Perilipin overexpression in mice protects against diet-induced obesity
2009; Elsevier BV; Volume: 51; Issue: 5 Linguagem: Inglês
10.1194/jlr.m002352
ISSN1539-7262
AutoresHideaki Miyoshi, Sandra C. Souza, Mikiko Endo, Takashi Sawada, James W. Perfield, Chikara Shimizu, Zlatina S. Stancheva, So Nagai, Katherine J. Strissel, Narihito Yoshioka, Martin S. Obin, Takao Koike, Andrew S. Greenberg,
Tópico(s)Plant biochemistry and biosynthesis
ResumoPerilipin A is the most abundant phosphoprotein on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Perilipin null mice exhibit diminished adipose tissue, elevated basal lipolysis, reduced catecholamine-stimulated lipolysis, and increased insulin resistance. To understand the physiological consequences of increased perilipin expression in vivo, we generated transgenic mice that overexpressed either human or mouse perilipin using the adipocyte-specific aP2 promoter/enhancer. Phenotypes of female transgenic and wild-type mice were characterized on chow and high-fat diets (HFDs). When challenged with an HFD, transgenic mice exhibited lower body weight, fat mass, and adipocyte size than wild-type mice. Expression of oxidative genes was increased and lipogenic genes decreased in brown adipose tissue of transgenic mice. Basal and catecholamine-stimulated lipolysis was decreased and glucose tolerance significantly improved in transgenic mice fed a HFD. Perilipin overexpression in adipose tissue protects against HFD-induced adipocyte hypertrophy, obesity, and glucose intolerance. Alterations in brown adipose tissue metabolism may mediate the effects of perilipin overexpression on body fat, although the mechanisms by which perilipin overexpression alters brown adipose tissue metabolism remain to be determined. Our findings demonstrate a novel role for perilipin expression in adipose tissue metabolism and regulation of obesity and its metabolic complications. Perilipin A is the most abundant phosphoprotein on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Perilipin null mice exhibit diminished adipose tissue, elevated basal lipolysis, reduced catecholamine-stimulated lipolysis, and increased insulin resistance. To understand the physiological consequences of increased perilipin expression in vivo, we generated transgenic mice that overexpressed either human or mouse perilipin using the adipocyte-specific aP2 promoter/enhancer. Phenotypes of female transgenic and wild-type mice were characterized on chow and high-fat diets (HFDs). When challenged with an HFD, transgenic mice exhibited lower body weight, fat mass, and adipocyte size than wild-type mice. Expression of oxidative genes was increased and lipogenic genes decreased in brown adipose tissue of transgenic mice. Basal and catecholamine-stimulated lipolysis was decreased and glucose tolerance significantly improved in transgenic mice fed a HFD. Perilipin overexpression in adipose tissue protects against HFD-induced adipocyte hypertrophy, obesity, and glucose intolerance. Alterations in brown adipose tissue metabolism may mediate the effects of perilipin overexpression on body fat, although the mechanisms by which perilipin overexpression alters brown adipose tissue metabolism remain to be determined. Our findings demonstrate a novel role for perilipin expression in adipose tissue metabolism and regulation of obesity and its metabolic complications. Obesity is associated with metabolic dysfunction and increased risk for diabetes, cardiovascular disease, cancer, and early mortality (1Reaven G. Metabolic syndrome: pathophysiology and implications for management of cardiovascular disease.Circulation. 2002; 106: 286-288Crossref PubMed Scopus (466) Google Scholar, 2Hjartaker A. Langseth H. Weiderpass E. Obesity and diabetes epidemics: cancer repercussions.Adv. Exp. Med. Biol. 2008; 630: 72-93Crossref PubMed Scopus (141) Google Scholar). In both lean and obese states, triacylglycerol is predominately stored within lipid droplets of adipocytes. 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Lapillojnne A. Hung-Junn Chang B. Quast M. Gorenstein D. Chen K-H. Chan L. Absence of perilipin results in leanness and reverses obesity in Leprdb/db mice.Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (495) Google Scholar, 8Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D. Zeo J. Gavrilova O. Reitman M. Deng C-X. Ki C. Kimmel A. et al.Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity.Proc. Natl. Acad. Sci. USA. 2001; 98: 6494-6499Crossref PubMed Scopus (609) Google Scholar). Perilipin null mice exhibit constitutively activated basal lipolysis and attenuated stimulated lipolysis and are characterized by a marked reduction in fat depots, which comprise small adipocytes. This phenotype occurs despite increased food intake, and the mice have been demonstrated to be resistant to both diet-induced and genetic obesity (7Martinez-Botas J. Andreson J. Tessler D. Lapillojnne A. Hung-Junn Chang B. 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Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium.J. Biol. Chem. 2002; 277: 32253-32257Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). In this article, we demonstrate that expression of human PeriA or mouse PeriA in perilipin null murine adipocytes regulates lipolysis in a similar manner. To understand the physiological consequences of increased PeriA expression in adipocytes and to determine if functional differences exist between mouse and human PeriA in vivo, we generated two lines of mice that overexpressed either human or mouse PeriA in adipose tissue. This study is to our knowledge the first to investigate the effects of PeriA overexpression in vivo. Results demonstrate that both lines of transgenic mice are protected against diet-induced obesity, coincident with increased oxidative gene expression in brown adipose tissue and an improved metabolic phenotype relative to wild-type mice. In general, the effects of the human PeriA transgene were more robust compared with the effects of the mouse PeriA transgene. Reagents were purchased from Sigma Chemicals (St. Louis, MO). Polyclonal antiperilipin antibody, ATGL antibody, and anti-HSL antibody were generated as previously described (9Souza S.C. Muliro K. Liscum L. Lien P. Yamamoto Y. Schaffer J. Dallal G. Wang X. Kraemer F. Obin M. et al.Modulation of hormone-sensitive lipase and protein kinase-A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 15Miyoshi H. Perfield 2nd, J.W. Souza S.C. Shen W.J. Zhang H.H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes.J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Phosphoserine antibody (1:1,000) was purchased from Zymed Laboratories (San Francisco, CA). We generated a mouse PeriA cDNA (mPeriA) with a Flag tag at the C terminus using a standard PCR method (28Zhang H. Souza S. Muliro K. Kraemer F. Obin M. Greenberg A.S. Lipase-selective functional domains of perililpin A differentially regulate constitutive and protein kinase A-stimulated lipolysis.J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We obtained the human PeriA cDNA (hPeriA) from Dr. Yusuke Nakamura. These cDNAs were then ligated into a pBluescript SK vector containing the aP2 enhancer/promoter region, the SV40 small tumor antigen splice site, and a polyadenylation signal sequence (provided by Dr. B. M. Spiegelman; Fig. 2A) (35Soloveva V. Graves R.A. Rasenick M.M. Spiegelman B.M. Ross S.R. Transgenic mice overexpressing the beta 1-adrenergic receptor in adipose tissue are resistant to obesity.Mol. Endocrinol. 1997; 11: 27-38Crossref PubMed Scopus (111) Google Scholar). A 7.7 kb SalI-NotI fragment containing the entire aP2-human PeriA or aP2-mouse PeriA-Flag transgenes was microinjected into fertilized eggs of C57BL/6J mice. Integration of the hPeriA or mPeriA-flag gene in newborn mice was determined by Southern blot analysis. Founder animals were bred with C57BL/6J mice, and two transgenic (Tg) mouse lines were established (hTg or mTg) on the C57BL/6J background. All Tg mice used for the study were hemizygous for the transgene. Littermates that lacked the transgene were used as controls (WT). hTg mice were generated at the Institute for Animal Experimentation at Hokkaido University and shipped to the JM-USDA Human Nutrition Research Center on Aging at Tufts, while mTg mice were generated at the Transgenic Core Facility at Tufts University. Mice were housed in a pathogen-free barrier facility at the JM-USDA HNRCA at Tufts in accordance with IACUC guidelines. All mice were housed at room temperature, maintained on a 12 h light/dark cycle, given free access to water, and fed a standard chow diet (ND) (Harlan #7012; Indianapolis, IN). For high-fat diet (HFD) (60% calories from fat; Research Diets #D12492; New Brunswick, NJ) experiments, mice were fed an HFD from the age of 5 weeks to 30 weeks. Food intake and body weight were monitored weekly. On the day prior to tissue harvest at 30 weeks, food was removed at 21:00 h for an overnight fast. Blood, white adipose tissue (WAT) from visceral (perigonadal, perirenal, and mesenteric) and subcutaneous (right side inguinal) depots, and interscapular brown adipose tissue (BAT) were rapidly dissected out, weighed, and processed for subsequent analysis. Adipocytes were isolated from the perigonadal depot using collagenase and centrifugation as previously described (36Honnor R.C. Dhillon G.S. Londos D. cAMP-dependent protein kinase and lipolysis in rat adipocytes I. Cell preparation, manipulation, and predictability in behavior.J. Biol. Chem. 1985; 260: 15122-15129Abstract Full Text PDF PubMed Google Scholar) with minor modifications. The samples were gassed (5% CO2 and 95% O2), capped, and incubated at 37°C with shaking until digestion was complete (30–40 min). Adipocytes were aliquoted in triplicate to measure lipolysis under basal (200 nM phenyl isopropyl adenosine) and stimulated (200 nM phenyl isopropyl adenosine + 10 µM isoproterenol) conditions. Lipolytic rate was assessed as glycerol released into the media for 2 h using a Free Glycerol Determination kit (Sigma) (22D'Eon T.M. Souza S.C. Aronovitz M. Obin M.S. Fried S.K. Greenberg A.S. Estrogen regulation of adiposity and fuel partitioning. Evidence of genomic and non-genomic regulation of lipogenic and oxidative pathways.J. Biol. Chem. 2005; 280: 35983-35991Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Lipolysis was quantified per cell. The number of adipocytes per incubation was determined according to previously published methods (37Di Girolamo M. Mendlinger S. Fertig J. A simple method to determine fat cell size and number in four mammalian species.Am. J. Physiol. 1971; 221: 850-858Crossref PubMed Scopus (271) Google Scholar). Lipid content (mg/ml) was determined by organic extraction of lipids from isolated adipocytes. Adipocytes were visually sized (100–150 adipocytes per mouse) using a light microscope and a 200 µm ocular micrometer. Cell number was then calculated using measures of lipid concentration and adipocyte diameter (37Di Girolamo M. Mendlinger S. Fertig J. A simple method to determine fat cell size and number in four mammalian species.Am. J. Physiol. 1971; 221: 850-858Crossref PubMed Scopus (271) Google Scholar). Intraperitoneal glucose tolerance tests (GTTs) and intraperitoneal insulin tolerance tests (ITTs) were performed on nonanesthetized mice that were fasted overnight or for 4 h. The ITT was performed 1 week subsequent to the GTT. For the GTT, blood glucose levels from whole-tail vein blood were measured at baseline and 30, 60, and 120 min following intraperitoneal injection of glucose (1 g/kg body weight) using an automated glucometer. For the ITT, mice received an intraperitoneal injection of human insulin (0.75 mU/kg body weight), and blood glucose was measured at baseline and 15, 30, 45, 60, and 90 min after injection. Glucose area under the curve (AUC0-120 or 0-90) was determined for HFD-fed and ND-fed cohorts and the difference (ΔAUC) reported (38Strissel K.J. Stancheva Z. Miyoshi H. Perfield II, J.W. DeFuria J. Jick Z. Greenberg A.S. Obin M.S. Adipocyte death, adipose tissue remodeling, and obesity complications.Diabetes. 2007; 56: 2910-2918Crossref PubMed Scopus (700) Google Scholar). WAT and BAT were dissected, fixed, embedded in paraffin, and sectioned (38Strissel K.J. Stancheva Z. Miyoshi H. Perfield II, J.W. DeFuria J. Jick Z. Greenberg A.S. Obin M.S. Adipocyte death, adipose tissue remodeling, and obesity complications.Diabetes. 2007; 56: 2910-2918Crossref PubMed Scopus (700) Google Scholar). Sections were stained with hematoxylin and eosin. Digital images were acquired with an Olympus DX51 light microscope. Total RNA was extracted from WAT and BAT using a commercial kit (RNeasy lipid tissue; Qiagen, Valencia, CA). RNA was quantified by RiboGreen Quantitation Assay (Molecular Probes, Eugene, OR), and cDNA was synthesized from 1 µg of total RNA (Reverse Transcription System; Promega, Madison, WI). Real-time PCR was performed in triplicate on an ABI PRISM® 7700 in 20 µl total volume reactions using SYBR® Green PCR Master MIX (Applied Biosystems, Foster City, CA). Primers were designed using Primer Express. Data were analyzed by comparative critical threshold (Ct) method (39Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (127155) Google Scholar) and normalized to an endogenous control gene (18S rRNA). Percentage of difference was calculated by 2−ΔΔCt. Stable lines of mouse embryonic fibroblasts (MEFs) were generated from embryos of Peri−/− mice as described (10Miyoshi H. Souza S.C. Zhang H.H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. et al.Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosp
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