actions via multiple and partly divergent central pathways (19, 20). For example, it has been suggested that leptin controls the hypothalamic melanocortin pathway and energy balance via STAT3-dependent1 signaling and the NPY pathway, reproductive function, and glucose homeostasis via STAT3-independent signaling (20, 21). Furthermore, leptin and insulin receptors appear to share mechanisms of signal transduction such as the activation of phosphatidylinositol 3-kinase (PI3K) (22, 23). These observations are intriguing because these two hormones have partly overlapping and partly divergent physiological actions (24–26). Of particular interest, the acute ICV administration of insulin markedly inhibits endogenous glucose production (GP) via a PI3K-dependent mechanism (27), whereas the acute administration of leptin stimulates gluconeogenesis and liver PEPCK expression (5, 6).

To delineate the contribution of the central melanocortin pathway to the acute effects of leptin on glucose homeostasis, we first examined whether and how the central activation of the melanocortin pathway modulates glucose fluxes, and then we examined the contribution of the central melanocortin pathway to the acute effects of systemic and ICV leptin on glucose kinetics. Our results reveal specific and potent melanocortin-dependent and melanocortin-independent effects of leptin on hepatic glucose fluxes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Design—Ten-week-old male Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilmington, MA) were housed in individual cages and subjected to a standard light-dark cycle (0600–1800/1800–0600). Three weeks prior to the in vivo studies (see Fig. 1B), all of the rats received implantations of ICV catheters by stereotaxic surgery as previously described (6). One week before the pancreatic insulin clamp protocols, the rats received additional catheters in the right internal jugular and left carotid artery as previously described (5, 6). The rats were fed a standard chow (catalog number 5001; Purina Mills Ltd.) and were allowed to recover completely before initiation of all in vivo studies.



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  		FIG. 1.
Effect of stimulation of the central melanocortin pathway on glucose metabolism. A, time line for the surgical implantation of catheters and ICV cannulae. B, experimental protocol for the ICV infusions and the pancreatic hyperinsulinemic clamp protocol. C, insulin action on peripheral glucose uptake and production in mg/kg·min. The glucose rate of disappearance (Rd) was measured during the hyperinsulinemic clamp by tracer dilution techniques, and the action of insulin on GP is expressed as a percentage of inhibition from basal values in the same animal. *, p < 0.01 versus vehicle. D, schematic representation of the effects of central melanocortin pathway activation on hepatic glucose metabolism. We postulate that the central activation of the melanocortin pathway will increase the hepatic expression of gluconeogenic enzymes and stimulate the rate of gluconeogenesis. We plan to test this hypothesis via central administration of the melanocortin agonist {alpha}-MSH in the absence (left panel) or presence (right panel) of the melanocortin receptor antagonist SHU9119.

 
Insulin Action Studies—The rats (n = 79) were implanted with chronic catheters as previously described (6). After full recovery from the catheterization, the animals were randomized into eight groups receiving the following infusions for the duration of the in vivo study (6H; see Fig. 1C): 1) ICV vehicle, 2) ICV {alpha}-MSH (3 µg/6 h), 3) ICV leptin (1.5 µg/6 h), and 4) IV leptin (3 µg/kg·min); each of the above infusions were performed in the absence or presence of ICV SHU9119 (1 µg/6 h). At t =–2 h (Fig. 1C), a primed continuous infusion of ICV solutions was initiated and maintained throughout the duration of the study. Plasma glucose was measured periodically initiating at the onset (t = 2h) of the ICV infusion and lasting throughout the duration of the study. Plasma samples for the determination of insulin, leptin, and nonesterified fatty acid concentration were obtained at the onset (t = –2 h) and at 30-min intervals during the study. At t = 0, a primed continuous infusion of high pressure liquid chromatography-purified [3-3H]glucose (PerkinElmer Life Sciences; 40 µCi of bolus, 0.4 µCi/min for duration of the study) was initiated and maintained for the last 4 h of the study. Samples for determination of [3H]glucose specific activity were obtained at 10-min intervals throughout the infusions. Finally, at t = 2 h, a pancreatic insulin clamp study was initiated and maintained for 2 h. During this procedure, a primed continuous infusion of regular insulin (3 milliunits/kg·min) was administered, and a variable infusion of 25% glucose solution was started at t = 2 and periodically adjusted to clamp the plasma glucose concentration at ~7mM. The rate of insulin infusion was designed to obtain physiological hyperinsulinemia (see Table I). To control for possible effects of the ICV injections on endocrine pancreas, somatostatin (3 µg/kg/min) was co-infused with insulin to inhibit endogenous islets secretion. At the end of the in vivo studies, the rats were anesthetized (pentobarbital 55 mg/kg body weight, intravenously), and tissue samples were freeze-clamped in situ with aluminum tongs precooled in liquid nitrogen. All of the tissue samples were stored at –80 °C for subsequent analysis.


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  		TABLE I
Characteristics of the experimental groups before and during the pancreatic clamp studies

The values during the clamp represent the steady-state levels obtained by averaging the results of at least four samples during the experimental period. Food intake was measured before the acute ICV infusions.

 
The ICV vehicle was artificial cerebrospinal fluid, whereas the IV vehicle was isotonic saline. The leptin used in these studies was recombinant mouse leptin (gift of Dr. M. McCaleb, Amgen, Inc., Thousand Oaks, CA; >95% pure by SDS-PAGE).

Analytical Procedures and Calculations—Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Inc., Fullerton, CA). Plasma insulin and leptin levels were determined by RIA (rat leptin RIA kit; Linco Research Inc., St. Charles, MO). Serum adiponectin was measured by RIA (Linco Research, Inc.). Plasma nonesterified fatty acid concentrations were determined an enzymatic method by an automated kit according to the manufacturer's specifications (Waco Pure Chemical Industries, Osaka, Japan). The radioactivity of [3-3H]glucose in plasma was measured from supernatants of Ba(OH)2 and from ZnSO4 precipitates (Somogyi procedure), after each was evaporated to dryness for the removal of tritiated water. The hepatic [14C]phosphoenolpyruvate (PEP) and [3H/14C]UDP glucose specific activities were obtained by two sequential chromatographic separations, as previously described (28–31). Calculations were performed as described (10). Briefly, the rates of PEP-gluconeogenesis, the flux through glucose-6-phosphatase (Glc-6-Pase flux) and glucose cycling (Glc-6-Pase flux–GP) were calculated as previously described (10). Gluconeogenesis was estimated from the specific activities of 14C-labeled hepatic UDP-glucose (assumed to reflect the specific activity of hepatic glucose-6-phosphate) and hepatic PEP following the infusion of [U-14C]lactate and [3-3H]glucose based on the following formula: gluconeogenesis = Glc-6-Pase flux x [14C]UDP-glucose specific activity/[14C]PEP specific activity x 2 (Table II). Net glycogenolysis was calculated as the difference between GP and gluconeogenesis.


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  		TABLE II
Specific activities of hepatic substrates used to calculate the "direct pathway" and the "indirect pathway" at the end of the [3-3H]glucose-[U-14C]lactate infusions

PEP, phosphoenolpyruvate; UUDPGlc, uridinediphosphoglucose; Indirect, percentage of the hepatic Glc-6-P pool derived from PEP-gluconeogenesis; Glc, plasma glucose; Direct, percentage of the hepatic Glc-6-P pool derived from plasma glucose.

 
Gene Expression—Glc-6-Pase and phosphoenolpyruvate carboxykinase (PEPCK) expression was measured by quantitative real time PCR. Briefly, total RNA was extracted from rat livers using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was then synthesized from 2 µg of total RNA using the Superscript III First Strand kit (Invitrogen). Samples of RNA in which the reverse transcriptase was omitted ("no reverse transcriptase controls") were also included. The cDNA and reverse transcriptase controls were diluted 1:20 with PCR grade water and 2 µl from each sample was used for PCR in a total volume of 20 µl. The FastStart DNA Master SYBR Green I kit (Roche Applied Science) and a LightCycler 2 instrument (Roche Applied Science) were used in all experiments. Either 18 S or glyceraldehyde-3-phosphate dehydrogenase mRNA expression was routinely measured for normalization purposes. The primers used were: 5'-GCGACCGTCCCCTTTGCATCTGTC-3' and 5'-CCACCAAACACTCCCCCTCCTCC-3' for Glc-6-Pase; 5'-GCCTCCTCAGCTGCATAATGGTCT-3' and 5'-GAATGCTTTCTCGAAGTCCTCTTCTG-3' for PEPCK; 5'-AGGGTTCGATTCCGGAGAGG-3' and 5'-CAACTTTAATATACGCTATTGG-3' for 18 S; and 5'-CGACCCCTTCATTGACCTCAAC-3' and 5'-ACTCCACGACATACTCAGCACC-3' for glyceraldehyde-3-phosphate dehydrogenase. The copy mRNA number was calculated from standard curves obtained using plasmid DNA standards of known copy number for the each gene of interest.

Statistical analysis was done using unpaired Student's t test or analysis of variance. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To investigate the mechanism by which leptin regulates glucose kinetics independently of its pleiotropic actions on energy balance and body composition, we examined whether and how acute modulation of the activity of central melanocortin receptors affect the action of leptin on hepatic and peripheral glucose fluxes. ICV catheters were implanted in male Sprague-Dawley rats by stereotaxic surgery (6). All of the infusion studies were performed ~3 weeks later following complete recovery from the operation (Fig. 1A).

Activation of Central Melanocortin Receptors Leads to a Marked Increase in Liver Gluconeogenesis—We first examined whether stimulation of central melanocortin receptors per se alters insulin action on glucose metabolism. To this end, four groups of conscious rats received an ICV infusion of either vehicle, {alpha}-MSH (agonist of the melanocortin receptors), SHU9119 (antagonist of the melanocortin receptors), or both {alpha}-MSH and SHU9119 (34) in combination with pancreatic insulin clamp studies (Fig. 1B). The plasma glucose, insulin, leptin, and FFA concentrations were similar in the four groups prior and during the clamp procedure. During physiologic hyperinsulinemia (plasma insulin ~400 pM), the rate of glucose infusion required to maintain the plasma glucose concentration at basal levels was decreased (by 25%) by ICV {alpha}-MSH, and this decrease was completely abolished by the concomitant ICV infusion of SHU9119. The two major effects of insulin on glucose metabolism are to stimulate the uptake of glucose into peripheral tissues (mostly in skeletal muscle and adipose tissue) and to diminish the production of glucose by the liver. The rate of tissue glucose uptake (Rd) in rats receiving ICV {alpha}-MSH (22.8 ± 1.7 mg/kg·min) was similar to that of rats receiving ICV vehicle (21.9 ± 1.4; p = not significant) (Fig. 1C). Conversely, ICV {alpha}-MSH impaired the action of insulin in inhibiting GP (Fig. 1D). Importantly, SHU9119 per se did not modify the effect of insulin on GP, but it blocked the effect of ICV {alpha}-MSH. In Fig. 1D, the effect of insulin on GP is expressed as a percentage of inhibition from basal levels. Insulin inhibited GP by 58 ± 4% in the presence of ICV vehicle but by only 20 ± 10% when ICV {alpha}-MSH was infused (p < 0.01 versus vehicle). When SHU9119 was co-infused ICV, {alpha}-MSH failed to alter the inhibitory action of insulin on GP (64 ± 11%), whereas SHU9119 alone did not alter hepatic insulin action (60 ± 11%). Thus, the stimulatory effect of the central administration of {alpha}-MSH on GP is specific because the selective inhibition of central melanocortin receptors is sufficient to negate the effects of ICV {alpha}-MSH on hepatic insulin action.

In the presence of physiological hyperinsulinemia, ICV {alpha}-MSH caused a 2-fold increase in the rate of GP (Fig. 2A) as compared with the ICV vehicle-infused group. This effect was abolished by the concomitant administration of SHU9119, whereas the ICV administration of an equal dose of the melanocortin antagonist per se did not modify GP. To further delineate the mechanisms responsible for the effects of {alpha}-MSH on GP, we estimated the in vivo flux through glucose-6-phosphatase (Fig. 2B) and the relative contributions of glycogenolysis (Fig. 2C) and gluconeogenesis (Fig. 2D) to glucose output. Glucose-6-phosphatase catalyzes the final step in the dephosphorylation of glucose-6-phosphate derived from either gluconeogenesis or glycogenolysis to glucose. The increase in GP caused by {alpha}-MSH was paralleled by a 2.4-fold increase in the flux through glucose-6-phosphatase, an effect that was also negated by the co-infusion of SHU9119. The marked effects of ICV {alpha}-MSH on GP and glucose-6-phosphatase flux were entirely accounted for by a 3.4-fold increase in the rate of gluconeogenesis when compared with the vehicle-infused group (9.3 ± 2.0 versus 2.7 ± 0.5 mg/kg·min; p < 0.01), whereas the rates of glycogenolysis were not significantly affected. The stimulatory effect of ICV {alpha}-MSH on gluconeogenesis was prevented by the ICV co-infusion of a melanocortin receptor antagonist. Finally, we examined whether changes in the expression of the liver gluconeogenic enzymes Glc-6-Pase and PEPCK could mediate these changes in hepatic glucose fluxes. Central activation of melanocortin receptors led to increased hepatic expression of Glc-6-Pase (2.4-fold; Fig. 2E) and PEPCK (2-fold; Fig. 2F). These transcriptional changes were also negated by the central administration of SHU9119 (Fig. 2, E and F). Overall, we describe a potent and rapid stimulatory effect of the central melanocortin pathway on gluconeogenesis and on hepatic Glc-6-Pase and PEPCK mRNA. Importantly, we have also identified a dose of the melanocortin receptor antagonist SHU9119 that specifically blocks the effects of ICV {alpha}-MSH but has no direct actions on liver glucose fluxes or gene expression. The latter observation provides an attractive experimental tool to delineate the contribution of central melanocortin receptors to the rapid metabolic actions of leptin.



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  		FIG. 2.
Effect of stimulation of the central melanocortin pathway on hepatic glucose fluxes and on the gene expression of gluconeogenic enzymes. GP (A) and glucose-6-phosphatase flux (B) were markedly increased in response to ICV {alpha}-MSH, and this effect was eliminated by the co-infusion of SHU9119; the rate of hepatic glycogenolysis (C) was not significantly altered by the ICV infusions. Conversely, the rate of gluconeogenesis (D) was markedly increased by the ICV infusion of {alpha}-MSH, and this effect was prevented by the co-infusion of SHU9119; {alpha}-MSH also increased the liver content of glucose-6-phosphatase (E) and PEPCK (F) mRNA. *, p < 0.01 versus vehicle.

 
Melanocortin-independent Effects of Systemic Leptin on Hepatic Glucose Fluxes—We next examined whether and how central antagonism of melanocortin receptors alters the effects of systemic leptin on hepatic glucose fluxes. To this end, three groups of conscious rats received a systemic infusion of either vehicle, leptin, or systemic leptin and ICV SHU9119 in combination with pancreatic insulin clamp studies (Fig. 3, A and B, and Table I). The plasma glucose, insulin, leptin, and FFA concentrations were similar in the three groups prior and during the clamp procedure (Table I). During physiologic hyperinsulinemia (~400 pM plasma insulin), the rate of glucose infusion required to maintain the plasma glucose concentration at basal levels was not affected by leptin alone, but it was significantly increased (by 75%) by the concomitant ICV infusion of SHU9119. The rates of glucose uptake (Rd) were similar in rats receiving systemic vehicle or leptin with or without ICV SHU9119 (p = not significant) (Fig. 3B). Conversely, the coinfusion of systemic leptin and ICV SHU9119 significantly enhanced the action of insulin in inhibiting GP (Fig. 3B). In Fig. 3B, the effect of insulin on GP is expressed as the percentage of inhibition from basal levels. Insulin inhibited GP by 55 ± 5% in the presence of vehicle. Although leptin alone did not alter hepatic insulin action (47 ± 4%), when ICV SHU9119 was co-infused systemic leptin resulted in enhanced suppression of GP (by 79 ± 6%; p < 0.01 versus vehicle). Thus, the central administration of an antagonist of the melanocortin receptors unveils a potent melanocortin-independent effect of systemic leptin on hepatic insulin action.



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  		FIG. 3.
Effect of systemic leptin on glucose metabolism. A, experimental protocol for the ICV infusions and the pancreatic hyperinsulinemic clamp protocol. B, insulin action on peripheral glucose uptake and production in mg/kg·min. The glucose rate of disappearance (Rd) was measured during the hyperinsulinemic clamp by tracer dilution techniques, and the action of insulin on GP is expressed as a percentage of inhibition from basal values in the same animal. *, p < 0.01 versus vehicle. C, schematic representation of the effects of systemic leptin on hepatic glucose metabolism. We postulate that the systemic administration of leptin leads to activation of hypothalamic leptin signaling, which in turn will increase the hepatic expression of gluconeogenic enzymes, stimulate the rate of gluconeogenesis, and decrease the rate of glycogenolysis. We plan to test the role of the melanocortin pathway in these effects of systemic leptin via central administration of the of the melanocortin receptor antagonist SHU9119. We postulate that the effects of systemic leptin on gluconeogenesis but not on glycogenolysis will depend on activation of the central melanocortin pathway.

 
To further delineate the mechanisms responsible for the effects of systemic leptin on GP, we estimated the in vivo flux through glucose-6-phosphatase (Fig. 4B) and the relative contributions of glycogenolysis (Fig. 4C and Table II) and gluconeogenesis (Fig. 4D) to glucose output. In the presence of physiological hyperinsulinemia, leptin did not modify the rate of GP as compared with the vehicle-infused group. However, the concomitant administration of systemic leptin and ICV SHU9119 markedly decreased GP (Fig. 3A). The decrease in GP caused by the combination of leptin and SHU9119 was paralleled by a marked decrease in the flux through Glc-6-Pase. Consistent with our previous report (5), the systemic infusion of recombinant leptin markedly increased gluconeogenesis by 2.3-fold (5.1 ± 0.7 versus 2.2 ± 0.3 mg/kg·min). However, leptin also markedly decreased the rate of glycogenolysis (from 4.0 ± 0.26 mg/kg·min in vehicle to 1.34 ± 0.26 mg/kg·min), and this effect was not modified by the central antagonism of melanocortin receptors (1.65 ± 0.51 mg/kg·min). Conversely, the stimulatory effect of leptin on gluconeogenesis was abolished by the ICV infusion of a melanocortin receptor antagonist (2.7 ± 0.5 mg/kg·min; p < 0.01). Thus, the marked inhibitory effects of systemic leptin and ICV SHU9119 on GP and glucose-6-phosphatase flux were entirely accounted for by a marked decrease in the rate of glycogenolysis when compared with the vehicle control group and by a marked decrease in the rate of gluconeogenesis when compared with the leptin-infused group. Finally, we examined whether changes in the liver expression of Glc-6-Pase (Fig. 4E) and PEPCK (Fig. 4F) could mediate these changes in hepatic glucose fluxes. Specifically, because central activation of melanocortin receptors as well as systemic leptin infusion led to increased hepatic expression of Glc-6-Pase and PEPCK, we postulated that the activation of central melanocortin receptors mediates this effect of leptin. Indeed, the central administration of SHU9119 also negated these hepatic transcriptional effects of systemic leptin (Fig. 4, E and F). Overall, the rapid stimulatory effect of the systemic leptin infusion on gluconeogenesis and on hepatic Glc-6-Pase and PEPCK mRNA requires activation of central melanocortin receptors. However, the potent inhibitory effect of leptin on hepatic glycogenolysis is independent of its activation of the central melanocortin pathway.



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  		FIG. 4.
Effect of systemic leptin on hepatic glucose fluxes and on the gene expression of gluconeogenic enzymes. Glucose production (A) and glucose-6-phosphatase flux (B) were markedly decreased by systemic leptin only when the central activation of the melanocortin pathway was prevented by the ICV infusion of SHU9119. The rate of hepatic glycogenolysis (C) was markedly suppressed by systemic leptin, but the ICV infusion of SHU9119 did not modify this effect. Conversely, the rate of gluconeogenesis (D) was markedly increased by the systemic infusion of leptin, and this effect was prevented by the ICV infusion of SHU9119; systemic leptin also increased the liver content of glucose-6-phosphatase (E) and PEPCK (F) mRNA, and these effects were prevented by the central administration of SHU9119. *, p < 0.01 versus vehicle.

 
Direct effects of leptin on hepatic insulin signaling and glycogenolysis have been previously reported in isolated hepatocytes or in perfused liver (35–37). Thus, it is important to delineate whether the potent melanocortin-independent effects of leptin on hepatic glucose fluxes can also be elicited in response to the central rather than systemic administration of leptin.

Melanocortin-independent Effects of Central Leptin on Hepatic Glucose Fluxes—We next examined whether and how central antagonism of melanocortin receptors modulates the effects of the central administration of leptin on hepatic glucose fluxes. To this end, three groups of conscious rats received either an ICV infusion of vehicle or leptin or ICV leptin and ICV SHU9119 in combination with pancreatic insulin clamp studies (Fig. 5A and Table I). The plasma glucose, insulin, leptin, and FFA concentrations were similar in the three groups prior and during the clamp procedure (Table I). During physiologic hyperinsulinemia (~450 pM plasma insulin), the rate of glucose infusion required to maintain the plasma glucose concentration at basal levels was not affected by ICV leptin alone, but it was significantly increased (by ~20%) by the concomitant ICV infusion of SHU9119. The rates of glucose uptake (Rd) were similar in rats receiving ICV vehicle or leptin with or without ICV SHU9119 (p = not significant) (Fig. 5B). Conversely, the ICV co-infusion of leptin and SHU9119 significantly enhanced the action of insulin in inhibiting GP (Fig. 5B). In Fig. 5B, the effect of insulin on GP is expressed as a percent of inhibition from basal levels. Insulin inhibited GP by 58 ± 4% in the presence of ICV vehicle. Although ICV leptin alone did not alter hepatic insulin action (58 ± 3%), when SHU9119 was co-infused ICV leptin resulted in enhanced suppression of GP (by 82 ± 6%; p < 0.01 versus vehicle). Thus, the central administration of an antagonist of the melanocortin receptors unveils a potent melanocortin-independent effect of central leptin on hepatic insulin action.



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  		FIG. 5.
Effect of central (ICV) leptin on glucose metabolism. A, experimental protocol for the ICV infusions and the pancreatic hyperinsulinemic clamp protocol. B, insulin action on peripheral glucose uptake and production in mg/kg·min. Glucose rate of disappearance (Rd) was measured during the hyperinsulinemic clamp by tracer dilution techniques, and the action of insulin on GP is expressed as a percentage of inhibition from basal values in the same animal. *, p < 0.01 versus vehicle. C, schematic representation of the effects of central leptin on hepatic glucose metabolism. We postulate that the central administration of leptin leads to activation of hypothalamic leptin signaling, which in turn will increase the hepatic expression of gluconeogenic enzymes, stimulate the rate of gluconeogenesis, and decrease the rate of glycogenolysis. We plan to test the role of the melanocortin pathway in these central effects of leptin via ICV administration of the of the melanocortin receptor antagonist SHU9119. We postulate that the effects of leptin on gluconeogenesis but not on glycogenolysis will depend on activation of the central melanocortin pathway.

 
To further delineate the mechanisms responsible for the effects of ICV leptin on GP, we estimated the in vivo flux through glucose-6-phosphatase (Fig. 6B) and the relative contributions of glycogenolysis (Fig. 6C) and gluconeogenesis (Fig. 6D) to glucose output. In the presence of physiological hyperinsulinemia, leptin did not modify the rate of GP as compared with the vehicle-infused group. However, the concomitant ICV administration of leptin and SHU9119 markedly decreased GP (Fig. 6A). The decrease in GP caused by the combination of leptin and SHU9119 was paralleled by a marked decrease in the flux through Glc-6-Pase. Consistent with our previous report (6), the ICV infusion of recombinant leptin markedly increased gluconeogenesis by ~2-fold (4.8 ± 0.6 versus 2.7 ± 0.5 mg/kg·min). However, leptin also markedly decreased the rate of glycogenolysis (from 4.0 ± 0.26 mg/kg·min in vehicle to 1.2 ± 0.5 mg/kg·min), and this effect was not modified by the central antagonism of melanocortin receptors (1.4 ± 0.5 mg/kg·min). Conversely, the stimulatory effect of leptin on gluconeogenesis was abolished by the ICV infusion of a melanocortin receptor antagonist (1.4 ± 0.6 mg/kg·min; p < 0.01). Thus, the marked inhibitory effects of central leptin and SHU9119 on GP and glucose-6-phosphatase flux were entirely accounted for by a marked decrease in the rate of glycogenolysis when compared with the vehicle control group and by a marked decrease in the rate of gluconeogenesis when compared with the leptin-infused group. Finally, we examined whether changes in the liver expression of Glc-6-Pase (Fig. 6E) and PEPCK (Fig. 6F) could mediate these changes in hepatic glucose fluxes. Because central activation of melanocortin receptors as well as ICV or systemic leptin infusion led to increased hepatic expression of Glc-6-Pase and PEPCK, we postulated that this effect of leptin is mediated via activation of central melanocortin receptors. Indeed, the central administration of SHU9119 also negated these hepatic transcriptional effects of ICV leptin (Fig. 6, E and F). Overall, the rapid stimulatory effect of the central administration of leptin on gluconeogenesis and on hepatic Glc-6-Pase and PEPCK mRNA requires activation of central melanocortin receptors. However, the potent inhibitory effect of ICV leptin on hepatic glycogenolysis is independent of its activation of the central melanocortin pathway.



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  		FIG. 6.
Effect of central leptin on hepatic glucose fluxes and on the gene expression of gluconeogenic enzymes. Glucose production (A) and glucose-6-phosphatase flux (B) were markedly decreased by systemic leptin only when the central activation of the melanocortin pathway was prevented by the ICV infusion of SHU9119; the rate of hepatic glycogenolysis (C) was markedly suppressed by ICV leptin, but the central infusion of SHU9119 did not modify this effect. Conversely, the rate of gluconeogenesis (D) was markedly increased by the ICV leptin and this effect was prevented by the central infusion of SHU9119; ICV leptin also increased the liver content of glucose-6-phosphatase (E) and PEPCK (F) mRNA, and these effects were prevented by the central administration of SHU9119. A and B,*, p < 0.01 versus leptin; C and D,*, p < 0.01 versus vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is increasing evidence for common central nervous system pathways regulating energy and glucose homeostasis (4–15, 38, 39). The adipose-derived hormone leptin is a major circulating signal of peripheral energy status, which acts mainly on hypothalamic receptors to regulate feeding behavior and energy expenditure (1, 40). Shortly after its cloning (41), leptin was also shown to regulate glucose tolerance, insulin signaling/action, and lipid metabolism independently of its anorectic effects (5, 6, 10, 38, 42, 43). These early observations generated great interest in the investigation of the mechanisms by which leptin interferes with metabolic processes. In this regard, leptin activates central melanocortin receptors partly via increased biosynthesis of the physiological ligand {alpha}-MSH and via decreased biosynthesis of a putative antagonist agouti-related protein (Agrp) at the level of the hypothalamus (44). The activation of the central melanocortin pathway appears to be a required step in lepin action on food intake, energy expenditure, sympathetic nervous system, insulin secretion, and body fat distribution (3, 10, 16, 45–48). Conversely, there is also recent evidence for the effects of leptin on energy balance and intermediate metabolism that may be independent of the activation of central melanocortin receptors (21). Here, we identify melanocortin-dependent and melanocortin-independent effects of leptin on hepatic glucose fluxes and gene expression. Specifically, leptin stimulates gluconeogenesis and the hepatic expression of PEPCK and Glc-6-Pase via central activation of melanocortin receptors. On the other hand, the systemic or central administration of leptin also restrains hepatic glycogenolysis via a central melanocortin-independent mechanism.

The acute and central activation of melanocortin receptors stimulated the expression of gluconeogenic enzymes within the liver, markedly increased the rate of gluconeogenesis, and diminished the suppressive effect of insulin on GP. These rapid metabolic consequences of the central modulation of the melanocortin pathway stand in sharp contrast with the insulin sensitizing effects of more prolonged manipulations of the central melanocortin receptors. In fact, week long activation of this central pathway led to decreased visceral adiposity and improved insulin action (15). Furthermore, genetic animal models in which the signaling via the central melanocortin receptors is defective display hyperphagia, adult onset diabetes or glucose intolerance, and hyperinsulinemia (32). The contrast between acute and chronic effects of central melanocortin modulation are likely due to the dramatic effects of this pathway on body fat mass and distribution, lipid oxidation and storage, and sympathetic nervous system activity. Acutely, the activation of the melanocortin pathway in the central nervous system is likely to enhance autonomic outflow to peripheral organs in the absence of changes in visceral adiposity and lipid storage (15, 32). In the liver, an increase in adrenergic tone leads to increased expression of Glc-6-Pase and PEPCK and to increased fat oxidation, which in turn can drive gluconeogenesis.

The effects of leptin on hepatic glucose fluxes appear to be more complex than those of {alpha}-MSH. The systemic infusion of leptin during insulin clamp studies similarly increased the hepatic expression of PEPCK and Glc-6-Pase and the rate of gluconeogenesis, but it also markedly suppressed the rate of glycogenolysis so that GP was not increased. These changes induced by a physiological elevation in circulating leptin levels recapitulate our previous findings in the presence of supraphysiological plasma levels of the hormone (5). When the stimulatory effect of systemic leptin on central melanocortin receptors was prevented, equal increases in circulating leptin concentrations failed to alter gluconeogenesis and liver PEPCK/Glc-6-Pase mRNA levels. However, under these experimental conditions, increasing the plasma leptin concentration markedly inhibited the rate of glycogenolysis and therefore enhanced the inhibition of GP by insulin. It should be pointed out that these insulin-like effects of systemic leptin on hepatic glucose fluxes could be mediated via direct effects on the liver as well as via extrahepatic (presumably hypothalamic) actions of leptin. In this regard, leptin has also been shown to directly modulate insulin signaling, lipid metabolism, and glycogenolysis in some studies conducted in isolated liver cells (35, 36) or perfused liver (37). Conversely, the effects of a physiological increase in systemic leptin on the hepatic expression of gluconeogenic enzymes as well as on gluconeogenesis are centrally mediated because they were abolished by the central antagonism of melanocortin receptors.

To discern whether the melanocortin-independent effects of leptin on glycogenolysis and GP require increased liver exposure to leptin or whether they are also centrally mediated, we next examined the effects of central leptin administration with or without activation of central melanocortin receptors. Once again leptin markedly suppressed GP via decreased glycogenolysis, when the activation of central melanocortin receptors was prevented. Thus, leptin can inhibit hepatic glycogenolysis and GP via a central pathway. This is consistent with the observation that neuron-specific deletion of leptin receptor expression largely recapitulates the behavioral and metabolic defects induced by the whole body loss-of-function in this receptor (33).

Although the central nervous system pathways and efferent signals mediating the melanocortin-independent effects of leptin on liver glycogenolysis are unknown, it is tempting to speculate that the activation of an "insulin-like" signaling pathway within the hypothalamus plays a key role. Leptin and insulin can both increase the activity of PI3K in the hypothalamus, and this signaling pathway is involved in their anorectic effects (22, 23). Importantly, central activation of insulin signaling markedly suppresses GP, and the effect of a physiological increase in circulating insulin on GP requires the central activation of PI3K (27). Here we report the metabolic effects of leptin via a melanocortin-independent signaling pathway that closely resemble the potent inhibitory effect of the central administration of insulin on GP. Taken together, these findings support the notion that activation of PI3K by leptin in the hypothalamus may mediate its insulin-like effect on GP and glycogenolysis.

It has long been recognized that the diminished ability of insulin to suppress glucose production by the liver (hepatic insulin resistance) is a main cause of hyperglycemia in type 2 diabetes mellitus. Our results indicate that leptin can modulate hepatic insulin action via melanocortin-dependent as well as via melanocortin-independent effects (Fig. 7). In fact, leptin stimulates hepatic gluconeogenesis and PEPCK/Glc-6-Pase expression via central activation of melanocortin receptors, but it also markedly suppresses hepatic glycogenolysis via a central melanocortin-independent mechanism (Fig. 7). Thus, selective signaling (or selective resistance) via either of these two central circuits mediating the rapid actions of leptin on the liver can lead to dramatic changes in hepatic insulin action and glucose metabolism. In this regard, it will be important to discern whether common forms of leptin resistance symmetrically impact on the melanocortin-dependent and melanocortin-independent effects of leptin on liver glucose metabolism.



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  		FIG. 7.
Schematic representation of how melanocortin and leptin modify hepatic glucose fluxes. The central administration of {alpha}-MSH markedly stimulated the rate of gluconeogenesis and doubled the rate of glucose production. The systemic or central administration of leptin also more than doubles the rate of gluconeogenesis, but GP does not change because glycogenolysis was markedly inhibited. Finally, the central administration of a melanocortin receptor antagonist completely abolished the effect of leptin on gluconeogenesis, but glycogenolysis was still markedly inhibited. As a result, leptin markedly inhibited the rate of glucose production when its activation of central melanocortin receptors was prevented. The bold numbers represent flux rates in mg/kg·min.

 

   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants DK 48321 and DK 45024 (to L. R.) and funds from the American Diabetes Association (to S. O. and L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a mentor-based fellowship from the American Diabetes Association. Back

§ To whom correspondence should be addressed: Depts. of Medicine & Molecular Pharmacology, Diabetes Research & Training Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4118/4215; Fax: 718-430-8557; E-mail: rossetti{at}aecom.yu.edu.

1 The abbreviations used are: STAT, signal transducers and activators of transcription; PI3K, phosphatidylinositol 3-kinase; GP, glucose production; ICV, intracerebroventricular; IV, intravenous; PEP, phosphoenolpyruvate; Glc-6-Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; FFA, free fatty acids; {alpha}-MSH, {alpha}-melanocyte-stimulating hormone. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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