Adipose-Specific Knockout of Seipin/Bscl2 Results in Progressive

2320 Diabetes Volume 63, July 2014

Lu Liu,1 Qingqing Jiang,1 Xuhong Wang,2 Yuxi Zhang,3 Ruby C.Y. Lin,3 Sin Man Lam,4 Guanghou Shui,4 Linkang Zhou,5 Peng Li,5 Yuhui Wang,1 Xin Cui,1 Mingming Gao,1 Ling Zhang,1 Ying Lv,6 Guoheng Xu,6 George Liu,1 Dong Zhao,2 and Hongyuan Yang3

Adipose-Speciﬁc Knockout of Seipin/Bscl2 Results in Progressive Lipodystrophy

Diabetes 2014;63:2320–2331 | DOI: 10.2337/db13-0729

Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) autosomal recessive disorder characterized by a near total is the most severe form of human lipodystrophy, char- loss of adipose tissue, severe insulin resistance, and fatty acterized by an almost complete loss of adipose tissue liver (1,2). To date, four genes have been linked to CGL/ and severe insulin resistance. BSCL2 is caused by loss- BSCL, including 1-acylglycerol-3-phosphate-O-acyl trans- of-function mutations in the BSCL2/SEIPIN gene, which ferase 2 (AGPAT2)/CGL1, SEIPIN/CGL2, CAVEOLIN/ is upregulated during adipogenesis and abundantly CGL3, and CAVIN/CGL4 (3). The most severe form of expressed in the adipose tissue. The physiological func- human CGL/BSCL is caused by mutations in SEIPIN/ tion of SEIPIN in mature adipocytes, however, remains BSCL2, which encodes an integral membrane protein of fi to be elucidated. Here, we generated adipose-speci c the endoplasmic reticulum (ER) with no recognizable Seipin knockout (ASKO) mice, which exhibit adipocyte functional domains (3–5). We and others have generated hypertrophy with enlarged lipid droplets, reduced lipoly- Seipin knockout (KO) mice (6–8), which have severe lipo- sis, adipose tissue inflammation, progressive loss of dystrophy and insulin resistance, thereby proving an es- METABOLISM white and brown adipose tissue, insulin resistance, and Seipin hepatic steatosis. Lipidomic and microarray analyses sential role of in adipogenesis in vivo. Interestingly, revealed accumulation/imbalance of lipid species, includ- SEIPIN and its orthologs also control the expansion of – ing ceramides, in ASKO adipose tissue as well as in- lipid droplets (LDs) and lipogenesis (9 12). Therefore, creased endoplasmic reticulum stress. Interestingly, the SEIPIN can regulate lipid storage at systemic (adipogene- ASKO mice almost completely phenocopy the fat-specific sis) and cellular (LD expansion) levels. peroxisome proliferator–activated receptor-g (Pparg) Exactly how ER-localized SEIPIN may regulate adipo- knockout (FKO-g) mice. Rosiglitazone treatment signifi- genesis remains an open question. The differentiation of cantly improved a number of metabolic parameters of the preadipocytes requires a transcriptional cascade that ulti- ASKO mice, including insulin sensitivity. Our results there- mately leads to the activation of the master regulator of fore demonstrate a critical role of SEIPIN in maintaining terminal adipogenesis: peroxisome proliferator–activated lipid homeostasis and function of adipocytes and reveal receptor-g (Pparg), which, together with its coactivators, an intimate relationship between SEIPIN and PPAR-g. stimulates the expression of a large number of gene products, including those that promote lipogenesis and glucose Congenital generalized lipodystrophy (CGL), also known transport (13–15). White adipose tissue (WAT) and brown as Berardinelli-Seip congenital lipodystrophy (BSCL), is an adipose tissue (BAT) are completely lost in a mouse model

1Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovas- 6Department of Physiology and Pathophysiology, School of Basic Medical Scien- cular Sciences, Ministry of Education, Peking University Health Science Center, ces, Peking University Health Science Center, Beijing, China Beijing, China Corresponding authors: George Liu, [email protected]; Dong Zhao, 2 Department of Endocrinology, Lu He Teaching Hospital of the Capital Medical [email protected]; and Hongyuan Yang, [email protected]. University, Beijing, China Received 7 May 2013 and accepted 9 March 2014. 3School of Biotechnology and Biomolecular Sciences, the University of New South Wales, Sydney, NSW, Australia This article contains Supplementary Data online at http://diabetes 4State Key Laboratory of Molecular Developmental Biology, Institute of Genetics .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0729/-/DC1. and Developmental Biology, Chinese Academy of Sciences, Beijing, China L.L., Q.J., and X.W. contributed equally to this work. 5MOE Key Laboratory of Bioinformatics and Tsinghua-Peking Center for Life © 2014 by the American Diabetes Association. See http://creativecommons.org Sciences, School of Life Sciences, Tsinghua University, Beijing, China /licenses/by-nc-nd/3.0/ for details. diabetes.diabetesjournals.org Liu and Associates 2321 lacking Pparg, and mutations in PPARg in the human pop- Glucose and Insulin Tolerance Tests ulation are associated with fat loss (14). It has been proposed Mice were fasted overnight for 16 or 4 h, respectively, that the absence of SEIPIN may lead to the accumulation of followed by intraperitoneal injection of glucose (2 g/kg certain phospholipid species, such as phosphatidic acid (PA), body weight) or insulin (0.75 mIU/g body weight; Humulin). which may serve as strong PPAR-g antagonists, thereby caus- Blood samples were collected before (time 0) and at 15, ing lipodystrophy (3). 30, 60, and 120 (90 for insulin tolerance test) min after Similar to PPARg, SEIPIN is highly expressed in adi- injection for glucose measurement. pose tissue, but its expression is low in liver and barely detectable in muscle. The expression of Seipin is dramat- Histological Studies ically increased at later stages of the differentiation of Liver was cryostat sectioned at a thickness of 7 mmfor fi 3T3-L1 cells (16,17). However, little is known about the Oil Red O staining. Paraf n-embedded WAT and BAT in vivo function of SEIPIN in mature adipocytes. Here, were sectioned at a thickness of 2 mm and stained we used the Cre-loxP system to generate adipose-specific with hematoxylin and eosin (H&E) or Sirius Red for fi Seipin KO (ASKO) mice, and our results reveal striking brosis analysis. Adipocyte area was measured using n n phenotypic similarities between the ASKO mice and the ImageJ software ( =200adipocytesperanimal, =5 fat PPAR-g–deficient (FKO-g) mice (18): both show severe animals per group). Immunodetections were performed adipocyte hypertrophy, progressive lipodystrophy, insulin with Mac2 antibody (Santa Cruz Biotechnology, Dallas, fi resistance, and fatty liver. Our results demonstrate that TX) to examine macrophage in ltration. TUNEL assay SEIPIN is required not only for the differentiation of wasdoneasdescribed(19). preadipocytes but also for the maintenance of lipid ho- Lipolysis meostasis and long-term survival of mature adipocytes. For in vivo lipolysis, mice were fasted for 4 h and given an – fi intraperitoneal injection of the b3-adrenergic speci c ag- RESEARCH DESIGN AND METHODS onist CL-316,243 (0.1 mg/kg, Sigma-Aldrich). Blood was All experiments involving mice were approved by the collected before and 15 min after injection for determi- Institutional Animal Care Research Advisory Committee nation of NEFA and glycerol levels. For ex vivo lipolysis, of Peking University Health Science Center. The Principles epididymal fat was removed, cut into 10-mg fat pads, and of Laboratory Animal Care (NIH Publication 85-23, stimulated with or without 1 mmol/L isoproterenol revised 1996) were followed. (Sigma-Aldrich) as described by Chen et al. (7). The me- dium was collected for determination of glycerol levels. Animals Intracellular cAMP concentrations were measured by im- Seipinfl/fl Homozygous mice were obtained as described munoassay (Enzo Life Sciences, Farmingdale, NY). (6). Adipose-specific deletion of Seipin exon 3 was induced fl fl by crossing Seipin / mice to transgenic mice expressing RNA Isolation and Quantitative Real-Time PCR Cre recombinase driven by an aP2 promoter (18). The Total tissue RNA was extracted using Trizol reagent genotyping was examined by PCR using the following (Invitrogen, Carlsbad, CA), and first-strand cDNA was primers: for the Cre transgene: 59-GCGGTCTGGCAGTAAA generated with a RT kit (Invitrogen). Quantitative real- AACTATC-39 and 59-GTGAAACAGCATTGCTGTCACTT-39; time PCR was performed using primers listed in Supple- for the upstream loxP site: 59-CTTGTCTCAAAGGGGTCT- mentary Table 1. All samples were quantitated by the 9 9 9 3 and 5 -TCAACAGAACAGACGCT-3 .Miceusedinmost comparative CT method for relative quantitation, normal- studies were maintained on a mixed genetic background of ized to Gapdh. 129 and C57BL/6. Mice for high-fat diet (HFD) treatment were from a C57BL/6 background after five generations of Western Blot Analysis backcrossing. The HFD (40% kilocalories from fat) was fed Mouse tissue was homogenized in radioimmunoprecipi- to 6-week-old mice for 6 weeks. For rosiglitazone (Rosi) tation assay buffer, and the protein content was deter- treatment, a chow diet containing Rosi (0.3 mg/g diet; mined using a bicinchoninic acid protein assay kit (Pierce, Sigma-Aldrich, St. Louis, MO) was fed to 6-month-old Rockford, IL). The following antibodies were used: Seipin mice for 10 weeks. (Abnova, Taipei, Taiwan); Akt, phospho-Akt (Ser473), hormone-sensitive lipase (HSL), phospho-HSL (Ser563), Blood Analysis adipocyte triglyceride lipase (ATGL), and phospho– Blood was obtained by retro-orbital bleed. Plasma choles- protein kinase-A (PKA) substrate (Cell Signaling Tech- terol, triacylglycerols (TAG), and glucose were determined nology, Beverly, MA); FSP27 (a gift from Prof. Peng Li); using enzymatic methods (Sigma-Aldrich kits). Plasma ADRP (Santa Cruz Biotechnology); perilipin A (Abcam, insulin, leptin, and adiponectin were measured by ELISA Cambridge, U.K.); and GAPDH (Millipore, Billerica, (Linco Research, St. Charles, MO). Free glycerol content MA). The protein bands were analyzed using densitom- (GPO-Trinder kit, Sigma-Aldrich) and the level of non- etry and ImageJ image analysis software. Arbitrary den- esterified fatty acids (NEFA) were measured by a colori- sitometry units were quantifiedandareexpressedas metric assay (Wako Chemical, Osaka, Japan). mean 6 SEM. 2322 SEIPIN and Adipocyte Maintenance Diabetes Volume 63, July 2014

Analysis of Liver Lipids under the control of the adipose-specific Fabp4/aP2 gene fl Liver (;100 mg wet weight) was weighed and homoge- promoter (aP2-CreTg/0) (18). The resulting Seipin /+aP2- fl fl nized in 1 mL PBS. Lipids were extracted as described by CreTg/0 progeny were then crossed with Seipin / mice to Folch et al. (20) and dissolved in 1 mL 3% Triton X-100. generate the ASKO mice. Litter mates lacking the Cre gene fl fl The determination of TAGs was done using enzymatic (Seipin / ) were used as controls and are referred to as methods, as described earlier. wild type (WT). Because aP2 is expressed only at late Lipid Extraction and Lipidomic Analysis stages of adipocyte differentiation, this strategy is Seipin Epididymal (Epi)-WAT (;100 mg) or BAT (50 mg) was expected to delete after formation of fat depots, weighed and homogenized in 1 mL PBS. Lipids were allowing normal differentiation of adipocytes. As revealed Seipin extracted by adding methanol/chloroform (1:2), and by real-time PCR, expression was almost completely lipidomic analysis was done as described (21). lost in adipose tissue (the residual expression is likely from nonadipocytes), but not in liver, kidney, heart, and Microarray Analysis skeletal muscle (Fig. 1A). Seipin expression was greatly Total RNA (100 ng) from fat was labeled and hybrid- diminished in the Epi-WAT and BAT of 3-, 6- and 10- ized onto Affymetrix GeneChip Mouse 430 2.0 arrays month-old ASKO mice (Fig. 1B and C). Seipin is not highly n n ( = 4 controls and = 4 KO, respectively) according to expressed in macrophages, and Seipin expression in intra- ’ the manufacturer s instructions. Data were analyzed as peritoneal macrophages from the ASKO mice appeared described (22). unchanged (Fig. 1A). Therefore, the metabolic defects of Statistical Analysis the ASKO mice (below) most likely result from SEIPIN All data are presented as means 6 SEM. Statistical com- loss in adipocytes. parison between groups was performed using the Student t test or two-way ANOVA. A value of P , 0.05 was con- Progressive Lipodystrophy in ASKO Mice sidered statistically significant. ASKO mice fed with a chow diet showed significant and progressive total WAT loss: ;25% loss at 3 months old, RESULTS ;50% at 6 months, and ;75% at 10 months (Fig. 2A). Generation of the ASKO Mice Notably, the loss of WAT at different fat depots pro- To examine the role of SEIPIN in mature adipocytes, we gressed at different rates (Supplementary Fig. 1A). Histo- used the Cre/loxP system to generate mice in which Seipin logical analyses showed that Epi-WAT from WT mice is specifically deleted from the adipose tissue. Homozy- contained normal mature adipocytes, which were charac- fl fl gous Seipin / mice were obtained as described (6) and terized by the presence of a unilocular LD. In contrast, crossed with transgenic mice expressing Cre recombinase adipocytes from 3-month-old ASKO mice showed signs of

Figure 1—Generation of ASKO mice. A: Detection of Seipin expression in various tissues of 6-month-old WT and ASKO mice by quantitative real-time PCR (top). The PCR products after 35 cycles are also resolved by agarose gel electrophoresis (bottom). M, month; SkM, skeletal muscle (soleus). Detection of Seipin mRNA (top) and protein (bottom) expression in Epi-WAT (B) and BAT (C) of 3-, 6- and 10-month-old WT and ASKO mice and 3-month-old WT and Seipin global KO (SKO) mice. Values are fold induction of gene expression normalized to the housekeeping gene Gapdh and expressed as mean 6 SEM, n =6.*P < 0.05, **P < 0.01, ***P < 0.001 for ASKO vs. WT. diabetes.diabetesjournals.org Liu and Associates 2323

Figure 2—Seipin ablation in adipose tissue leads to progressive lipodystrophy. A: Weight changes of total fat (left) and gross morphology of Epi-WAT (arrows)/testis (right) from 3-, 6-, and 10-month-old WT and ASKO mice. Fat weight is normalized to body weight (BW). M, month. Values are the mean 6 SEM (n = 9). ***P < 0.001 for ASKO vs. WT. B: H&E staining of Epi-WAT from 3-, 6-, and 10-month-old WT and ASKO mice. Scale bar is 50 mm. C: Measurements of adipocyte area of Epi-WAT from 3-, 6-, and 10-month-old WT and ASKO mice. The quantiﬁcation is performed using ImageJ software. Each plot represents a distribution of an individual adipocyte population according to size (area). Each distribution is obtained from ﬁve mice in each group and at least 200 adipocytes in each mouse. D: Weight changes (top) and gross morphology (bottom) of BAT from 3-, 6-, and 10-month-old WT and ASKO mice. The weight is normalized to body weight. Values are the mean 6 SEM, n = 9. ***P < 0.001 for ASKO vs. WT. E: H&E staining of BAT from 3-, 6-, and 10-month-old WT and ASKO mice. Scale bar is 50 mm.

hypertrophy, and adipocytes from 6-month-old ASKO C/ebpa, and Acc were downregulated in 6-month-old mice. mice were highly hypertrophic (Fig. 2B and C). The adi- In 10-month-old ASKO mice, multiple genes were down- pocytes in 10-month-old ASKO mice were vastly variable regulated dramatically. The expression of adipocytokines in size, displaying very large unilocular vacuoles (LDs) or (adiponectin and resistin) was also downregulated in Epi- very small adipocytes containing brightly eosinophilic cy- WAT of ASKO mice, but Leptin mRNA did not change toplasm. The subcutaneous fat of the ASKO mice showed signiﬁcantly (Supplementary Fig. 2B). similar changes (Supplementary Fig. 1B). BAT mass also decreased progressively, although not as We next examined the effect of Seipin deletion on the severely as WAT. There was no diminution of the expression of adipocyte genes (Supplementary Fig. 2A). interscapular fat pads in 3-month-old ASKO mice. How- For Epi-WAT of ASKO mice, the expression of genes in- ever, BAT mass in 6- and 10-month-old ASKO mice volved in lipogenesis, fatty acid uptake, and storage did decreased by ;40% and ;50%, respectively (Fig. 2D). not change in 3-month-old mice, and only Pparg, Fabp4, Strikingly, brown adipocytes from 3- and 6-month-old 2324 SEIPIN and Adipocyte Maintenance Diabetes Volume 63, July 2014

ASKO mice displayed giant, white adipocyte–like droplets Lipodystrophy often leads to insulin resistance and (Fig. 2E). In 10-month-old ASKO mice, BAT adipocytes glucose intolerance in humans and mice. The glucose were replaced by a coagulum of amorphous eosinophilic tolerance test revealed delayed glucose clearance in 6- and material and cytoplasmic debris, implying necrosis (Fig. 10-month-old ASKO mice and also dramatically increased 2E). Despite these morphological changes, the expression of insulin levels during glucose infusion (Fig. 3A and B). An BAT-specific Ucp1 was unchanged in 3- and 6-month-old insulin tolerance test showed that 10-month-old ASKO animals (Supplementary Fig. 2C). Nevertheless, the ASKO mice had impaired insulin sensitivity (Fig. 3C). To further mice were cold-sensitive (Supplementary Fig. 2D). assess tissue-specific sites of insulin resistance, we examined the expression of Akt, Glut4, insulin-receptor Metabolic Characterization of the ASKO Mice substrate 1 (Irs1), and Irs2 in WAT and liver. For 10- Metabolic parameters of ASKO mice were examined in fed month-old ASKO mice, the expression of all four genes and fasting states (Supplementary Table 2). When fed, was markedly decreased in Epi-WAT (Supplementary Fig. ASKO mice showed significantly increased plasma TAG 3A), whereas only the expression of Irs2 was decreased in and NEFA and decreased adiponectin. Leptin was signif- liver (Supplementary Fig. 3B). To determine whether insu- icantly decreased, whereas insulin increased only at 10 lin signaling was impaired in WAT and liver of ASKO mice, months. Short-term fasting caused little change, but fast- we detected AKT phosphorylation (S473). As expected, the ing for 16 h led to significant decreases in plasma TAG in ratio of phospho-AKT to total AKT was markedly reduced old ASKO mice. Plasma glucose increased in all three age in WAT of 6- and 10-month-old ASKO mice (Fig. 3D), and groups upon a 16-h fast. in the liver (Fig. 3E) of 10-month-old ASKO mice.

Figure 3—Insulin resistance and hepatic steatosis in ASKO mice. Glucose tolerance (A) and plasma insulin concentrations (B) during the glucose tolerance tests (GTT) and insulin tolerance tests (ITT) (C) in 3-, 6-, and 10-month-old WT and ASKO mice. M, month. Values are the mean 6 SEM (n = 6). Representative Western blot images (left) for the indicated proteins in Epi-WAT (D) and liver (E) and the quantiﬁcation (right) by densitometry of phosphorylated AKT (P-AKT) normalized to total AKT for 3-, 6-, and 10-month-old WT and ASKO mice. Values are the mean 6 SEM, n =4.F: Weight changes of the liver in 3-, 6-, and 10-month-old WT and ASKO mice. Liver weight is normalized to body weight. Values are the mean 6 SEM (n = 9). G: Oil Red O staining of the liver from 3-, 6-, and 10-month-old WT and ASKO mice. Nuclei are counterstained with hematoxylin. The red-colored droplets represent the LD. Scale bar is 50 mm. H: Liver TAG contents from 3-, 6-, and 10- month-old WT and ASKO mice. TAG content is normalized to liver weight. Values are the mean 6 SEM, n =4–5. *P < 0.05, **P < 0.01, ***P < 0.001 for ASKO vs. WT. diabetes.diabetesjournals.org Liu and Associates 2325

Lipodystrophy is often accompanied by fatty liver. ASKO-B6. When fed a chow diet, these mice showed Liver weight, gross morphology, and histology from similar metabolic properties as the ASKO mice, in- 3-month-old ASKO mice were similar to those of WT mice. cluding lipodystrophy (Fig. 4A). When fed the HFD for With aging, liver weight progressively increased (Fig. 3F). 6 weeks, WT-B6 mice gained ;20% body weight (Fig. Oil Red O staining of cryosections showed the liver of 4B)and;100% total fat weight (Fig. 4C). In contrast, 6-month-old ASKO mice contained more LDs than WT ASKO-B6micegainedlittlefatpadandbodyweight, mice, and the liver of 10-month-old ASKO mice showed except the gonadal fat (Fig. 4B–D). Total plasma cho- significant steatosis (Fig. 3G). Consistent with the histo- lesterol, glucose, and especially insulin levels were sig- logical observations, the amount of liver TAG of 6- and nificantly higher in ASKO-B6 mice after fasting for 4 h 10-month-old ASKO mice was 20% and 50% higher than (Fig. 4E and F). Fatty liver is apparent in the KO but not that of WT mice (Fig. 3H). The expression of Fas, Scd1, theWTmiceaftertheHFD(Fig.4G and H). These and Pparg was significantly increased in liver of 6- results suggest that although ASKO-B6 mice are resis- and 10-month-old ASKO mice (Supplementary Fig. 3C tant to diet-induced obesity, they appear to be more and D), although SREBP-1c mRNA and protein levels susceptible to HFD-induced insulin resistance and appeared unchanged (Supplementary Fig. 3C and E). No- fatty liver. tably, no obvious changes in fat metabolism and insulin Macrophage Infiltration and Inflammation in Adipose signaling were detected in ASKO muscle (Supplementary Tissue of ASKO Mice F G Fig. 3 and ). An early and striking change of the ASKO mice is the ASKO Mice Are Resistant to Diet-Induced Obesity but enlargement of LDs and adipocyte hypertrophy (Fig. 2B). Susceptible to HFD-Induced Insulin Resistance Adipocyte hypertrophy may promote adipocyte death, To test the effects of HFD on ASKO mice, ASKO mice of macrophage infiltration, and chronic inflammation mixedbackgroundwerebackcrossedwithC57BL/6for (22). Indeed, there is more apoptotic cell death in five generations, and the resulting mice were named ASKO adipose tissue (Supplementary Fig. 4A). H&E

Figure 4—Effects of HFD on ASKO-B6 mice. A: Fat pad weight of 6-month-old WT-B6 and ASKO-B6 mice. Fat weight is normalized to body weight (BW). B: Body weight of WT-B6 and ASKO-B6 mice after 6 weeks of normal diet (ND) or HFD from 6 weeks old. C: Total fat weight of WT-B6 and ASKO-B6 mice fed with the ND or HFD. D: Fat pad weight of WT-B6 and ASKO-B6 mice fed with the ND or HFD. E: Plasma total cholesterol (TC), TAG, and glucose (GLU) content in mice fasted for 4 h. F: Plasma insulin content in mice fasted for 4 h. G: Oil Red O staining of livers from WT-B6 and ASKO-B6 mice fed with the ND or HFD. Nuclei are counterstained with hematoxylin. The red- colored droplets represent the LD. Scale bar is 50 mm. H: TAG content of livers from WT-B6 and ASKO-B6 mice fed with the ND or HFD. TAG content is normalized to liver weight. Values are the mean 6 SEM, n =5–7. *P < 0.05, **P < 0.01, ***P < 0.001 for HFD vs. ND. #P < 0.05, ##P < 0.01, ###P < 0.001 for ASKO-B6 vs. WT-B6. 2326 SEIPIN and Adipocyte Maintenance Diabetes Volume 63, July 2014 staining of WAT and BAT had suggested infiltration of Impaired Lipolysis in ASKO Mice inflammatory cells in 6- and 10-month-old ASKO mice Because impaired lipolysis can contribute to the enlarge- (Fig. 2B and E). Mac2-stained macrophages were almost ment of LDs and adipocyte hypertrophy (22), we exam- absent in the Epi-WAT of WT and 3-month-old ASKO ined lipolysis in WT and ASKO mice. In vivo lipolysis in mice but prominent in 6-month-old ASKO mice and ASKO mice was evaluated by measuring plasma NEFA and abundant in 10-month-old ASKO mice (Fig. 5A). Macro- glycerol before and after administering the b3-adrenergic phages occurred individually or surrounding dead adipo- agonist CL-316,243. Baseline NEFA and glycerol levels fi cytes to form crown-like structures (arrows in Fig. 5A). were not signi cantly different between WT and ASKO The number of infiltrating macrophages in the BAT of mice. After 15 min of CL-316,243 treatment, WT mice showed a normal increase in glycerol (;twofold) and ASKO mice also increased dramatically with age (Fig. NEFA (;1.7-fold) levels, indicative of increased lipolysis, 5B). Consistent with the histological observations, whereas little change was observed in 3- and 6-month-old Mac2 expression was increased in Epi-WAT and BAT of ASKO mice (Fig. 6A). Isoproterenol-stimulated glycerol 6- and 10-month-old ASKO mice (Fig. 5C and D). The release was also markedly diminished in fat explants expression F4/80,anothermarkerofmacrophages,was from ASKO mice compared with WT mice, although the also elevated in Epi-WAT and BAT of 10-month-old basal levels of glycerol were almost the same (Fig. 6B). The fl – ASKO mice. A subset of proin ammatory M1 macrophage level of cAMP was also reduced in ASKO mice after iso- Mcp1 Tnf fi associated genes ( and a)wassigni cantly upreg- proterenol treatment (Fig. 6C). Lipolytic rates are tightly ulated in the BAT of 6-month-old ASKO mice, and the regulated by PKA-mediated phosphorylation of HSL and – M1- and the prorepair M2 macrophage associated genes PERILIPIN1, and the interplay among PERILIPIN1, were both upregulated in WAT and BAT of 10-month- CGI58, HSL, and ATGL (23,24). Under basal conditions, old ASKO mice (Fig. 5C and D). These findings suggest ASKO mice exhibited reduced ATGL expression and HSL that macrophages (especially M1) were increased in phosphorylation (Fig. 6D). Isoproterenol-induced phos- older ASKO mice, reflecting chronic inflammation. Fi- phorylation of HSL was attenuated in fat explants of nally, fibrosis was evident in the adipose tissue from ASKO mice compared with WT mice (Fig. 6D). We further 6- and 10-month-old ASKO mice (Supplementary Fig. detected pan–phospho-Ser/Thr PKA substrates and found 4B and C). that isoproterenol-stimulated phosphorylation of PKA

Figure 5—Increased adipose tissue inﬂammation in ASKO mice. Mac2 immunostaining in Epi-WAT (A) and BAT (B) from 3-, 6-, and 10-month-old WT and ASKO mice, with arrows indicating crown-like structures. M, month. Scale bar is 50 mm. Relative mRNA levels of macrophage markers and inﬂammatory cytokines in Epi-WAT (n =5)(C) and BAT (n =7)(D) from 6- and 10-month-old WT and ASKO mice. IL, interleukin; TGF, transforming growth factor. Values are expressed as mean 6 SEM. *P < 0.05, **P < 0.01 for ASKO vs. WT. diabetes.diabetesjournals.org Liu and Associates 2327

Figure 6—Impaired lipolysis in ASKO mice. A: Circulating levels of glycerol (left) and NEFA (right) response to the b3-adenoreceptor agonist CL-316,243 (CL) (0.1 mg/kg intraperitoneally [i.p.]) in 3- and 6-month-old WT and ASKO mice. Values are expressed as mean 6 SEM, n =6 for 3-month-old mice and n = 4 for 6-month-old mice. Basal and isoproterenol (Iso)-stimulated glycerol release (B) and intracellular cAMP content (C) in Epi-WAT explants from 3-month-old WT and ASKO mice. Values are expressed as mean 6 SEM, n =4.D: Representative Western blot images for the indicated proteins and phosphoproteins in basal and Iso-stimulated Epi-WAT explants from 3-month-old WT and ASKO mice. E: Circulating levels of glycerol response to b3-adenoreceptor agonist CL-316,243 (0.1 mg/kg, i.p.) in 10-month-old WT and ASKO mice. Values are expressed as mean 6 SEM, n =4.*P < 0.05, **P < 0.01, ***P < 0.001 for ASKO vs. WT.

substrates was broadly reduced in ASKO mice compared were also consistent with those from lipodystrophy patients with WT mice (Fig. 6D). Lipolysis in 10-month-old ASKO with TZDs treatment (26). The expression of PPAR-g and its mice was similarly reduced as in 3-month-old mice (Fig. target genes increased in most of the examined genes in 6E). Moreover, reduced lipolysis was also detected in both genotypes after Rosi treatment (Fig. 7D). BAT also in- ASKO-B6 mice (Supplementary Fig. 4D and E). creased in mass (Fig. 7E). As a result of expanded fat storage capacity after Rosi treatment, plasma TAG and NEFA were Effects of Thiazolidinedione Treatment of ASKO Mice signiﬁcantly reduced in WT and ASKO mice (Fig. 7F). Im- To investigate the functional relationship between SEIPIN portantly, Rosi improved glucose tolerance and insulin sen- and PPAR-g, we determined the effects of PPAR-g ago- sitivity in ASKO mice (Fig. 7G and H), resulting in markedly nists, the thiazolidinediones (TZDs) (14). WT and ASKO decreased plasma fasting glucose and insulin level (Fig. 7F mice (6 months old) were treated with or without Rosi for and I). Plasma adiponectin and leptin were increased in re- 10 weeks. For WT mice, Rosi treatment resulted in an in- sponse to Rosi administration in both genotypes (Fig. 7J). crease in body weight as well as in WAT mass (Fig. 7A). Histologically, the Rosi-treated livers showed decreased However, ASKO mice did not show such an increase in body lipid deposition in ASKO mice (Supplementary Fig. 5A). weight or in total WAT mass even though adipose mass in Liver mass (Supplementary Fig. 5B) and TAG (Supplemen- the subcutaneous and inguinal areas was signiﬁcantly in- tary Fig. 5C) were also decreased in ASKO mice after Rosi creased (Fig. 7B). Many small and newly differentiated administration. The expression of most transcription fac- adipocytes appeared in subcutaneous WAT after Rosi treat- tors and metabolic enzymes involved in lipid synthesis ment (arrows in Fig. 7C). Studies have reported differences (Supplementary Fig. 5D), b-oxidation (Supplementary between subcutaneous and visceral adipose tissues in func- Fig. 5E), and glucose homeostasis (Supplementary Fig. tion as well as in sensitivity to TZDs (25). These results 5F) were upregulated after Rosi treatment in the liver. 2328 SEIPIN and Adipocyte Maintenance Diabetes Volume 63, July 2014

Figure 7—Effects of Rosi treatment on ASKO mice. Rosi (0.03%) or vehicle was administered to 6-month-old male WT and ASKO mice for 10 weeks. A: Body weight curve (left) and total fat weight (right) of WT and ASKO mice with or without Rosi. B: Fat pad weight of WT and ASKO mice with or without Rosi. C: H&E staining of subcutaneous WAT from WT and ASKO mice with or without Rosi. Arrows indicate putative newly formed adipocytes. Scale bar is 50 mm. D: Expression of PPAR-g and its target genes in Epi-WAT. E: Weight changes of BAT in WT and ASKO mice with or without Rosi. F: Plasma total cholesterol (TC), TAG, glucose (GLU) (left), and NEFA (right) in mice fasted for 4 h. Plasma glucose concentrations for glucose tolerance tests (GTT) (G) and insulin tolerance tests (ITT) (H) in mice fasted for 16 h and 4 h, respectively. Area under curve (AUC) is also quantiﬁed. Plasma levels of insulin (I), leptin, and adiponectin (J) in mice fasted for 4 h. Values are the mean 6 SEM, n =5–8. *P < 0.05, **P < 0.01, ***P < 0.001 for Rosi diet vs. normal diet (ND). #P < 0.05, ##P < 0.01, ###P < 0.001 for ASKO vs. WT. diabetes.diabetesjournals.org Liu and Associates 2329

SEIPIN and Adipocyte Lipid Homeostasis were significantly enriched from pathway ANOVA analysis A key finding of this work is the progressive loss of (P , 8.22E-03) using quantitative real-time PCR (Fig. 8A). mature adipocytes with aging when SEIPIN function is Moreover, from pathway ANOVA analysis (Supplementary compromised in adipocytes. We performed microarray Table 5), the following metabolic pathways are prominently (Supplementary Table 3) and lipidomic analyses to inves- implicated: linoleic acid, sphingolipid, glycerolipid, and fatty tigate the molecular basis for the observed fat loss in acid elongation. Importantly, lipidomic analyses revealed ASKO mice. From gene and pathway enrichment analyses, significant changes of TAG, phospholipid, sphigomyelin, we found many significantly enriched pathways belonging andceramidespeciesintheASKOmice(Fig.8),consistent to inflammation (Supplementary Table 4), confirming the with the microarray data. Finally, ER stress was activated upregulation of inflammatory markers in the ASKO adi- in the ASKO mice (Fig. 8I). pose tissue (Fig. 5). Interestingly, sphingolipid metabolism is the first significantly enriched pathway other DISCUSSION than those affecting the immune system. We validated Mutations in SEIPIN are associated with the most severe four genes in the sphingolipid metabolism pathway that form of human fat loss (i.e., CGL2/BSCL2). We and others

Figure 8—Lipid accumulation/imbalance and ER stress in ASKO mice. A: Relative mRNA levels of sphingolipid metabolism pathway genes in Epi-WAT from 6-month-old WT and ASKO mice. Sptlc3, serine palmitoyltransferase, long chain base subunit 3; Sgpp1, sphingosine-1- phosphate phosphatase 1; Acer3, alkaline ceramidase 3; Gla, galactosidase-a. Values are expressed as mean 6 SEM, n = 5. Lipidomic analysis of Epi-WAT (B–E) and BAT (F–H) from 6-month-old WT and ASKO mice using liquid chromatography mass spectroscopy. Different lipid groups (B and F) and different classes of Cer (C, D, G, and H) and TAG (E) are compared. D and H: Sphingolipids are the sum of all ceramide and sphingomyelin species. Values are expressed as mean 6 SEM, n =4.*P < 0.05, **P < 0.01, ***P < 0.001 for ASKO vs. WT. Cer, ceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin. I: Representative Western blot images for the indicated proteins and phosphoproteins related to ER stress in Epi-WAT from 10- month-old WT and ASKO mice. 2330 SEIPIN and Adipocyte Maintenance Diabetes Volume 63, July 2014 have established an essential role of SEIPIN in adipogen- which may cause ER stress and eventual cell death (Fig. esis both in vitro and in vivo (6–8,16,17). However, SEIPIN 8). New adipocytes are continually formed from a preex- is highly expressed in mature adipocytes, where its func- isting stem cell or preadipocyte pool. However, cell loss tion is completely unknown. Here, we report the genera- eventually outpaces replenishment as ASKO mice age, tion of the ASKO mice that lack SEIPIN only in mature causing progressive lipodystrophy (18,30). adipocytes. The ASKO mice exhibit adipocyte hypertrophy, progressive loss of WAT and BAT, insulin resistance, SEIPIN and PPAR-g and hepatic steatosis. Interestingly, to our knowledge, the The similar tissue distribution patterns of SEIPIN and only other adipose-specific KO mice via aP2-Cre that ex- PPAR-g, as well as the fact that SEIPIN and PPAR-g are hibit adipocyte hypertrophy and progressive lipodystro- required for adipogenesis and for the maintenance of adi- phy are the FKO-g mice (18). Our results therefore pocytes, strongly suggest that they are closely connected. fi uncover a critical role of SEIPIN in the maintenance of PPAR-g function appears to be signi cantly impaired in adipose tissue and also reveal an intimate relationship ASKO adipose tissue. Indeed, although the expression of between SEIPIN and PPAR-g. PPAR-g is decreased by ;20% in 6-month-old ASKO adipose tissue, the expression of PPAR-g target genes is almost identical between ASKO mice and FKO-g mice SEIPIN, Adipocyte Hypertrophy, and Progressive Lipodystrophy (Supplementary Fig. 2). SEIPIN is abundantly expressed in adipose tissue, and we How might SEIPIN, an ER resident protein, regulate show here that its absence from mature adipocytes results the activity of PPAR-g, a ligand-activated transcription in adipocyte hypertrophy, progressive fat loss, and factor? Previous results and data in this work suggest associated metabolic disorders. Therefore, SEIPIN is a fundamental role for SEIPIN/Fld1p in lipid metabolism required not only for adipogenesis but also for the normal (3,21). Loss of SEIPIN function can change the quantity function and survival of mature adipocytes. The hyper- and/or distribution of certain lipids, such as PA (21). PAs trophy of adipocytes is likely associated with the role of could inhibit adipocyte differentiation by serving as high- fi SEIPIN in LD expansion. Although aggregates of small af nity PPAR-g antagonists (31). In support of this hy- fi LDs were found in SEIPIN/Fld1-deficient cells (9–11,27), pothesis, Rosi treatment signi cantly improved a number fi the most striking change was the formation of giant/su- of metabolic pro les of the ASKO mice and also rescued Seipin2/2 fi persized LDs (8,10,12). Upregulation of PA, a fusogenic the adipogenic defect in mouse embryonic bro- lipid, is believed to contribute to the formation of super- blasts, as shown recently (Fig. 7) (8). It should also be AGPAT2 LPIN1 sized LDs (21,28), and indeed, PA is increased in ASKO noted that and are key mammalian genes adipose tissue (Fig. 8B and E). Increased lipogenesis and linked to severe generalized lipodystrophy, and genetic reduced lipolysis can also contribute to the formation of ablation of either gene also causes accumulation of PA, – supersized LDs. In the ASKO adipocytes, the level of TAG which could account for the failure in adipogenesis (32 is significantly increased, whereas hormone-stimulated li- 34). Therefore, PA toxicity appears to be a common theme polysis is decreased. A recent study showed unrestrained in a few models of mammalian lipodystrophy. 2/2 fi lipolysis in Seipin mouse embryonic fibroblast cells, In summary, our ndings reveal an essential role of which accounts for the failure of adipogenesis (7). SEIPIN SEIPIN in adipocyte lipid homeostasis and maintenance may differentially regulate lipolysis in preadipocytes and and, therefore, provide important insights into the phys- adipocytes. It should be noted that SEIPIN overexpression iological function of SEIPIN. Understanding the molecular in adipocytes increased lipolysis (29), consistent with our function of SEIPIN may lead to novel therapeutic strategies current finding. Together, these changes (increased PA, against human obesity and insulin resistance. TAG, and reduced lipolysis) may underscore the striking enlargement of LDs in adipocytes: ASKO brown adipo- Acknowledgments. The authors thank Dr. Nigel Turner and members of cytes, which usually contain multiple small LDs, are now the Liu and Yang laboratories for helpful discussions. full of giant LDs (Fig. 2E). Therefore, we hypothesize that Funding. This work is supported in part by Major National Basic Research the increased lipid storage in the form of supersized LDs Program of the People’s Republic of China (2011CB503900 and 2012CB517505) may form the basis of hypertrophic ASKO adipocytes. to G.L., National Natural Science Foundation of the People’s Republic of China The loss of adipose mass in ASKO mice indicates that to G.L. (30930037 and 81121061), Y.W. (30971102), and H.Y. (31228014), a large number of fat cells die with aging. Indeed, adipose and a grant from the National Health and Medical Research Council of Aus- tissue inflammation becomes evident as ASKO mice age tralia (1027387) to H.Y. H.Y. is a Future Fellow of the Australian Research Council. (Fig. 5). The severe hypertrophy of surviving adipocytes, Duality of Interest. No potential conflicts of interest relevant to this article which are known to be susceptible to apoptosis, leads to were reported. further fat loss (22). Our results from lipidomic and Author Contributions. L.L., Q.J., and X.W. generated the bulk of the microarray analyses suggest that SEIPIN may directly reg- results, conceived and designed the experiments, drafted the manuscript, and ulate fatty acid/sphingolipid/phospholipid metabolism in contributed equally to this work. Y.Z. and R.C.Y.L. contributed to research data. adipose tissue. Compromised SEIPIN function leads to S.M.L. and G.S. performed lipidomics analysis. L.Zho. and Y.W. contributed to accumulation of toxic lipid species such as ceramides, discussion. X.C. researched data and provided advice. M.G., L.Zha., and Y.L. diabetes.diabetesjournals.org Liu and Associates 2331

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