Adpla Ablation Increases Lipolysis and Prevents Obesity Induced by High-Fat Feeding Or Leptin Deficiency
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ARTICLES AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency Kathy Jaworski1,4, Maryam Ahmadian1,4, Robin E Duncan1,4, Eszter Sarkadi-Nagy1, Krista A Varady1, Marc K Hellerstein1, Hui-Young Lee2, Varman T Samuel2, Gerald I Shulman2, Kee-Hong Kim3, Sarah de Val1, Chulho Kang3 & Hei Sook Sul1 A main function of white adipose tissue is to release fatty acids from stored triacylglycerol for other tissues to use as an energy source. Whereas endocrine regulation of lipolysis has been extensively studied, autocrine and paracrine regulation is not well understood. Here we describe the role of the newly identified major adipocyte phospholipase A2, AdPLA (encoded by Pla2g16, also called HREV107), in the regulation of lipolysis and adiposity. AdPLA-null mice have a markedly higher rate of lipolysis owing to increased cyclic AMP levels arising from the marked reduction in the amount of adipose prostaglandin E2 that binds the Gai-coupled receptor, EP3. AdPLA-null mice have markedly reduced adipose tissue mass and triglyceride content but normal adipogenesis. They also have higher energy expenditure with increased fatty acid oxidation within adipocytes. AdPLA-deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and is crucial for the development of obesity. 8 Triacylglycerol in adipose tissue is the major energy storage form hydrolysis of the sn-2 ester bond of phospholipids . The PLA2 in mammals. An imbalance between energy intake and expenditure enzymes function in remodeling of membrane phospholipids by can result in excess triacylglycerol accumulation in this tissue, acylation and deacylation cycles8. Additionally, because the sn-2 resulting in obesity1.Inmorbidobesityincreasedadipocyte position of phospholipids is typically enriched in arachidonic acid number (hyperplasia) may occur through adipocyte differentiation and other unsaturated fatty acids, PLA2 enzymes catalyze the initial of precursor cells present in adipose tissue2. However, obesity is rate-limiting step in the production of eicosanoids9. Eicosanoids, largely attributed to adipocyte hypertrophy that occurs when including prostaglandins, are potent local mediators of signal trans- triacylglycerol synthesis exceeds breakdown (lipolysis), resulting duction and are known to modulate many physiological systems. © All rights reserved. 2009 Inc. Nature America, in elevated triacylglycerol storage1,3. Indeed, unlike the triacylgly- These signaling molecules exert autocrine action, paracrine action or 4,5 cerol synthesis that occurs at high levels in other tissues, includ- both through binding to specific G-coupled stimulatory (Gas)or ing in the liver for very low density lipoprotein production, inhibitory (Gai) receptors that can, in turn, modulate a host of effects, lipolysis for the liberation of fatty acids that can then be used including lipolysis through regulation of cAMP levels10. Although as an energy source by other tissues is unique to adipocytes6. their physiological importance is unclear, there are some reports that Furthermore, lipolysis in adipocytes is tightly regulated by hor- suggest prostaglandins may modulate adipocyte differentiation mones that are secreted according to nutritional status. During in vitro11–14. In mature adipocytes, depending on the concentrations fasting,lipolysisisstimulatedbycatecholaminesthatincreasecyclic used, some prostaglandins have been reported to stimulate, inhibit or AMP (cAMP) concentrations, whereas, in the fed state, lipolysis is exert no effect on lipolysis15,16. Regardless, because AdPLA is highly inhibited by insulin1,3. Although regulation of lipolysis by these expressed only in adipose tissue, we hypothesized that it could have a endocrine factors has been extensively studied, the local regulation key role in adipose-specific processes such as lipolysis through of lipolysis in adipose tissue by autocrine and paracrine factors is modulation of arachidonic acid metabolism and its provision for not well understood. prostaglandin biosynthesis. We recently identified an adipocyte phospholipase A2 (PLA2)by Here we show that AdPLA is the major PLA2 enzyme in adipose microarray analysis that we named AdPLA7 (also called PLA2G16, tissue and that it regulates lipolysis in an autocrine and paracrine HRASLS3, HREV107, HREV107-3, MGC118754 or H-REV107-1). manner through PGE2. We report that ablation of AdPLA prevents AdPLA belongs to a newly discovered group of intracellular calcium- obesity from high fat feeding or leptin deficiency by regulating 7 dependent PLA2s . The enzymes in the PLA2 superfamily catalyze lipolysis through the PGE2-EP3-cAMP pathway. 1Department of Nutritional Science and Toxicology, 220 Morgan Hall, University of California, Berkeley, California 94720, USA. 2Department of Internal Medicine, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06510, USA. 3Department of Molecular and Cell Biology, 447 Life Sciences Addition #2751, University of California, Berkeley, California 94720, USA. 4These authors contributed equally to this work. Correspondence should be addressed to H.S.S. ([email protected]). Received 29 September 2008; accepted 19 November 2008; published online 11 January 2009; doi:10.1038/nm.1904 NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1 ARTICLES RESULTS specifically expressed in adipocytes and is upregulated by feeding and AdPLA is highly expressed primarily in adipose tissue insulin, and in obesity. As we have shown previously7, the 1.3-kilobase AdPLA messenger RNA and the 18-kDa AdPLA protein are expressed in mice in large AdPLA-null mice are resistant to diet-induced obesity amounts only in white adipose tissue (WAT) and brown adipose tissue To elucidate the physiological role of AdPLA, we used gene targeting (BAT) depots (Fig. 1a). AdPLA is found exclusively in adipocytes and to generate AdPLA-null mice (Supplementary Fig. 1 online) that we not in the stromal vascular fraction of adipose tissue that contains compared with wild-type littermates on a mixed genetic background preadipocytes (Fig. 1a). In humans, similar to mice, ADPLA mRNA (C57BL/6J and 129 SVJ) as well as on a C57BL/6J background (genetic was undetectable by RT-PCR in skeletal muscle and barely detectable backgrounds of mice used in specific experiments are indicated in the in liver but was abundantly expressed in WAT (Fig. 1a). We measured Methods). Although total body weights did not differ at weaning, at 7 expression of all currently known intracellular PLA2 enzymes .We 11 weeks of age, AdPLA-null mice fed a high-fat diet (HFD) began to detected AdPLA at approximately 1,000–100,000-fold higher levels gain weight at a slower rate than wild-type littermates (Fig. 1d). This (Fig. 1b), indicating that AdPLA is the major PLA2 in adipose tissue. disparity in body weight was exacerbated as mice aged, such that by We investigated AdPLA regulation in different nutritional and hor- 64 weeks of age, AdPLA-null mice fed a HFD weighed only 39.1 ± monal states. The AdPLA mRNA level in epididymal WAT was low in 0.2 g (n ¼ 3) versus wild-type littermates who weighed 73.7 ± 0.3 g fasted mice but markedly rose (by eightfold) after feeding the mice a (n ¼ 3), P o 0.001. The decreased weight gain was also observed in high-carbohydrate, fat-free diet (P o 0.01) (Fig. 1c). AdPLA mRNA AdPLA-null mice fed a standard chow diet (SD), albeit to a lesser expression was also low in adipose tissue of streptozotocin-diabetic extent (Fig. 1d). Food intakes in AdPLA-null and wild-type mice were mice, but it was similarly increased, by approximately tenfold, after equivalent, despite the differences in body weights (0.09 grams per day insulin administration (Fig. 1c). We observed substantially higher per gram body weight, n ¼ 6). AdPLA mRNA expression in WATof genetically obese ob/ob mice that The lower body weight of AdPLA-null mice was largely accounted are hyperinsulinemic and have markedly higher adipose tissue tri- for by a reduction in WAT weight (Fig. 2a). Body composition acylglycerol storage (Fig. 1c) and higher AdPLA protein expression in analysis indicated that AdPLA-null mice had decreased triacylgly- obese ob/ob and db/db mice than in lean wild-type mice in both the cerol content (Supplementary Table 1 online). At 18 weeks of fasted and refed states (Fig. 1c). AdPLA is therefore highly and age, AdPLA-null mice fed a HFD had substantially smaller WAT a Mouse Human b c Diabetes 40,000 Fasting FeedingDiabetes + Insulin WT ob/ob WATBrainHeartSI KidneyLiverLungSM BATEpi Ing SVFAd.F SM Liver WAT 30,000 AdPLA ADPLA 20,000 AdPLA AdPLA 1.3 kb ACTB 200 28S 28S mRNA / 28S 2 18S 18S 18S 100 PLA Gapdh mRNA WT db/db ob/ob ND ND ND 0 α β γ δ ε ξ β γ Ren Ing Ren Ing Ren Ing 2 2 2 2 2 2 2 2 HeartLungSpleenLiverPancreasKidneySM Epi Ren Ing BAT AdPLA Fasted AdPLAcPLAcPLAcPLAcPLAcPLAcPLAiPLA iPLA AdPLA 18 kDa AdPLA Refed © All rights reserved. 2009 Inc. Nature America, d 18 weeks 32 weeks WT KO WT KO Figure 1 AdPLA tissue distribution, regulation of WT-HFD expression and body weights of AdPLA-null mice. KO-HFD (a) Top left, northern blot analysis of 10 mg of total WT-SD RNA from various mouse tissues. SI, small intestine; KO-SD SM, skeletal muscle; Epi, epididymal fat; Ing, 50 inguinal fat; SVF, stromal vascular fraction; Ad.F, *** adipocyte fraction. Top right, RT-PCR analysis of RNA 18 weeks 18 weeks *** (2.5 mg) from human SM, liver or WAT for expression WT KO WT KO ** 40 of AdPLA or b-actin. Bottom, western blot analysis Kidney for AdPLA protein in various mouse tissues. 80 mgof WAT Liver * protein was subjected to SDS-PAGE and probed with 30 antibodies to AdPLA. Ren, renal fat. (b) Quantitative Dorsal RT-PCR of RNA from wild-type (WT) renal WAT. Ventral Body weight (g) Values for PLA2 enzymes were normalized to Gapdh ISc WAT 20 Liver mRNA and then expressed relative to cPLA2a mRNA (n ¼ 5).