Effect of leptin infusion on insulin sensitivity and lipid metabolism in diet-induced lipodystrophy model mice
© Nagao et al; licensee BioMed Central Ltd. 2008
Received: 12 February 2008
Accepted: 18 March 2008
Published: 18 March 2008
Lipodystrophies are rare acquired and genetic disorders characterized by the complete or partial absence of body fat with a line of metabolic disorders. Previous studies demonstrated that dietary conjugated linoleic acid (CLA) induces hepatic steatosis and hyperinsulinemia through the drastic reduction of adipocytokine levels due to a paucity of adipose tissue in mice and the pathogenesis of these metabolic abnormalities in CLA-fed mice is similar to that in human lipodystrophy. The present study explores the effect of leptin infusion on the pathogenesis of diet-induced lipodystrophy in mice. C57BL/6N mice were assigned to three groups: (1) mice were fed a semisynthetic diet supplemented with 6% corn oil and infused PBS intraperitoneally (normal group), (2) mice were fed a semisynthetic diet supplemented with 4% corn oil plus 2% CLA and infused PBS intraperitoneally (lipodystrophy-control group), and (3) mice were fed a semisynthetic diet supplemented with 4% corn oil plus 2% CLA and infused recombinant murine leptin intraperitoneally (lipodystrophy-leptin group). All mice were fed normal or lipodystrophy model diets for 4 weeks and were infused intrapeneally 0 or 5 μ g of leptin per day from third week of the feeding period for 1 week.
The results indicate that leptin infusion can attenuate hepatic steatosis and hyperinsulinemia through the reduction of hepatic triglyceride synthesis and the improvement of insulin sensitivity in diet-induced lipodystrophy model mice.
We expect the use of this model for clarifying the pathophysiology of lipodystrophy-induced metabolic abnormalities and evaluating the efficacy and safety of drug and dietary treatment.
Recent advances in molecular and cell biology have shown that adipose tissue not only stores excess energy in the form of fat, but also secretes physiologically active substances called adipocytokines . In obesity, it is well known that adipocytes, cells of adipose tissues, are increased and enlarged, and they secrete excess amounts of inflammatory adipocytokines, such as tumor necrosis factor-alpha  and monocyte chemoattractant protein-1 . This induces insulin resistance, hyperinsulinemia, and fatty liver [4, 5]. On the other hand, it is reported that the deficiency of adipocytes also induces type-2 diabetes due to a paucity of normally functioning adipocytokines such as leptin  and adiponectin [7, 8]. This symptom is known as lipodystrophy in humans. Lipodystrophies are rare acquired and genetic disorders characterized by the complete or partial absence of body fat with a line of metabolic disorders [9, 10]. Recent reports indicated that the clinical treatment of HIV-infected patients by using HIV-1 protease inhibitors also induces acquired lipodystrophy .
In the present study, we investigated the effects of leptin infusion on insulin sensitivity and lipid metabolism in diet-induced lipodystrophy model mice. Previous studies demonstrated that leptin treatment attenuated insulin resistance in genetically diabetic mice (such as ob/ob mice and MKR mice) [23, 24] and in lipodystrophy model transgenic mice (such as aP2-SREBP-1c mice) . In addition, Tsuboyama-Kasaoka, Takahashi, Tnemura, Kim, Tnage, Okuyama, Kasai, Ikemoto, and Ezaki  showed preliminary data indicating that leptin infusion lowers the levels of serum insulin and attenuates hepatocyte fat deposition in CLA-fed lipodystrophy model mice. To clarify the precise effect of leptin infusion, we measured hepatic enzyme activities in relation to lipid metabolism and tested insulin sensitivities in these model mice.
Results and Discussion
Effect of leptin infusion on growth parameters in diet-induced lipodystrophy model mice
Effect of leptin infusion on growth parameters in C57BL/6N mice
Final body weight (g)
21.7 ± 0.5
22.1 ± 0.3
21.3 ± 0.2
Food intake (g)
61.2 ± 1.3
58.4 ± 2.1
59.6 ± 1.2
Liver (g/100 g body weight)
4.11 ± 0.05 a
6.47 ± 0.77 b
5.65 ± 0.29 b
Whit adipose tissue (g/100 g body weight)
1.87 ± 0.22 a
0.290 ± 0.029 b
0.228 ± 0.030 b
0.836 ± 0.104 a
0.199 ± 0.018 b
0.178 ± 0.018 b
1.13 ± 0.09 a
0.652 ± 0.022 b
0.740 ± 0.029 b
1.95 ± 0.16 a
0.329 ± 0.014 b
0.344 ± 0.017 b
Effect of leptin infusion on hepatic triglyceride metabolism in diet-induced lipodystrophy model mice
Effect of leptin infusion on adipocytokine levels and insulin sensitivities in diet-induced lipodystrophy model mice
The present study explored the effect of leptin infusion on the pathogenesis of diet-induced lipodystrophy in mice. The results indicate that leptin infusion can attenuate hepatic steatosis and hyperinsulinemia through the reduction of hepatic triglyceride synthesis and the improvement of insulin sensitivity in diet-induced lipodystrophy model mice. We expect the use of this model for clarifying the pathophysiology of lipodystrophy-induced metabolic abnormalities and evaluating the efficacy and safety of drug and dietary treatment.
Animals and diets
All aspects of the experiment were conducted according to the guidelines provided by the Ethical Committee of Experimental Animal Care at Saga University. C57BL/6N mice (Kyudo Co., Ltd., Saga, Japan) were housed individually in metal cages in a temperature-controlled room (24°C) under a 12-hour light/dark cycle. Mice were assigned to three groups (3–6 mice each): (1) mice were fed a semisynthetic diet supplemented with 6% corn oil and infused PBS (Gibco, Tokyo, Japan) intraperitoneally (normal group), (2) mice were fed a semisynthetic diet supplemented with 4% corn oil plus 2% CLA and infused PBS intraperitoneally (lipodystrophy-control group), and (3) mice were fed a semisynthetic diet supplemented with 4% corn oil plus 2% CLA and infused recombinant murine leptin (5 μ g/day, PeproTech EC, London, United Kingdom) intraperitoneally (lipodystrophy-leptin group). The semisynthetic diets were prepared according to recommendations of the AIN-93G  and contained (in weight %) casein, 20; fat, 6; alpha-cornstarch, 13.2; vitamin mixture (AIN-93™), 1; mineral mixture (AIN-93G™), 3.5; L-cystein, 0.3; choline bitartrate, 0.25; cellulose, 5; sucrose, 10; tert-buthyhydroquinone, 0.0014; and beta-cornstarch, 40.7486. The mice received the diets ad libitum using Rodent CAFE (KBT Oriental Co. Ltd., Saga, Japan) for 4 weeks. The mice were killed by exsanguination of the heart, and serum was separated from the blood. Liver and WATs (perirenal, epididymal, omental, and waist subcutaneous) were also excised for analysis.
Analysis of hepatic triglyceride and serum parameters
Liver lipids were extracted according to the method of Folch, Lee, and Sloane-Stanley , and the concentrations of triglyceride were measured using the methods of Fletcher . Serum insulin, adiponectin, and leptin levels were measured using commercial mouse ELISA kits (Shibayagi Co. Ltd., Gunma, Japan; Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan; and Morinaga Co. Ltd., Yokohama, Japan, respectively). Activities of AST in serum were measured using commercial enzyme assay kits (Wako Pure Chemicals, Tokyo, Japan).
Measurement of hepatic enzyme activities
A piece of liver was homogenized insix volumes of a 0.25-M sucrose solution that contained 1 mM EDTA in a 10-mM Tris-HCL buffer (pH 7.4). Fractions of cytosol and microsomes were obtained as previously described . The protein concentration was determined according to the method of Lowry, rosebrough, Farr, and Randall , with bovine serum albumin used as the standard. The enzyme activities of ME (EC 184.108.40.206)  and FAS (EC 220.127.116.11)  in the liver cytosol fraction and phosphatidate phosphohydrolase (EC 18.104.22.168)  in the liver microsomal fraction were determined as described.
Insulin tolerance test
At the end of the feeding period, human insulin (Humulin R; Eli Lilly Japan K.K., Kobe, Japan) was injected intraperitoneally (0.75 mU/g body weight) to all mice. Blood glucose was measured on samples obtained from tail tip before and 30, 60, 90, and 120 min after insulin injection. Blood glucose concentrations were measured using the GLUCOCARD™ G meter (Arkray, Kyoto, Japan).
All values are expressed as means ± SE. Data were analyzed by one-way ANOVA, and all differences were inspected by Duncan's new multiple-range test . Differences were considered to be significant at P < 0.05.
List of abbreviations
conjugated linoleic acid
fatty acid synthase
white adipose tissue.
This work was supported by a research grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology. We thank Miho Nakagawa for technical assistance and the Nisshin OilliO Group Ltd. (Yokosuka, Japan) for providing sample oils.
- Matsuzawa Y, Funahashi T, Nakamura T: Molecular Mechanism of Metabolic Syndrome X: Contribution of Adipocytokines and adipocyte-derived bioactive substances. Ann NY Acad Sci. 1999, 892: 146-154.View ArticlePubMedGoogle Scholar
- Hotamisligil GS, Shargill NS, Spiegelman BM: Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science. 1993, 259: 87-91.View ArticlePubMedGoogle Scholar
- Sartipy P, Loskutoff DJ: Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci USA. 2003, 100: 7265-7270.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang B, Jenkins JR, Trayhurn P: Expression and secretion of inflammation-related adipokines by human adipocytes differentiated in culture: integrated response to TNF-alpha. Am J Physiol Endocrinol Metab. 2005, 288: E731-E740.View ArticlePubMedGoogle Scholar
- Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira K, Kasuga M: MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest. 2006, 116: 1494-1505.PubMed CentralView ArticlePubMedGoogle Scholar
- Ahima RS, Flier JS: Leptin. Annu Rev Physiol. 2000, 62: 413-437.View ArticlePubMedGoogle Scholar
- Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y: Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun. 1999, 257: 79-83.View ArticlePubMedGoogle Scholar
- Hotta K, Funahashi T, Bodkin NL, Ortmeyer HK, Arita Y, Hansen BC, Matsuzawa Y: Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys. Diabetes. 2001, 50: 1126-1133.View ArticlePubMedGoogle Scholar
- Yu YH, Ginsberg HN: Adipocyte signaling and lipid homeostasis: sequelae of insulin-resistant adipose tissue. Circ Res. 2005, 96: 1042-1052.View ArticlePubMedGoogle Scholar
- Simha V, Garg A: Lipodystrophy: lessons in lipid and energy metabolism. Curr Opin Lipidol. 2006, 17: 162-169.View ArticlePubMedGoogle Scholar
- Shimomura I, Hammer RE, Richardson JA, Ikemoto S, Bashmakov Y, Goldstein JL, Brown MS: Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev. 1998, 12: 3182-3194.PubMed CentralView ArticlePubMedGoogle Scholar
- Shimomura I, Bashmakov Y, Horton JD: Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem. 1999, 274: 30028-30032.View ArticlePubMedGoogle Scholar
- Horton JD, Shimomural I, Ikemoto S, Bashmakov Y, Hammer RE: Overexpression of sterol regulatory element-binding protein-1a in mouse adipose tissue produces adipocyte hypertrophy, increased fatty acid secretion, and fatty liver. J Biol Chem. 2003, 278: 36652-36660.View ArticlePubMedGoogle Scholar
- Moitra J, Mason MM, Olive M, Krylov D, Gavrilova O, Marcus-Samuels B, Feigenbaum L, Lee E, Aoyama T, Eckhaus M, Reitman ML, Vinson C: Life without white fat: a transgenic mouse. Genes Dev. 1998, 12: 3168-3181.PubMed CentralView ArticlePubMedGoogle Scholar
- Reitman ML, Gavrilova O: A-ZIP/F-1 mice lacking white fat: a model for understanding lipoatrophic diabetes. Int J Obes Relat Metab Disord. 2000, 24: S11-14.View ArticlePubMedGoogle Scholar
- Gavrilova O, Marcus-Samuels B, Reitman ML: Lack of responses to a beta3-adrenergic agonist in lipoatrophic A-ZIP/F-1 mice. Diabetes. 2000, 49: 1910-1916.View ArticlePubMedGoogle Scholar
- Clement L, Pirier H, Niot I, Bocher V, Guerre-Millo M, Krief S, Staels B, Besnard P: Dietary trans-10, cis-12 conjugated linoleic acid induces hyperinsulinemia and fatty liver in the mouse. J Lipid Res. 2002, 43: 1400-1409.View ArticlePubMedGoogle Scholar
- Pariza MW: Perspective on the safety and effectiveness of conjugated linoleic acid. Am J Clin Nutr. 2004, 79: 1132S-1136S.PubMedGoogle Scholar
- Ohashi A, Matsushita Y, Shibata H, Kimura K, Miyashita K, Saito M: Conjugated linoleic acid deteriorates insulin resistance in obese/diabetic mice in association with decreased production of adiponectin and leptin. J Nutr Sci Vitaminol. 2004, 50: 416-421.View ArticlePubMedGoogle Scholar
- Poirier H, Rouault C, Clement L, Niot I, Monnot MC, Guerre-Millo M, Besnard P: Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse. Diabetologia. 2005, 48: 1059-1065.View ArticlePubMedGoogle Scholar
- Wang YM, Nagao K, Ujino Y, Sakata K, Higa K, Inoue N, Yanagita T: Short-term feeding of conjugated linoleic acid does not induce hepatic steatosis in C57BL/6J mice. J Nutr Sci Vitaminol. 2005, 51: 440-444.View ArticlePubMedGoogle Scholar
- Tsuboyama-Kasaoka N, Miyazaki H, Kasaoka S, Ezaki O: Increasing the amount of fat in a conjugated linoleic acid-supplemented diet reduces lipodystrophy in mice. J Nutr. 2003, 133: 1793-1799.PubMedGoogle Scholar
- Harris RB, Zhou J, Redmann SM, Smagin GN, Smith SR, Rodgers E, Zachwieja JJ: A leptin dose-response study in obese (ob/ob) and lean (+/?) mice. Endocrinology. 1998, 139: 8-19.View ArticlePubMedGoogle Scholar
- Toyoshima Y, Gavrilova L, Yakar S, Jou W, Pack S, Asghar Z, Wheeler MB, LeRoith D: Leptin improves insulin resistance and hyperglycemia in a mouse model of type 2 diabetes. Endocrinology. 2005, 146: 4024-4035.View ArticlePubMedGoogle Scholar
- Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL: Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature. 1999, 401: 73-76.View ArticlePubMedGoogle Scholar
- Tsuboyama-Kasaoka N, Takahashi M, Tanemura K, Kim HJ, Tnage T, Okuyama H, Kasai M, Ikemoto S, Ezaki O: Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes. 2000, 49: 1534-1542.View ArticlePubMedGoogle Scholar
- Takahashi Y, Kushiro M, Shinohara K, Ide T: Activity and mRNA levels of enzymes involved in hepatic fatty acid synthesis and oxidation in mice fed conjugated linoleic acid. Biochim Biophys Acta. 2003, 1631: 265-273.View ArticlePubMedGoogle Scholar
- Vance DE: Glycerolipid biosynthesis in eukaryotes. Biochemistry of lipids, lipoproteins and membranes. Edited by: Vance DE, Vance J. 1996, 31: 153-181. Elsevier Science Publishers.View ArticleGoogle Scholar
- Purushotham A, Wendel AA, Liu LF, Belury MA: Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice. J Lipid Res. 2007, 48: 444-452.View ArticlePubMedGoogle Scholar
- Liu LF, Purushotham A, Wendel AA, Belury MA: Combined effects of rosiglitazone and conjugated linoleic acid on adiposity, insulin sensitivity, and hepatic steatosis in high-fat-fed mice. Am J Physiol Gastrointest Liver Physiol. 2007, 292: G1671-1682.View ArticlePubMedGoogle Scholar
- Reeves PG, Nielsen FH, Fahey GC: AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993, 123: 1939-1951.PubMedGoogle Scholar
- Folch J, Lee M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957, 226: 497-509.PubMedGoogle Scholar
- Fletcher MJ: A colorimetric method for estimation of serum triglycerides. Clin Chem Acta. 1968, 22: 393-397.View ArticleGoogle Scholar
- Nagao K, Inoue N, Wang YM, Shirouchi B, Yanagita T: Dietary conjugated linoleic acid alleviates nonalcoholic fatty liver disease in Zucker (fa/fa) rats. J Nutr. 2005, 135 (1): 9-13.PubMedGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem. 1951, 193: 265-275.PubMedGoogle Scholar
- Ochoa S: Malic enzyme: malic enzymes from pigeon and wheat germ. Methods in Enzymology. Edited by: Colowick SP, Kaplan NO. 1955, 1: 323-326. New York: Academic Press.Google Scholar
- Kelley DS, Nelson GJ, Hunt JE: Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes. Biochem J. 1986, 235: 87-90.PubMed CentralView ArticlePubMedGoogle Scholar
- Walton PA, Possmayer F: Mg dependent phosphatidate phosphohydrolase of rat lung: Development of an assay employing a defined chemical substrate which reflects phosphohydrolase activity measured using membrane-bound and substrate. Anal Biochem. 1985, 151: 479-486.View ArticlePubMedGoogle Scholar
- Duncan DB: Multiple range and multiple F tests. Biometrics. 1955, 11: 1-42.View ArticleGoogle Scholar
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