Artigo Acesso aberto Revisado por pares

Short Term Effects of Leptin on Hepatic Gluconeogenesis and in Vivo Insulin Action

1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês

10.1074/jbc.272.44.27758

ISSN

1083-351X

Autores

Luciano Rossetti, Duna Massillon, Nir Barzilai, Patricia Vuguin, Wei Chen, Meredith Hawkins, Jie Wu, Jali Wang,

Tópico(s)

Adipokines, Inflammation, and Metabolic Diseases

Resumo

Long term administration of leptin decreases caloric intake and fat mass and improves glucose tolerance. Here we examine whether leptin acutely regulates peripheral and hepatic insulin action. Recombinant mouse leptin (0.3 mg/kg·h, Leptin +) or vehicle (Leptin −) were administered for 6 h to 4-month-old rats (n = 20), and insulin (3 milliunits/kg·min) clamp studies were performed. During physiologic hyperinsulinemia (plasma insulin ∼65 microunits/ml), the rates of whole body glucose uptake, glycolysis, and glycogen synthesis and the rates of 2-deoxyglucose uptake in individual tissues were similar inLeptin − and Leptin +. Post-absorptive hepatic glucose production (HGP) was similar in the two groups. However, leptin enhanced insulin's inhibition of HGP (4.1 ± 0.7 and 6.2 ± 0.7 mg/kg·min; p < 0.05). The decreased HGP in theLeptin + group was due to a marked suppression of hepatic glycogenolysis (0.7 ± 0.1 versus 4.1 ± 0.6 mg/kg·min, in Leptin + versus Leptin −, respectively;p < 0.001), whereas the % contribution of gluconeogenesis to HGP was markedly increased (82 ± 3%versus 36 ± 4% in Leptin + andLeptin −, respectively; p < 0.001). At the end of the 6-h leptin infusion, the hepatic abundance of glucokinase mRNA was decreased, whereas that of phosphoenolpyruvate carboxykinase mRNA was increased compared with Leptin −. We conclude that an acute increase in plasma leptin 1) enhances insulin's ability to inhibit HGP, 2) does not affect peripheral insulin action, and 3) induces a redistribution of intrahepatic glucose fluxes and changes in the gene expression of hepatic enzymes that closely resemble those of fasting. Long term administration of leptin decreases caloric intake and fat mass and improves glucose tolerance. Here we examine whether leptin acutely regulates peripheral and hepatic insulin action. Recombinant mouse leptin (0.3 mg/kg·h, Leptin +) or vehicle (Leptin −) were administered for 6 h to 4-month-old rats (n = 20), and insulin (3 milliunits/kg·min) clamp studies were performed. During physiologic hyperinsulinemia (plasma insulin ∼65 microunits/ml), the rates of whole body glucose uptake, glycolysis, and glycogen synthesis and the rates of 2-deoxyglucose uptake in individual tissues were similar inLeptin − and Leptin +. Post-absorptive hepatic glucose production (HGP) was similar in the two groups. However, leptin enhanced insulin's inhibition of HGP (4.1 ± 0.7 and 6.2 ± 0.7 mg/kg·min; p < 0.05). The decreased HGP in theLeptin + group was due to a marked suppression of hepatic glycogenolysis (0.7 ± 0.1 versus 4.1 ± 0.6 mg/kg·min, in Leptin + versus Leptin −, respectively;p < 0.001), whereas the % contribution of gluconeogenesis to HGP was markedly increased (82 ± 3%versus 36 ± 4% in Leptin + andLeptin −, respectively; p < 0.001). At the end of the 6-h leptin infusion, the hepatic abundance of glucokinase mRNA was decreased, whereas that of phosphoenolpyruvate carboxykinase mRNA was increased compared with Leptin −. We conclude that an acute increase in plasma leptin 1) enhances insulin's ability to inhibit HGP, 2) does not affect peripheral insulin action, and 3) induces a redistribution of intrahepatic glucose fluxes and changes in the gene expression of hepatic enzymes that closely resemble those of fasting. The recent discovery of the ob gene (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11935) Google Scholar) and preliminary analysis of the properties of its product, leptin (2Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3914) Google Scholar, 3Mantzoros C.S. Qu D. Frederich R.C. Susulic V.S. Lowell B.B. Maratos-Flier E. Flier J.S. Diabetes. 1996; 45: 909-914Crossref PubMed Scopus (318) Google Scholar, 4Weigle D.S. Bukowski T.R. Foster D.C. Holderman S. Kramer J.M. Lasser G. Lofton-Day C.E. Prunkard D.E. Raymond C. Kuijper J.L. J. Clin. Invest. 1995; 96: 2065-2070Crossref PubMed Scopus (449) Google Scholar, 5Rentsch J. Levens N. Chiesi M. Biochem. Biophys. Res. Commun. 1995; 214: 131-136Crossref PubMed Scopus (137) Google Scholar, 6Frederich R.C. Hamann A. Anderson S. Lollmann B. Lowell B.B. Flier J.S. Nat. Med. 1995; 1: 1311-1314Crossref PubMed Scopus (1438) Google Scholar, 7Campfield L. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3089) Google Scholar), have shed new light on the regulation of energy homeostasis (8Flier J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4242-4245Crossref PubMed Scopus (195) Google Scholar). Since the most common alteration in energy balance, obesity, is tightly associated with insulin resistance, it has been proposed that leptin may play a role in carbohydrate metabolism and insulin action (8Flier J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4242-4245Crossref PubMed Scopus (195) Google Scholar, 9Cohen B. Novick D. Rubinstein M. Science. 1996; 274: 1185-1188Crossref PubMed Scopus (656) Google Scholar, 10Considine R.V. Sinha M.K. Heiman M.L. Kriauciunas A. Stephens T.W. Nyce M.R. Ohannesian J.P. Marco C.C. McKee L.J. Bauer T. Caro J. N. Engl. J. Med. 1996; 334: 324-325Crossref PubMed Scopus (5641) Google Scholar, 11Kolaczynski J.W. Considine R.V. Ohannesian J. Marco C. Opentanova I. Nyce M.R. Myint M. Caro J.F. Diabetes. 1996; 45: 1511-1515Crossref PubMed Google Scholar). Indeed, recent work in cultured adipose cells (12Muller G. Ertl J. Gerl M. Preibisch G. J. Biol. Chem. 1997; 272: 10585-10593Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 13Bai Y. Zhang S. Kim K.-S. Lee J.-K. Kim K.-H. J. Biol. Chem. 1996; 271: 13939-13942Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) and hepatocytes (9Cohen B. Novick D. Rubinstein M. Science. 1996; 274: 1185-1188Crossref PubMed Scopus (656) Google Scholar) has suggested that leptin may antagonize insulin action in these cells. Conversely, early work on the effect of exogenous leptin inob/ob mice had demonstrated a marked decrease in both plasma insulin and glucose concentrations following prolonged administration of this protein (2Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3914) Google Scholar, 4Weigle D.S. Bukowski T.R. Foster D.C. Holderman S. Kramer J.M. Lasser G. Lofton-Day C.E. Prunkard D.E. Raymond C. Kuijper J.L. J. Clin. Invest. 1995; 96: 2065-2070Crossref PubMed Scopus (449) Google Scholar, 5Rentsch J. Levens N. Chiesi M. Biochem. Biophys. Res. Commun. 1995; 214: 131-136Crossref PubMed Scopus (137) Google Scholar, 7Campfield L. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3089) Google Scholar, 14Schwartz M.W. Baskin D.G. Bukowski T.R. Kuijper J.L. Foster D. Lasser G. Prunkard D.E. Porte D.J. Woods S.C. Seeley R. Weigle D. Diabetes. 1996; 45: 531-535Crossref PubMed Google Scholar, 15Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4280) Google Scholar). Since the decline in plasma glucose and insulin was greater in leptin-treated mice than in pair-fed control mice (2Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3914) Google Scholar, 14Schwartz M.W. Baskin D.G. Bukowski T.R. Kuijper J.L. Foster D. Lasser G. Prunkard D.E. Porte D.J. Woods S.C. Seeley R. Weigle D. Diabetes. 1996; 45: 531-535Crossref PubMed Google Scholar), it has been proposed that leptin may directly or indirectly improve in vivo insulin action. However, it is well established that changes in food intake, body weight, fat mass and/or fat distribution similar to those associated with long term leptin administration can independently alter insulin action, particularly in insulin-resistant and obese animal models (16Kissebah A.H. Int. J. Obes. 1991; 15: 109-115PubMed Google Scholar,17Bjorntorp P. Diabetes Care. 1991; 14: 1132-1143Crossref PubMed Scopus (903) Google Scholar). Thus, it is presently unknown whether the short term administration of exogenous leptin modulates insulin's ability to promote glucose disposal and/or to regulate hepatic glucose fluxes. Furthermore, a recent report in cultured hepatocytes indicates that leptin may also regulate the gene expression of a key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (9Cohen B. Novick D. Rubinstein M. Science. 1996; 274: 1185-1188Crossref PubMed Scopus (656) Google Scholar), and may thus regulate the intrahepatic distribution of glucose fluxes. To delineate whether leptin acutely regulates hepatic and peripheral insulin action in vivo, we examined the effects of systemic infusion of recombinant leptin on whole body glucose disposal, hepatic glucose fluxes, and glucokinase and PEPCK 1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; FFA, free fatty acids; GK, glucokinase; HGP, hepatic glucose production; MOPS, 4-morpholinepropanesulfonic acid. mRNA in the presence of euglycemic hyperinsulinemic clamp studies in conscious rats. Two groups of male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were studied. Group 1 consisted of 8 rats receiving an infusion of vehicle for 6 h (Leptin −) and group 2 consisted of 12 rats receiving an infusion of recombinant mouse leptin (Amgen, Inc., Thousand Oaks, CA; >95% pure by SDS-polyacrylamide gel electrophoresis) at the rate of 5 μg/kg·min for 6 h (Leptin +). Rats were housed in individual cages and subjected to a standard light (6 a.m. to 6 p.m.)/dark (6 p.m. to 6 a.m.) cycle. One week before the in vivostudy, rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body weight), and indwelling catheters were inserted in the right internal jugular vein and in the left carotid artery. 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 (23Kieffer T.J. Heller R.S. Habener J.F. Biochem. Biophys. Res. Commun. 1996; 224: 522-527Crossref PubMed Scopus (320) Google Scholar, 24Kraegen E. James D. Jenkins A. Chisolm P. Am. J. Physiol. 1985; 248: E353-E362PubMed Google Scholar, 25Giaccari A. Rossetti L. J. Chromatogr. 1989; 497: 69-78Crossref PubMed Scopus (27) Google Scholar, 26Rossetti L. Barzilai N. Chen W. Harris T. Yang D. Rogler C.E. J. Biol. Chem. 1996; 271: 203-208Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Studies were performed in awake, unstressed, and chronically catheterized rats (18Rossetti L. Smith D. Shulman G.I. Papachristou D. DeFronzo R.A. J. Clin. Invest. 1987; 79: 1510-1515Crossref PubMed Scopus (722) Google Scholar, 19Rossetti L. Giaccari A. J. Clin. Invest. 1990; 85: 1785-1792Crossref PubMed Scopus (237) Google Scholar). Fat-free mass and fat mass were calculated from the whole body volume of distribution of water, estimated by tritiated water bolus injection in each experimental rat (20Barzilai N. Massillon D. Rossetti L. Biochem. J. 1995; 310: 819-826Crossref PubMed Scopus (20) Google Scholar). Briefly, boluses of 20 μCi of 3H20 (NEN Life Science Products) were injected intra-arterially on the morning of the study (9 a.m.). Steady state for 3H20-specific activity in rats is generally achieved within 45 min; 5 samples were collected between 1 and 2 h after injection. The distribution space of water was obtained by dividing the total radioactivity injected by the steady-state specific activity of plasma water which was assumed to be 93% of the total plasma volume. Fat-free mass was calculated from the whole body water distribution space divided by 0.73. Studies were performed in unrestrained rats using the insulin clamp technique (18Rossetti L. Smith D. Shulman G.I. Papachristou D. DeFronzo R.A. J. Clin. Invest. 1987; 79: 1510-1515Crossref PubMed Scopus (722) Google Scholar,19Rossetti L. Giaccari A. J. Clin. Invest. 1990; 85: 1785-1792Crossref PubMed Scopus (237) Google Scholar, 21Rossetti L. Giaccari A. Barzilai N. Howard K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (178) Google Scholar), in combination with high pressure liquid chromatography purified [3-3H]glucose, [U-14C]2-deoxyglucose, and [U-14C]lactate infusions, as described previously (21Rossetti L. Giaccari A. Barzilai N. Howard K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (178) Google Scholar, 22Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar). Food was removed for ∼5 h before the in vivo protocol. All studies lasted 360 min and included a 120-min equilibration period for assessment of the space of distribution of water, a 120-min basal period for assessment of the post-absorptive rates of glucose turnover, and a 120-min hyperinsulinemic clamp period. At the beginning of the basal period and 120 min before starting the glucose/insulin infusions, a primed continuous infusion of high pressure liquid chromatography purified [3-3H]glucose (NEN Life Science Products; 20 μCi bolus, 0.2 μCi/min) was initiated and maintained throughout the remaining 4 h of the study. A bolus of [U-14C]2-deoxyglucose (20 μCi) was injected 30 min before the end of the study. [U-14C]Lactate (5 μCi bolus/0.25 μCi/min) was infused during the last 10 min of the study. The protocol followed during the insulin clamp study was similar to that described previously (18Rossetti L. Smith D. Shulman G.I. Papachristou D. DeFronzo R.A. J. Clin. Invest. 1987; 79: 1510-1515Crossref PubMed Scopus (722) Google Scholar, 19Rossetti L. Giaccari A. J. Clin. Invest. 1990; 85: 1785-1792Crossref PubMed Scopus (237) Google Scholar, 21Rossetti L. Giaccari A. Barzilai N. Howard K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (178) Google Scholar, 22Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar). Briefly, a primed continuous infusion of regular insulin (3 milliunits/kg·min) was 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 mm. To control for possible effects of leptin on the endocrine pancreas (23Kieffer T.J. Heller R.S. Habener J.F. Biochem. Biophys. Res. Commun. 1996; 224: 522-527Crossref PubMed Scopus (320) Google Scholar), somatostatin (1.5 μg/kg·min) was also infused to inhibit endogenous insulin secretion in both groups. Plasma samples for determination of [3H]glucose- and [3H]water-specific activities were obtained at 10-min intervals during the basal and clamp periods. Steady-state conditions for the plasma glucose concentration and specific activity were achieved within 90 min in both the basal and clamp periods of the studies (Fig. 1). Plasma samples for determination of plasma insulin and FFA concentrations were obtained at 30-min intervals during the study. Plasma samples for determination of plasma [U-14C]2-deoxyglucose-specific activity were obtained at 90, 91, 93, 95, 98, 100, 105, 110, 115, and 120 min during the clamp studies. The total volume of blood withdrawn was ∼3.0 ml/study; to prevent volume depletion and anemia, a solution (1:1 v/v) of ∼4.0 ml of fresh blood (obtained by heart puncture from littermates of the test animals) and heparinized saline (10 units/ml) was infused at a rate of 20 μl/min. Furthermore, following larger samples, red blood cells were resuspended in saline and immediately returned through the sampling catheter. All determinations were also performed on portal vein blood obtained at the end of the experiment. At the end of the in vivo studies, rats were anesthetized (pentobarbital 60 mg/kg body weight, intravenously); the abdomen was quickly opened; portal vein blood was obtained, and rectus abdominal and hindlimb muscle and liver were freeze-clamped in situwith aluminum tongs pre-cooled in liquid nitrogen. The time from the injection of the anesthetic until freeze-clamping of the tissues was less than 45 s. All tissue samples were stored at −80 °C for subsequent analysis. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Palo Alto, CA). Plasma insulin, glucagon, and leptin (rat Leptin radioimmunoassay kit, Linco Research Inc., St. Charles, MO) concentrations were measured by radioimmunoassay. The plasma concentration of free fatty acids was determined by an enzymatic method with 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 ZnSO4precipitates (Somogyi procedure) of plasma samples (20 μl) after evaporation to dryness to eliminate tritiated water. The rates of glycolysis were estimated as described previously (19Rossetti L. Giaccari A. J. Clin. Invest. 1990; 85: 1785-1792Crossref PubMed Scopus (237) Google Scholar). Briefly, plasma-tritiated water-specific activity was determined by liquid scintillation counting of the protein-free supernatant (Somogyi filtrate) before and after evaporation to dryness. Because tritium on C-3 of glucose is lost to water during glycolysis, it can be assumed that plasma tritium is present either in tritiated water or [3-3H]glucose. To measure plasma [U-14C]2-deoxyglucose, samples were deproteinized as described above, and an aliquot of the supernatant was counted in a double channel beta-counter after addition of 500 μl of water and 5 ml of liquid scintillation mixture. To measure muscle [U-14C]2-deoxyglucose, frozen tissue samples were weighed and dissolved in 0.5 ml of 1 m NaOH kept in a shaking water bath at 60 °C for 1 h. Following neutralization with 0.5 ml of 1 m HCl, 2 aliquots were taken. One was deproteinized with Ba(OH)2 and ZnSO4 and the other with 6% HClO4. The HClO4 supernatant contains both phosphorylated and unphosphorylated 2-deoxyglucose, whereas the Ba(OH)2 and ZnSO4 supernatant contains only the unphosphorylated form. The difference in dpm between the two supernatants measures the muscle content of 2-deoxyglucose-phosphate (24Kraegen E. James D. Jenkins A. Chisolm P. Am. J. Physiol. 1985; 248: E353-E362PubMed Google Scholar). Uridine-diphosphoglucose (UDP-Glc), uridine-diphosphogalactose (UDP-galactose), and phosphoenolpyruvate (P-enolpyruvate) concentrations and specific activities in the liver were obtained through two sequential chromatographic separations, as previously reported (21Rossetti L. Giaccari A. Barzilai N. Howard K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (178) Google Scholar, 22Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar, 25Giaccari A. Rossetti L. J. Chromatogr. 1989; 497: 69-78Crossref PubMed Scopus (27) Google Scholar, 26Rossetti L. Barzilai N. Chen W. Harris T. Yang D. Rogler C.E. J. Biol. Chem. 1996; 271: 203-208Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Hepatic glucokinase (GK) and phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance was assessed by Northern blot analysis (27Barzilai N. Rossetti L. J. Biol. Chem. 1993; 268: 25019-25025Abstract Full Text PDF PubMed Google Scholar, 28Massillon D. Barzilai N. Chen W. Hu M. Rossetti L. J. Biol. Chem. 1996; 271: 9871-9874Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 29Massillon D. Barzilai N. Hawkins M. Prus-Wertheimer D. Rossetti L. Diabetes. 1997; 46: 153-157Crossref PubMed Google Scholar). Total RNA was isolated on freeze-clamped liver tissues according to the RNA-STAT kit (Tel-TEST "B" Inc. Friendswood, TX). The isolated RNA was assessed for purity by the 260/280 ratio absorbency. Twenty μg of total RNA were electrophoresed on a 1.2% formaldehyde denatured agarose gel in 1 × MOPS running buffer. The RNA was visualized with ethidium bromide and transferred to a hybond-N membrane (Amersham Corp.). The GK and PEPCK cDNA probes were labeled with 32P using the Megaprime labeling system kit (Amersham Corp.). Prehybridization and hybridization were carried out using the rapid hybridization buffer (Amersham Corp.). The filters were then exposed to Fuji x-ray films for 12–48 h at −80 °C with intensifying screens. Quantification of GK and PEPCK mRNA was done by scanning densitometry, normalized for ribosomal RNA signal to correct for loading irregularities. The term total glucose output is intended as total in vivo flux through Glc-6-Pase. The term hepatic glucose production (HGP) is intended as the net rates of Glc-6-P dephosphorylation to glucose. Finally, glucose cycling is defined as the input of extracellular glucose into the Glc-6-P pool followed by exit of plasma-derived Glc-6-P back into the extracellular pool. Under steady-state conditions for plasma glucose concentrations, the rate of glucose disappearance (Rd) equals the rate of glucose appearance (Ra). The latter was calculated as the ratio of the rate of infusion of [3-3H]glucose (dpm/min) and the steady-state plasma [3H]glucose-specific activity (dpm/mg). When exogenous glucose was given, the rate of endogenous glucose production was calculated as the difference between Ra and the infusion rate of glucose. The rate of 2-deoxyglucose uptake was calculated as described by Kraegen et al. (24Kraegen E. James D. Jenkins A. Chisolm P. Am. J. Physiol. 1985; 248: E353-E362PubMed Google Scholar). The percent of the hepatic glucose 6-phosphate pool directly derived from plasma glucose was calculated as the ratio of [3H]UDP-Glc- and plasma [3H]glucose-specific activities. The percent of the hepatic glucose 6-phosphate pool derived from P-enolpyruvate gluconeogenesis was calculated as the ratio of the specific activities of [14C]UDP-Glc and 2 × [14C]P-enolpyruvate following in vivo labeling with [U-14C]lactate (21Rossetti L. Giaccari A. Barzilai N. Howard K. Sebel G. Hu M. J. Clin. Invest. 1993; 92: 1126-1134Crossref PubMed Scopus (178) Google Scholar, 22Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar). Comparisons between groups were made with Student's t test for unpaired samples, and all values are presented as the mean ± S.E. of the means. The discovery by positional cloning of the murine obgene (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11935) Google Scholar) and of its product, a 16-kDa protein named leptin (15Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4280) Google Scholar), has dramatically altered our understanding of energy metabolism (8Flier J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4242-4245Crossref PubMed Scopus (195) Google Scholar). However, although it is now well established that circulating leptin is produced by adipose cells (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11935) Google Scholar, 2Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3914) Google Scholar, 3Mantzoros C.S. Qu D. Frederich R.C. Susulic V.S. Lowell B.B. Maratos-Flier E. Flier J.S. Diabetes. 1996; 45: 909-914Crossref PubMed Scopus (318) Google Scholar, 6Frederich R.C. Hamann A. Anderson S. Lollmann B. Lowell B.B. Flier J.S. Nat. Med. 1995; 1: 1311-1314Crossref PubMed Scopus (1438) Google Scholar, 15Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4280) Google Scholar, 30Cinti S. Frederich R.C. Zingaretti M.C. De Matteis R. Flier J.S. Lowell B.B. Endocrinology. 1997; 138: 797-804Crossref PubMed Scopus (171) Google Scholar) and exerts marked anorectic effects through specific hypothalamic receptors (7Campfield L. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3089) Google Scholar, 14Schwartz M.W. Baskin D.G. Bukowski T.R. Kuijper J.L. Foster D. Lasser G. Prunkard D.E. Porte D.J. Woods S.C. Seeley R. Weigle D. Diabetes. 1996; 45: 531-535Crossref PubMed Google Scholar, 31Schwartz M.W. Seeley R.J. Campfield L.A. Burn P. Baskin D.G. J. Clin. Invest. 1996; 98: 1101-1106Crossref PubMed Scopus (1386) Google Scholar, 32Schwartz M.W. Peskind E. Raskind M. Boyko E.J. Porte D.J. Nat. Med. 1996; 2: 589-593Crossref PubMed Scopus (902) Google Scholar, 33Jacob R.J. Dziura J. Medwick M.B. Leone P. Caprio S. During M. Shulman G.I. Sherwin R.S. Diabetes. 1997; 46: 150-152Crossref PubMed Scopus (103) Google Scholar, 34Considine R.V. Considine E.L. Williams C.J. Hyde T.M. Caro J.F. Diabetes. 1996; 45: 992-994Crossref PubMed Google Scholar), there is minimal information on putative metabolic roles of leptin in carbohydrate or lipid homeostasis. Studies of the interactions between leptin and insulin action have been limited to isolated cell systems in culture (9Cohen B. Novick D. Rubinstein M. Science. 1996; 274: 1185-1188Crossref PubMed Scopus (656) Google Scholar, 12Muller G. Ertl J. Gerl M. Preibisch G. J. Biol. Chem. 1997; 272: 10585-10593Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Although the nutritional state and insulin levelsper se can regulate the gene expression of ob in adipose cells and leptin levels in plasma (6Frederich R.C. Hamann A. Anderson S. Lollmann B. Lowell B.B. Flier J.S. Nat. Med. 1995; 1: 1311-1314Crossref PubMed Scopus (1438) Google Scholar, 8Flier J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4242-4245Crossref PubMed Scopus (195) Google Scholar, 10Considine R.V. Sinha M.K. Heiman M.L. Kriauciunas A. Stephens T.W. Nyce M.R. Ohannesian J.P. Marco C.C. McKee L.J. Bauer T. Caro J. N. Engl. J. Med. 1996; 334: 324-325Crossref PubMed Scopus (5641) Google Scholar, 11Kolaczynski J.W. Considine R.V. Ohannesian J. Marco C. Opentanova I. Nyce M.R. Myint M. Caro J.F. Diabetes. 1996; 45: 1511-1515Crossref PubMed Google Scholar), it is presently unknown whether leptin plays any role in the regulation ofin vivo insulin action and hepatic glucose fluxes. To examine the effect of the acute administration of leptin on whole body, skeletal muscle, and liver insulin action, 12 rats were infused with 5 μg/kg·min leptin for 6 h and were compared with 8 control rats receiving a vehicle infusion. There were no differences in the mean body weights, average food intake, and fat-free mass between the two groups of rats. Similarly, following ∼5-h fast (post-absorptive state), the plasma insulin, glucose, and FFA concentrations were similar in the rats assigned to the two experimental groups.Table IBase-line characteristics of rats receiving infusions of vehicle (Leptin −) or leptin (Leptin +)GroupLeptin−Leptin +n812Body weight (g)322 ± 19311 ± 11Food intake (g)20 ± 120 ± 1FFM (% of body wt)90 ± 487 ± 3Plasma glucose (mm)7.6 ± 0.27.9 ± 0.2Plasma insulin (microunits/ml)34 ± 433 ± 4Plasma FFA (meq/liter)0.90 ± 0.020.89 ± 0.03Rats were fasted for 5 h prior to plasma sampling. Biochemical parameters represent the average ± S.E. of at least three basal measurements in each rat. Food intake represents the average ± S.E. of the last 3 days, and fat-free mass (FFM) was estimated from the steady-state specific activity of 3H2O (five plasma samples at 20-min intervals) 1–2 h following the injection of a bolus of 3H2O (20 μCi). Open table in a new tab Rats were fasted for 5 h prior to plasma sampling. Biochemical parameters represent the average ± S.E. of at least three basal measurements in each rat. Food intake represents the average ± S.E. of the last 3 days, and fat-free mass (FFM) was estimated from the steady-state specific activity of 3H2O (five plasma samples at 20-min intervals) 1–2 h following the injection of a bolus of 3H2O (20 μCi). To assess the metabolic effects of insulin in vivo, a physiologic increase in the plasma insulin concentrations was generated for 120 min, and the plasma glucose concentrations were maintained at the basal levels (∼7 mm) by a variable glucose infusion. Thus, conscious rats were compared in the presence of similar steady-state hyperinsulinemia and normoglycemia in the presence or absence of exogenous leptin (TableII). Steady-state conditions for plasma glucose concentration and specific activity were achieved within 90 min during both the basal and clamp periods (Fig. 1). The plasma FFA concentration did not change significantly from its post-absorptive levels following the vehicle infusion (Table II and Fig. 1). However, there was a transient increase (by ∼35%) in the plasma FFA concentrations between 120 and 180 min during the leptin infusion. Regardless of their basal levels, the plasma FFA concentrations were decreased at a similar level during the hyperinsulinemic clamp studies. The plasma leptin concentrations during the leptin infusions averaged 458 ± 49 ng/ml. The rates of glucose infusion required to maintain the plasma glucose concentration at the target level during the hyperinsulinemic clamp studies were similar in the rats receiving vehicle and leptin (Table II).Table IISteady-state plasma glucose, insulin, and free fatty acids (FFA) concentrations and average rate of glucose infusion (GIR) in Leptin − and Leptin + rats during the basal period (Basal) and during the hypeinsulinemic clamp studies (Insulin).GroupLeptin−Leptin +BasalInsulinBasalInsulinGlucose (mm)7.7 ± 0.27.4 ± 0.27.7 ± 0.47.4 ± 0.2Insulin (microunits/ml)25 ± 164 ± 22-ap < 0.05 versus basal.23 ± 265 ± 22-ap < 0.05 ver

Referência(s)