Liporegulation in Diet-induced Obesity

To test the hypothesis that the physiologic liporegulatory role of hyperleptinemia is to prevent steatosis during caloric excess, we induced obesity by feeding normal Harlan Sprague-Dawley rats a 60% fat diet. Hyperleptinemia began within 24 h and increased progressively to 26 ng/ml after 10 weeks, correlating with an ∼150-fold increase in body fat (r = 0.91, p < 0.0001). During this time, the triacylglycerol (TG) content of nonadipose tissues rose only 1–2.7-fold implying antisteatotic activity. In rodents without leptin action (fa/fa rats and ob/ob anddb/db mice) receiving a 6% fat diet, nonadipose tissue TG was 4–100 times normal. In normal rats on a 60% fat diet, peroxisome proliferator-activated receptor α protein and liver-carnitine palmitoyltransferase-1 (l-CPT-1) mRNA increased in liver. In their pancreatic islets, fatty-acid oxidation increased 30% without detectable increase in the expression of peroxisome proliferator-activated receptor-α or oxidative enzymes, whereas lipogenesis from 14Cglucose was slightly below that of the 4% fat-fed rats (p < 0.05). Tissue-specific overexpression of wild-type leptin receptors in the livers offa/fa rats, in which marked steatosis is uniformly present, reduced TG accumulation in liver but nowhere else. We conclude that a physiologic role of the hyperleptinemia of caloric excess is to protect nonadipocytes from steatosis and lipotoxicity by preventing the up-regulation of lipogenesis and increasing fatty-acid oxidation. To test the hypothesis that the physiologic liporegulatory role of hyperleptinemia is to prevent steatosis during caloric excess, we induced obesity by feeding normal Harlan Sprague-Dawley rats a 60% fat diet. Hyperleptinemia began within 24 h and increased progressively to 26 ng/ml after 10 weeks, correlating with an ∼150-fold increase in body fat (r = 0.91, p < 0.0001). During this time, the triacylglycerol (TG) content of nonadipose tissues rose only 1–2.7-fold implying antisteatotic activity. In rodents without leptin action (fa/fa rats and ob/ob anddb/db mice) receiving a 6% fat diet, nonadipose tissue TG was 4–100 times normal. In normal rats on a 60% fat diet, peroxisome proliferator-activated receptor α protein and liver-carnitine palmitoyltransferase-1 (l-CPT-1) mRNA increased in liver. In their pancreatic islets, fatty-acid oxidation increased 30% without detectable increase in the expression of peroxisome proliferator-activated receptor-α or oxidative enzymes, whereas lipogenesis from 14Cglucose was slightly below that of the 4% fat-fed rats (p < 0.05). Tissue-specific overexpression of wild-type leptin receptors in the livers offa/fa rats, in which marked steatosis is uniformly present, reduced TG accumulation in liver but nowhere else. We conclude that a physiologic role of the hyperleptinemia of caloric excess is to protect nonadipocytes from steatosis and lipotoxicity by preventing the up-regulation of lipogenesis and increasing fatty-acid oxidation. triacylglycerol fatty acid Zucker Diabetic Fatty polymerase chain reaction adenocytomegalovirus acyl-CoA oxidase liver-carnitine palmitoyltransferase-1 peroxisome proliferator-activated receptor magnetic nuclear resonance spectroscopy Compelling theoretical considerations coupled with corroborating experimental evidence argue against the conventional view that the physiologic role of leptin is to prevent obesity. First, plasma leptin levels of rodents and humans are low in the lean and high in the obese (1Maffei M. Halaas J. Ravussin E. Pratley R.E. Lee G.H. Zhang Y. Fei H. Kim S. Lallone R. Ranganathan S. Kern P.A. Friedman J.M. Nat. Med. 1995; 1: 1155-1161Crossref PubMed Scopus (3327) Google Scholar), hardly the credentials of an antiobesity hormone. Second, diet-induced obesity is not prevented in hypoleptinemic mice by restoring their plasma leptin levels to normal with recombinant leptin (2Surwit R.S. Edwards C.L. Murthy S. Petro A.E. Diabetes. 2000; 49: 1203-1208Crossref PubMed Scopus (32) Google Scholar). Third, there is no evidence that overnutrition and obesity have ever posed a serious survival threat in evolution. On the contrary, the principal survival threat throughout evolution has been famine, against which obesity provides a measure of protection as the “thrifty gene” hypothesis maintains (3Neel J.V. Nutr. Rev. 1997; 57: S2-S9Crossref Scopus (268) Google Scholar). Finally, it seems implausible to suggest that hormones evolve for the purpose of preventing the clinical consequences of their own deficiency. Just as insulin evolved to confer advantages in nutrient metabolism rather than to prevent diabetic ketoacidosis, leptin must have evolved, not to prevent its deficiency syndrome, obesity (4Halaas J.L. Gajiwaia K.S. Maffei M. Cohen S.L. Chalt B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4230) Google Scholar), but rather to confer a metabolic advantage that has not as yet been identified. We previously had suggested that the metabolic advantage conferred by the hyperleptinemia of obesity might be the prevention of overaccumulation of triacylglycerols (TG)1 in nonadipose tissues (5Unger R.H. Zhou Y.-T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (376) Google Scholar). Clearly, leptin does have powerful antilipogenic activity in some such tissues (6Shimabukuro M. Koyama K. Chen G. Wang M.-Y. Trieu F. Lee Y. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4637-4641Crossref PubMed Scopus (618) Google Scholar). In the absence of leptin action, lipogenesis is increased and fatty-acid (FA) oxidation is reduced (7Lee Y. Hirose H. Zhou Y.-T. Esser V. McGarry J.D. Unger R.H. Diabetes. 1997; 46: 408-413Crossref PubMed Scopus (175) Google Scholar), accounting for the steatosis and lipotoxicity that occur in such circumstances (7Lee Y. Hirose H. Zhou Y.-T. Esser V. McGarry J.D. Unger R.H. Diabetes. 1997; 46: 408-413Crossref PubMed Scopus (175) Google Scholar, 8Lee Y. Hirose H. Ohneda M. Johnson J.H. McGarry J.D. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10878-10882Crossref PubMed Scopus (719) Google Scholar, 9Zhou Y.-T. Grayburn P. Karim A. Shimabukuro M. Higa M. Baetens D. Orci L. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1784-1789Crossref PubMed Scopus (1077) Google Scholar). For example, in Zucker Diabetic Fatty (ZDF) rats with a loss-of-function mutation in the leptin receptors (10Iida M. Murakami T. Ishida K. Mizuno A. Kuwajima M. Shima K. Biochem. Biophys. Res. Commun. 1996; 224: 597-604Crossref PubMed Scopus (168) Google Scholar, 11Phillips M.S. Liu O. Hammond H. Dugan V. Hey P. Caskey C.T. Hess J.F. Nat. Genet. 1996; 13: 18-19Crossref PubMed Scopus (759) Google Scholar), tissue TG ranges from 10 to 50 times the normal content (8Lee Y. Hirose H. Ohneda M. Johnson J.H. McGarry J.D. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10878-10882Crossref PubMed Scopus (719) Google Scholar) and is associated with functional impairment of pancreatic β-cells (12Hirose H. Lee Y.H. Inman L.R. Nagasawa Y. Johnson J.H. Unger R.H. J. Biol. Chem. 1996; 271: 5633-5637Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 13Wang M.-Y. Koyama K. Shimabukuro M. Newgard C. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 714-718Crossref PubMed Scopus (90) Google Scholar) and myocardium (9Zhou Y.-T. Grayburn P. Karim A. Shimabukuro M. Higa M. Baetens D. Orci L. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1784-1789Crossref PubMed Scopus (1077) Google Scholar) and insulin resistance (14McGarry J.D. Science. 1992; 258: 766-770Crossref PubMed Scopus (573) Google Scholar). Ultimately, the progressive overaccumulation of lipids causes death of cells in pancreatic islets and myocardium, resulting in diabetes and myocardial failure, which are the most serious complications of obesity. It has been proposed that the lipid overaccumulation enlarges the intracellular pool of fatty acyl-CoA beyond the oxidative requirements of the cell (15Unger R.H. Trends Endocrinol. Metab. 1998; 7: 276-282Google Scholar), thereby providing substrate for potentially destructive nonoxidative pathways, such as de novo ceramide formation (16Shimabukuro M. Zhou Y.-T. Levi M. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2498-2502Crossref PubMed Scopus (1016) Google Scholar) and lipid peroxidation (17Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1612) Google Scholar, 18Vincent H.K. Powers S.K. Stewart D.J. Shanely R.A. Demirel H. Naito H. Int. J. Obes. Relat. Metab. Disord. 1999; 23: 67-74Crossref PubMed Scopus (118) Google Scholar). If the foregoing abnormalities develop in the absence of leptin action, it follows that leptin must be able to prevent them. Certainly hyperleptinemia induced by adenoviral transfer of the leptin gene has remarkable lipopenic and antilipogenic activity in tissues of normal rats, down-regulating the expression of genes involved in lipogenesis while up-regulating those genes involved in β-oxidation and thermogenesis (19Zhou Y.-T. Wang Z.-W. Higa M. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2391-2395Crossref PubMed Scopus (208) Google Scholar). Although they are consistent with putative antisteatotic activity of hyperleptinemia, such studies do not prove that the actual physiologic role of adipocyte-derived hyperleptinemia in obesity is to prevent the ectopic accumulation of TG in nonadipose tissues. This study was designed to test this premise. Three groups of rodents were employed. Obese homozygous (fa/fa) ZDF-Drt rats, which are unresponsive to leptin because of a loss-of-function mutation in their leptin receptor (10Iida M. Murakami T. Ishida K. Mizuno A. Kuwajima M. Shima K. Biochem. Biophys. Res. Commun. 1996; 224: 597-604Crossref PubMed Scopus (168) Google Scholar, 11Phillips M.S. Liu O. Hammond H. Dugan V. Hey P. Caskey C.T. Hess J.F. Nat. Genet. 1996; 13: 18-19Crossref PubMed Scopus (759) Google Scholar), and lean wild-type (+/+) ZDF controls were bred in our laboratory from ZDF/Drt-fa (F10) rats purchased from Dr. R. Peterson (University of Indiana School of Medicine, Indianapolis, IN). Two groups of mice, C57BL/6J-ob/ob and C57BL/KS-J-db/db, and their wild-type controls, C57BL/6J +/+ and C57BL/KS-J +/+ mice, were purchased from the Jackson Laboratory (Bar Harbor, ME). To achieve diet-induced obesity in normal rats, Harlan Sprague-Dawley rats, purchased from Charles River Laboratories (Raleigh, NC) were employed. They were housed in individual metabolic cages (Nalgene, Rochester, NY) with a constant temperature and 12 h of light altering with 12 h of darkness. Body weight and food intake were measured weekly. Initially, all rats were fed standard chow (Teklad 4% mouse/rat diet, Teklad Madison, WI) ad libitum and had free access to water. At 4 weeks of age they either continued on this diet, which contains 24.8% protein, 4% fat, and 3.94 Kcal/g, or they were switched to a high fat diet (Purina Test Diet, Purina Mills, Inc., Richmond, IN) containing 60% fat, 7.5% carbohydrate, 24.5% protein, and 6.7 Kcal/g to produce diet-induced obesity. In in vivo experiments containing a total of 1 × 1012 plaque-forming units of recombinant adenovirus containing the cDNA of the leptin receptor OB-Rb (AdCMV-OB-Rb) or as a control β-galactosidase (AdCMV-β-galactosidase), prepared as described previously (20Chen G.X. Koyama K. Yuan X. Lee Y. Zhou Y.-T. O'Doherty R. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14795-14799Crossref PubMed Scopus (285) Google Scholar), were infused into conscious animals over a 10-min period through polyethylene tubing (PE-50, Becton Dickinson) previously anchored in the left jugular vein of 9-week-old ZDF fa/fa rats under sodium pentobarbital anesthesia (20Chen G.X. Koyama K. Yuan X. Lee Y. Zhou Y.-T. O'Doherty R. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14795-14799Crossref PubMed Scopus (285) Google Scholar). To compare the expression of wild-type OB-Rb in fa/fa rats with mutated OB-Rb, total RNA of rat liver and hypothalamus were extracted using TRIzol reagent (Life Technologies, Inc.). Reverse transcription of total RNA was carried out after treating RNA samples with RNase-free DNase I. The first strand cDNA was then used to PCR-amplify an OB-RbcDNA fragment with OB-Rb-specific primers encompassing the region with thefa/fa mutation as described previously (11Phillips M.S. Liu O. Hammond H. Dugan V. Hey P. Caskey C.T. Hess J.F. Nat. Genet. 1996; 13: 18-19Crossref PubMed Scopus (759) Google Scholar). The conditions of the PCR were as follows: denaturation for 45 s at 92 °C, annealing for 45 s at 55 °C, and elongation for 1 min at 72 °C. The amplified PCR products were digested with MspI at 37 °C 1 h and then run on a 1.2% agarose gel. Total RNA was extracted by the TRIzol isolation method, and Northern blot analysis was carried out as described previously (21Kakuma T. Lee Y. Higa M. Wang Z.-W. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). cDNA for the oxidative enzymes, acyl-CoA oxidase and liver-carnitine were prepared by using the and and The fragment after M.-Y. Koyama K. Shimabukuro M. Newgard C. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 714-718Crossref PubMed Scopus (90) Google Scholar) with only the intracellular of OB-Rb was used as a of The were by were to the with an RNA gene The used was on described by J. P. C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) and O'Doherty P. H.K. D. Newgard C.B. J. 2000; PubMed Scopus Google Scholar). Total RNA was with RNase-free DNase and cDNA was with the in the cDNA was carried out in with of the cDNA reaction as with of PCR containing units of polymerase and containing and of = 37 and of The standard was as follows for mRNA and and gene mRNA and denaturation of °C for 1 annealing at 55 °C for 1 and at 72 °C for 1 min for 24 in liver and for 26 in for in a products were on 1 and 6% and were by a protein mRNA was as an and were as to its To by individual we the of individual in products were in which mRNA and protein in pancreatic islets were the of individual products and their of as by the within the from a 50 of liver from the rats were in of with total of of protein in of were used for with from were used for was carried out with from at the of McGarry J.D. Int. J. Obes. 1995; Scholar), and magnetic resonance were with a with a rats were within the and in the of the of the rat were into and fat the of which were using the nuclear magnetic resonance for were from the region of were with the = = a of and a × were using of were under sodium pentobarbital were and in Total lipids were extracted from of tissue by the of J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and under TG was by the of H. Y. S. Y. S. J. Nutr. Sci. 1992; PubMed Scopus Google Scholar). vein was in with was at °C. leptin was using the leptin was measured by the oxidase using the free fatty were using the TG levels were measured by the of by islets was as described of islets were in with 1 for oxidation was by in the was by with an of acid and in a were in a containing and at 50 °C for was measured as described for of J.L. Hirose H. Lee Y.H. Nagasawa Y. A. Ohneda M. H. Newgard C.B. Johnson J.H. Unger R.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of into lipids was measured in islets as described previously in S. A. Ohneda M. Unger R.H. McGarry J.D. Diabetes. 1994; PubMed Scopus Google Scholar). islets were for in containing of in lipids were extracted from the islets to the of and J. Biochem. PubMed Scopus Google Scholar), and into total lipid were are as analysis was by test by analysis of If the of leptin during caloric excess is to the accumulation of lipids in nonadipose hyperleptinemia at the of overnutrition and increase progressively as the overnutrition To test this a of 10 normal Harlan Sprague-Dawley rats was fed a diet in which 60% of the were from control rats a 4% fat diet. groups were for leptin levels in control rats were by only to a of only ng/ml on the of the in rats on a 60% fat diet, plasma leptin rose to ng/ml (p < within 24 h and increased progressively by to a of 26 ng/ml at 1 In this the in plasma leptin levels the in body fat by 1 there was a body fat and the plasma leptin (r = 0.91, p < leptin levels to to a caloric excess, and they increase in to of the which is consistent with the If the hyperleptinemia induced by high fat feeding does in protect nonadipose tissues of normal rats from overaccumulation of their tissue TG content low during the of obesity the of the tissue and the in plasma lipid To test this we measured tissue TG content of nonadipose tissues after the of the high fat diet at which the total body fat measured by had increased an ∼150-fold the 1 and plasma TG and free fatty-acid levels were TG content in nonadipose tissues increased only 1–2.7-fold the nonadipose tissues of rats only a of the total increase in body fat over of fat during which the animals had obese In the protection against steatosis might not only increased of low but oxidation. In the an increase in the expression of and its and might be Y.-T. Shimabukuro M. Wang M.-Y. Lee Y. Higa M. J.L. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). To the in vivo protection against overaccumulation of TG in normal rats on a high fat diet is by this we protein and and in livers of normal rats receiving either a or a 4% fat protein and mRNA were in the but mRNA was not islets excess which for their in obesity. To the of the protection against lipid overaccumulation that in the of we measured the of oxidation of in islets of Harlan Sprague-Dawley rats receiving either a 4 or 60% fat diet. was 30% in pancreatic islets of rats on the 60% fat diet than in controls that were on the 4% fat diet in no in or be by not suggest that the oxidative of the islets was able to this increase in oxidation without an increase in expression of genes the We had previously that in the absence of leptin activity as ZDF rats, increased lipogenesis was the most in the ectopic overaccumulation of lipids in islets (7Lee Y. Hirose H. Zhou Y.-T. Esser V. McGarry J.D. Unger R.H. Diabetes. 1997; 46: 408-413Crossref PubMed Scopus (175) Google M.S. T. K. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar). in normal rats the high fat diet not the increase in lipogenesis and that had been in islets of the in and there was no increase in the of 14Cglucose to lipids or in the expression of In a but in lipogenesis and in fatty acid mRNA was and This was in to the ZDF fa/fa rats in which lipogenesis was times If the antilipogenic protection in normal rats during caloric excess in the action of the hyperleptinemia, with either a leptin deficiency mice) or a loss-of-function mutation in their leptin receptors mice and ZDF fa/fa be from lipid measured the plasma leptin levels and the TG content of islets, and liver of rodents Although their diet only 6% fat, the TG content of their nonadipose tissues from to normal controls on the diet. leptin action is protection from lipid overaccumulation in nonadipocytes is the fat intake is normal. If the marked steatosis and of obese ZDF fa/fa rats are the of a of leptin action on the overexpression of the wild-type leptin receptor in the liver of leptin animals protect them. we infused into 9-week-old ZDF fa/fa rats units of recombinant adenovirus containing the cDNA of wild-type OB-Rb, the of the leptin receptor was infused into rats as a The wild-type OB-Rb in vivo with an adenovirus was in the liver of the ZDF fa/fa rats was in tissues the after with TG levels of rats slightly below levels and below the controls for weeks after TG content was than that of β-galactosidase controls and controls the of a in the increase in liver TG with the TG of and were intake was in the groups the liver was the only of expression of the normal OB-Rb in rats and the only of antisteatotic action, we must that the leptin which 24 ng/ml in rats and ng/ml in controls, a antisteatotic action on the liver. This that the of hyperleptinemia of obesity is to prevent steatosis in tissues with suggest that a physiologic role of leptin during overnutrition is to protect nonadipocytes from the consequences of lipid This protection at the of as the of progressively increasing hyperleptinemia that to for the of This to overaccumulation of lipids by preventing the increase in lipogenesis that in the absence of leptin action M.S. T. K. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar) and through the up-regulation of metabolism of the fatty (7Lee Y. Hirose H. Zhou Y.-T. Esser V. McGarry J.D. Unger R.H. Diabetes. 1997; 46: 408-413Crossref PubMed Scopus (175) Google Scholar). in the liver there was an increase in protein and no such be in pancreatic islets a 30% increase in the of oxidation. The of in liver than in tissues a by M.S. T. K. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar). In islets the antilipogenic action of hyperleptinemia to be at as as the increase in oxidation in islets from the lipid leptin action is as in fa/fa ZDF rats, the islets have a high of 14Cglucose into lipids in with increased and fatty acid expression on a 6% fat intake Y.-T. Shimabukuro M. Lee Y. Koyama K. Higa M. T. Unger R.H. Diabetes. 1998; 49: Scopus Google Scholar). in normal rats receiving the 60% fat diet, in the low normal and the the antilipogenic of leptin is lipogenesis is and is not reduced by lipid excess, as in normal islets The most evidence in of the antisteatotic role for leptin was the in vivo in ZDF rats that overexpression of the wild-type receptor in their livers prevented the steatosis and that are with evidence of the antisteatotic action of recombinant leptin I. R.E. S. M.S. J.L. 1999; PubMed Scopus Google Scholar) and of fat tissue in mice with O. D. Kim C. M. J. 2000; PubMed Scopus Google Scholar). in our experiments the wild-type leptin receptors were only in the liver and not in the hypothalamus or else. it follows that the hyperleptinemia of those rats must have the OB-Rb to prevent the lipid The of plasma leptin levels on the first of the high fat diet and their high of with the body fat are all consistent with the of an antilipogenic with a physiologic liporegulatory to in nonadipocytes during This protection for the that in rats and humans the complications of diet-induced obesity do not in leptin H. Proc. Biol. Med. 1998; PubMed Scopus Google Scholar, Lee Y. T. Zhou Y.-T. Unger R.H. J. 13: Scholar). leptin is as in I. R.E. S. M.S. J.L. 1999; PubMed Scopus Google Scholar) or leptin receptors are as in ZDF rats, complications in in It be that we do not suggest that the antisteatotic activity to the hyperleptinemia of obesity in normal lean It to be a only during overnutrition plasma leptin levels or the for the which is in the of 10 ng/ml Z.-W. Zhou Y.-T. T. Lee Y. Higa M. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). In the absence of plasma levels are below and leptin action is to be on the for control of food intake and J.M. 2000; PubMed Scopus Google Scholar). We for We and for of this We Dr. for with