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Short Title: SEIPIN and maintenance

Adiposespecific knockout of Seipin/Bscl2 results in progressive lipodystrophy

Lu Liu1,a, Qingqing Jiang1,a, Xuhong Wang2,a, Yuxi Zhang3, Ruby CY Lin3, Sin Man

Lam4, Guanghou Shui4, Linkang Zhou5, Peng Li5, Yuhui Wang1, Xin Cui1, Mingming

Gao1, Ling Zhang1, Ying Lv6, Guoheng Xu6, George Liu1,b, Dong Zhao2,band

Hongyuan Yang3,b

1Institute of Cardiovascular Sciences and Key Laboratory of Molecular

Cardiovascular Sciences, Ministry of Education, Peking University Health Science

Center, Beijing, 100191, China; 2Department of Endocrinology, Lu He Teaching

Hospital of the Capital Medical University, Beijing, 101149, China ; 3School of

Biotechnology and Biomolecular Sciences, the University of New South Wales,

Sydney, NSW, 2052, Australia; 4State Key Laboratory of Molecular Developmental

Biology, Institute of Genetics and Developmental Biology, Chinese Academy of

Sciences, Beijing, 100101,China; 5MOE key laboratory of Bioinformatics and

TsinghuaPeking Center for Life Sciences, School of Life Sciences, Tsinghua

University,Beijing,100084, China; 6Department of Physiology and Pathophysiology,

School of Basic Medical Sciences, Peking University Health Science Center, Beijing,

100191, China.

aL.L., Q. J. and X.W. contributed equally to this work.

1 For Peer Review Only Diabetes Publish Ahead of Print, published online March 12, 2014 Page 3 of 64 Diabetes

bCorresponding authors:

George Liu, Email: [email protected], Tel: 861082802769

Dong Zhao, Email: [email protected], Tel: 861069543901

Hongyuan Yang, Email: [email protected], Tel: 61293858133,

Fax:61293851483

Word count of the main text: 3994

Number of tables: 0

Number of figures: 8

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ABSTRACT:

BerardinelliSeip congenital lipodystrophy type 2 (BSCL2) is the most severe form of human lipodystrophy, characterized by an almost complete loss of and severe resistance. BSCL2 is caused by lossoffunction mutations in the

BSCL2/SEIPIN , which is upregulated during adipogenesis and abundantly expressed in the adipose tissue. The physiological function of SEIPIN in mature , however, remains to be elucidated. Here, we generated adiposespecific

Seipin knockout mice (ASKO mice), which exhibit adipocyte hypertrophy with enlarged lipid droplets, reduced lipolysis, adipose tissue inflammation, progressive loss of both white and , insulin resistance and hepatic steatosis.

Lipidomic and microarray analyses revealed accumulation/imbalance of lipid species including ceramides in ASKO adipose tissue, as well as increased endoplasmic reticulum stress. Interestingly, the ASKO mice almost completely phenocopy the fatspecific Pparγ knockout (FKOγ) mice. Rosiglitazone treatment significantly improved a number of metabolic parameters of the ASKO mice, including insulin sensitivity. Our results therefore demonstrate a critical role of SEIPIN in maintaining lipid homeostasis and function of adipocytes, and reveal an intimate relationship between SEIPIN and PPARγ.

Keywords: seipin, lipid droplets, BerardinelliSeip congenital lipodystrophy type 2,

BSCL2, PPARγ, fatty liver, insulin resistance

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Congenital generalized lipodystrophy (CGL, also known as BerardinelliSeip

congenital lipodystrophy/BSCL), is an autosomal recessive disorder characterized by

a near total loss of adipose tissue, severe insulin resistance and fatty liver (1; 2). To

date, four have been linked to CGL/BSCL, including

1acylglycerol3phosphateOacyl transferase 2 (AGPAT2)/CGL1; SEIPIN/CGL2;

CAVEOLIN/CGL3 and CAVIN/CGL4 (3). The most severe form of human

CGL/BSCL is caused by mutations in SEIPIN/BSCL2, which encodes an integral

membrane of the endoplasmic reticulum (ER) with no recognizable functional

domains (35). We and others have generated Seipin knockout mice (68), which

suffer from severe lipodystrophy and insulin resistance, thereby proving an essential

role of Seipin in adipogenesis in vivo. Interestingly, SEIPIN and its orthologs also

control the expansion of lipid droplets (LDs) and lipogenesis (912). Therefore,

SEIPIN can regulate lipid storage at both systemic (adipogenesis) and cellular (LD

expansion) levels.

Exactly how the ERlocalized SEIPIN may regulate adipogenesis remains an open

question. The differentiation of preadipocytes requires a transcriptional cascade that

ultimately leads to the activation of the master regulator of terminal adipogenesis:

peroxisome proliferatoractivated receptorγ (Pparγ), which, together with its

coactivators, stimulates the expression of a large number of gene products including

those that promote lipogenesis and glucose transport (1315). Both white adipose

tissue (WAT) and brown adipose tissue (BAT) are completely lost in a mouse model

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lacking Pparγ, and mutations in PPARγ in human population are associated with fat loss (14). It has been proposed that the absence of SEIPIN may lead to the accumulation of certain phospholipid species, such as phosphatidic acid (PA), which may serve as strong PPARγ antagonists, thereby causing lipodystrophy (3).

Similar to PPARγ, SEIPIN is highly expressed in adipose tissue, but low in liver and barely detectable in muscle. The expression of Seipin is dramatically increased at later stages of the differentiation of 3T3L1 cells (16; 17). However, little is known about the in vivo function of SEIPIN in mature adipocytes. Here, we used the CreloxP system to generate adiposespecific Seipin knockout (ASKO) mice, and our results reveal striking phenotypic similarities between the ASKO mice and the fat

PPARγdeficient (FKOγ) mice (18): both show severe adipocyte hypertrophy, progressive lipodystrophy, insulin resistance and fatty liver. Our results demonstrate that SEIPIN is required not only for the differentiation of preadipocytes, but also for the maintenance of lipid homeostasis and longterm survival of mature adipocytes.

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Research Design and Methods

Animals. Homozygous Seipinfl/fl mice were obtained as described (6).

Adiposespecific deletion of Seipin exon 3 was induced by crossing Seipinfl/fl mice to

transgenic mice expressing Cre recombinase driven by an aP2 promoter (18). The

genotyping was examined by PCR using the following primers: for the Cre transgene:

5’GCGGTCTGGCAGTAAAAACTATC3’ and 5’

GTGAAACAGCATTGCTGTCACTT3’; for the upstream loxP site:

5’CTTGTCTCAAAGGGGTCT3’ and 5’TCAACAGAACAGACGCT3’. All

experiments involving mice were approved by the Institutional Animal Care Research

Advisory Committee of Peking University Health Science Center. The Principles of

Laboratory Animal Care (NIH publication no. 85–23, revised 1996) were followed.

Mice used in most studies were maintained on a mixed genetic background of 129 and

C57BL/6. Mice for HFD treatment were from a C57BL/6 background after five

generations of backcrossing. HFD (40% kilocalories from fat) was fed to 6weekold

mice for 6 weeks. For rosiglitazone treatment, rosiglitazone (Sigma, St. Louis,

MO)containing chow diet (0.3 mg/g diet) was fed to 6monthold mice for 10 weeks.

Blood analysis. Blood was obtained by retroorbital bleed. Plasma cholesterol,

triacylglycerols and glucose were determined using enzymatic methods (Sigma kits).

Plasma insulin, leptin and adiponectin were measured by ELISA (Linco Research, St

Charles, MO), free glycerol content (GPOTrinder , Sigma) and the level of

nonesterified fatty acids (NEFA) was measured by a colorimetric assay (Wako

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Chemical, Osaka, Japan).

Glucose and insulin tolerance tests. Mice were fasted overnight for 16 h or 4 h, respectively, followed by intraperitoneal injection of glucose (2 g/kg body weight) or insulin (0.75 mIU/g body weight, Humulin). Blood samples were collected before

(time 0) and at 15, 30, 60 and 120 (90 for ITT) min after injection for glucose measurement.

Histological studies. Liver was cryostat sectioned at a thickness of 7 m for Oil red

O staining. Paraffinembedded WAT and BAT were sectioned at a thickness of 2m and stained with hematoxylin and eosin (HE) or sirius red for analysis.

Adipocyte area was measured using Image J software (n=200 adipocytes/animal, n=5 animals/group). Immunodetections were performed with Mac2 antibody (Santa Cruz

Biotechnology, Dallas, Texas) to examine infiltration. TUNEL assay was carried out as described (19).

Lipolysis. For in vivo lipolysis, mice were fasted for 4 h and given an intraperitoneal injection of the β3specific agonist CL316,243 (0.1 mg/kg, Sigma). Blood was collected before and 15 min after injection for determination of NEFA and glycerol levels. For ex vivo lipolysis, epididymal fat was removed, cut into 10 mg fat pads and stimulated with or without 1 M isoproterenol (Sigma) as described by Chen et al.

(20). The medium was collected for determination of glycerol levels. Intracellular

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cAMP concentrations were measured by immunoassay (Enzo Life Sciences,

Farmingdale, NY).

RNA isolation and quantitative realtime PCR. Total tissue RNA were extracted

using Trizol reagent (Invitrogen, Carlsbad, CA) and firststrand cDNA was generated

with a RT kit (Invitrogen). Quantitative realtime PCR was performed using primers

shown in Table S1. All samples were quantitated by comparative CT method for

relative quantitation, normalized to Gapdh.

Western blot analysis. Mouse tissue were homogenized in RIPA buffer and the

protein content was determined using a bicinchoninic acid protein assay kit (Pierce,

Rockford, IL). The following antibodies were used: Seipin (Abnova, Taipei, Taiwan);

Akt, PhosphoAkt (Ser473), HSL, PhosphoHSL(Ser563), ATGL and PhosphoPKA

substrate (Cell Signaling Technology, Beverly, MA); FSP27 (a gift from Prof. Peng

Li); ADRP (Santa Cruz Biotechnology); Perilipin A (Abcam, Cambridge, UK);

GAPDH (Millipore, Billerica, MA). The protein bands were analyzed using

densitometry and Image J image analysis software. Arbitrary densitometry units were

quantified and are expressed as mean ± SEM.

Analysis of liver lipids. Approximately 100 mg of liver (wet weight) was weighed

and homogenized in 1 ml PBS. Lipids were extracted as described by Folch (20) and

dissolved in 1ml 3% Triton X100. The determination of triacylglycerols was carried

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out using enzymatic methods as described earlier.

Lipid extraction and lipidomic analysis. Approximately 100mg EpiWAT or 50mg

BAT was weighed and homogenized in 1 ml PBS. Lipids were extracted by adding methanol/chloroform (1:2), and lipidomic analysis was carried out as described (21).

Microarray analysis

100 ng of total RNA from fat was labelled and hybridised onto AffymetrixGeneChip®

Mouse 430 2.0 arrays (n=4 Controls and n = 4 KO, respectively) according to the manufacturer’s instructions. Data analyses were carried out as described (22).

Statistical analysis. All data are presented as means±SEM. Statistical comparison between groups was performed using Student’s ttest or twoway ANOVA. A value of

P <0.05 was considered statistically significant.

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Results

Generation of the Adiposespecific Seipin Knockout (ASKO) mice.

To examine the role of SEIPIN in mature adipocytes, we utilized the Cre/loxP system

to generate mice in which Seipin is specifically deleted from the adipose tissue.

Homozygous Seipinfl/fl mice were obtained as described (6), and were crossed with

transgenic mice expressing Cre recombinase under the control of the adiposespecific

Fabp4/aP2 gene promoter (aP2CreTg/0) (18). The resulting seipinfl/+aP2CreTg/0

progeny were then crossed with seipinfl/fl mice to generate adiposespecific SEIPIN

knockout mice (ASKO mice). Littermates lacking the Cre gene (seipinfl/fl) were used

as controls, and referred to as wild type (WT). Because aP2 is expressed only at late

stages of adipocyte differentiation, this strategy is expected to delete Seipin after

formation of fat depots, allowing normal differentiation of adipocytes. As revealed by

real timePCR, Seipin expression was almost completely lost in adipose tissue (the

residual expression is likely from nonadipocytes), but not in liver, kidney, heart and

skeletal muscle (Fig. 1A). Seipin expression was greatly diminished in the epididymal

WAT (EpiWAT) and BAT of 3, 6 and 10monthold ASKO mice (Fig. 1B and C).

Seipin is not highly expressed in and Seipin expression in intraperitoneal

macrophages from the ASKO mice appeared unchanged (Fig. 1A). Therefore the

metabolic defects of the ASKO mice (below) most likely result from SEIPIN loss in

adipocytes.

Progressive lipodystrophy in ASKO mice

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On chow diet, ASKO mice showed significant and progressive total WAT loss: ~25% loss at 3 months’ old, ~50% at 6 months and ~75% at 10 months (Fig. 2A). Notably, the loss of WAT at different fat depots progressed at different rates (Fig. S1A).

Histological analyses showed that EpiWAT from WT mice contained normal mature adipocytes, which were characterized by the presence of a unilocular LD. In contrast, adipocytes from 3monthold ASKO mice showed signs of hypertrophy, and adipocytes from 6monthold ASKO mice were highly hypertrophic (Fig. 2B and C).

In 10monthold ASKO mice, the adipocytes were vastly variable in size, displaying either very large unilocular vacuoles (LDs) or very small adipocytes containing brightly eosinophilic cytoplasm. The subcutaneous fat of the ASKO mice showed similar changes (Fig. S1B). We next examined the effect of Seipin deletion on the expression of adipocyte genes (Fig. S2A). For EpiWAT of ASKO mice, the expression of genes involved in lipogenesis, fatty acid uptake and storage did not change in 3monthold mice, and in 6monthold mice, only Pparγ, Fabp4, C/ebpα and Acc were downregulated. In 10monthold ASKO mice multiple genes were downregulated dramatically. The expression of adipocytokines (adiponectin and resistin), was also downregulated in EpiWAT of ASKO mice, but Leptin mRNA did not change significantly (Fig. S2B).

BAT mass also decreased progressively, although not as severe as WAT. There was no diminution of the interscapular fat pads in 3monthold ASKO mice. However, BAT mass in 6 and 10month old ASKO decreased by ~40% and ~50%, respectively (Fig.

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2D). Strikingly, brown adipocytes from 3 and 6monthold ASKO mice displayed

giant, white adipocytelike droplets (Fig. 2E). In 10monthold ASKO mice, BAT

adipocytes were replaced by a coagulum of amorphous eosinophilic material and

cytoplasmic debris, implying necrosis (Fig. 2E). Despite these morphological changes,

the expression of BATspecific Ucp1 was unchanged at 3 and 6monthold animals

(Fig. S2C). Nevertheless, the ASKO mice were coldsensitive (Fig. S2D).

Metabolic characterization of the ASKO mice

Metabolic parameters of ASKO mice were examined in fed and fasting states (Table

S2). When fed, ASKO mice showed significantly increased plasma triacylglycerols

(TAG) and nonesterified fatty acids (NEFA) and decreased adiponectin. Leptin was

significantly decreased whereas insulin increased only at 10 month. Short term fasting

caused little change but fasting for 16 hours led to significant decreases in plasma

TAG in old ASKO mice. Plasma glucose increased in all three age groups upon a

16hour fasting.

Lipodystrophy often leads to insulin resistance and glucose intolerance in both human

and mice. Glucose tolerance test (GTT) revealed delayed glucose clearance in both 6

and 10monthold ASKO mice, and also dramatically increased insulin levels during

glucose infusion (Fig. 3A, 3B). Insulin tolerance test (ITT) showed that 10monthold

ASKO mice had impaired insulin sensitivity (Fig. 3C). To further assess

tissuespecific sites of insulin resistance, we examined the expression of Akt, Glut4,

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insulin receptor substrate 1 (Irs1) and Irs2 in WAT and liver. For 10monthold ASKO mice, the expression of all four genes was markedly decreased in EpiWAT (Fig. S3A), while only the expression of Irs2 was decreased in liver (Fig. S3B). To determine whether insulin signaling was impaired in WAT and liver of ASKO mice, we detected

AKT phosphorylation (S473). As expected, the ratio of phosphoAKT to total AKT was markedly reduced in WAT of 6 and 10monthold ASKO mice (Fig. 3D), and in the liver (Fig. 3E) of 10monthold ASKO mice.

Lipodystrophy is often accompanied by fatty liver. Liver weight, gross morphology and from 3monthold ASKO mice were similar to those of WT mice. With ageing, liver weight progressively increased (Fig. 3F). OilredO staining of cryosections showed the liver of 6monthold ASKO mice contained more LDs than

WT mice, and the liver of 10monthold ASKO mice showed significant steatosis (Fig.

3G). Consistent with the histological observations, the amount of liver TAG of 6 and

10monthold ASKO mice was 20% and 50% higher than that of WT mice (Fig. 3H).

The expression of Fas, Scd1 and Pparγ was significantly increased in 6 and

10monthold ASKO liver (Fig. S3C, D), although SREBP1c mRNA and protein levels appear unchanged (S3C, E). Notably, no obvious changes in fat metabolism and insulin signaling were detected in ASKO muscle (Fig. S3F&G).

ASKO mice are resistant to dietinduced obesity but susceptible to HFDinduced insulin resistance.

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To test the effects of HFD on ASKO mice, ASKO mice of mixed background were

backcrossed with C57BL/6 for 5 generations and the resulting mice were named

ASKOB6. On chow diet, these mice showed similar metabolic properties as the

ASKO mice including lipodystrophy (Fig. 4A). On HFD for 6 weeks, WTB6 mice

gained ~20% body weight (Fig. 4B), and ~100% total fat weight (Fig. 4C). In contrast,

ASKOB6 mice gained little fat pad and body weight except the gonadal fat (Fig.

4BD). Total plasma cholesterol, glucose and especially insulin levels were

significantly higher in ASKOB6 mice upon fasting for 4 hours (Fig. 4E and F). Fatty

liver is apparent in the knockout but not the WT mice after HFD (Fig. 4G and H).

These results suggest that although ASKOB6 mice are resistant to dietinduced

obesity, they appear to be more susceptible to HFDinduced insulin resistance and

fatty liver.

Macrophage infiltration and inflammation in adipose tissue of ASKO mice

An early and striking change of the ASKO mice is the enlargement of LDs and

adipocyte hypertrophy (Fig. 2B). It is known that adipocyte hypertrophy may promote

adipocyte death, macrophage infiltration and chronic inflammation (22). Indeed, there

is more apoptotic cell death in ASKO adipose tissue (Fig. S4A). HE staining of WAT

and BAT had suggested infiltration of inflammatory cells in 6 and 10monthold

ASKO mice (Fig. 2B and E). MAC2stained macrophages were almost absent in

EpiWAT of WT and 3monthold ASKO mice, but prominent in 6monthold ASKO

mice and abundant in 10monthold ASKO mice (Fig. 5A). Macrophages occurred

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individually or surrounding dead adipocytes to form crownlike structures (CLS)

(arrows) (Fig. 5A). The number of infiltrating macrophages in the BAT of ASKO mice also increased dramatically with age (Fig. 5B). Consistent with the histological observations, Mac2 expression was increased in EpiWAT and BAT of 6 and

10monthold ASKO mice (Fig. 5C and D). The expression F4/80, another marker of macrophages, was also elevated in EpiWAT and BAT of 10monthold ASKO mice.

A subset of proinflamatory M1 macrophageassociated genes (Mcp1 and Tnfα) was significantly upregulated in the BAT of 6monthold ASKO mice, and both M1 and the prorepair M2 macrophageassociated genes were upregulated in WAT and BAT of

10monthold ASKO mice (Fig. 5C and D). These findings suggest that macrophages

(especially M1) were increased in older ASKO mice, reflecting chronic inflammation.

Finally, fibrosis was evident in the adipose tissue from 6 and 10monthold ASKO mice (Fig. S4B&C).

Impaired lipolysis in ASKO mice

As impaired lipolysis can contribute to the enlargement of LDs and adipocyte hypertrophy (22), we examined lipolysis in WT and ASKO mice. In vivo lipolysis in

ASKO mice was evaluated by measuring plasma NEFA and glycerol before and after administering β3adrenergic agonist CL316,243. Baseline NEFA and glycerol levels were not significantly different between WT and ASKO mice. After 15 min of

CL316,243 treatment, WT mice showed a normal increase in glycerol (~2fold) and

NEFA (~1.7fold) levels, indicative of increased lipolysis, whereas little change was

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observed in 3 and 6monthold ASKO mice (Fig. 6A). Isoproterenolstimulated

glycerol release was also markedly diminished in fat explants from ASKO mice as

compared with WT mice, although the basal levels of glycerol were almost the same

(Fig. 6B). The level of cAMP was also reduced in ASKO mice after isoproterenol

treatment (Fig. 6C). Lipolytic rates are tightly regulated by proteinkinaseA

(PKA)mediated phosphorylation of hormonesensitivelipase (HSL) and

PERILIPIN1, and the interplay between PERILIPIN1, CGI58, HSL and adipocyte

triglyceride lipase (ATGL) (23; 24). Under basal conditions, ASKO mice exhibited

reduced ATGL expression and HSL phosphorylation (Fig. 6D). Isoproterenolinduced

phosphorylation of HSL was attenuated in fat explants of ASKO mice as compared

with WT mice (Fig. 6D). We further detected panphosphoSer/Thr PKA substrates

and found that isoproterenolstimulated phosphorylation of PKA substrates was

broadly reduced in ASKO mice when compared with WT mice (Fig. 6D). Lipolysis in

10month old ASKO mice was similarly reduced as 3month mice (Fig. 6E).

Moreover, reduced lipolysis was also detected in ASKOB6 mice (Fig. S4D&E).

Effects of TZD treatment of ASKO mice

To investigate the functional relationship between SEIPIN and PPARγ, we determined

the effects of PPARγ agonists, the thiazolidinediones (TZDs) (14). Both WT and

ASKO mice (6 month old) were treated with or without rosiglitazone (Rosi) for 10

weeks. For WT mice, Rosi treatment resulted in an increase in body weight as well as

in WAT mass (Fig. 7A). However, ASKO mice did not show such increase in

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bodyweight nor in total WAT mass even though adipose mass in subcutaneous and inguinal area was significantly increased (Fig. 7B). In subcutaneous WAT, many small and newly differentiated adipocytes (arrows) appeared after Rosi treatment (Fig. 7C).

Past studies have reported differences between subcutaneous and visceral adipose tissues in function as well as in sensitivity to TZDs (25). These results were also consistent with those from lipodystrophy patients with TZDs treatment(26). The expression of PPARγ and its target genes increased in most of the examined genes in both genotypes after Rosi treatment (Fig. 7D). BAT also increased in mass (Fig. 7E).

As a result of expanded fat storage capacity after Rosi treatment, plasma TAG and

NEFA in both WT and ASKO mice were significantly reduced (Fig. 7F). Importantly,

Rosi improved glucose tolerance and insulin sensitivity in ASKO mice (Fig. 7G and

H), resulting in markedly decreased plasma fasting glucose and insulin level (Fig. 7F and I). Plasma adiponectin and leptin were both increased in response to Rosi administration in both genotypes (Fig. 7J).

Histologically, the Rositreated livers showed decreased lipid deposition in ASKO mice (Fig. S5A). Liver mass (Fig. S5B) and TAG (Fig. S5C) were also decreased in

ASKO mice after Rosi administration. The expression of most transcription factors and metabolic enzymes involved in lipid synthesis (Fig. S5D), βoxidation (Fig. S5E) and glucose homeostasis (Fig. S5F) were upregulated after Rosi treatment in the liver.

SEIPIN and adipocyte health

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A key finding of this work is the progressive loss of mature adipocytes with aging

when SEIPIN function is compromised in adipocytes. We carried out microarray

(Table S3) and lipidomic analyses to investigate the molecular basis for the observed

fat loss in ASKO mice. From gene and pathway enrichment analyses, we found many

significantly enriched pathways belonging to inflammation (Table S4), confirming the

upregulation of inflammatory markers in the ASKO adipose tissue (Fig. 5).

Interestingly, sphingolipid metabolism is the first significantly enriched pathway other

than those affecting the immune system. We validated 4 genes in the sphingolipid

metabolism pathway which were significantly enriched from pathway ANOVA

analysis (P < 8.22E03) using qRTPCR (Fig 8A). Moreover, from pathway ANOVA

analysis (Table S5), the following metabolic pathways are prominently implicated:

linoleic acid, sphingolipid, glycerolipid and fatty acid elongation. Importantly,

lipidomic analyses revealed significant changes of TAG, phospholipid, sphigomyelin

and ceramide species in the ASKO mice (Fig.8), consistent with the microarray data.

Finally, ER stress was activated in the ASKO mice (Fig.8I).

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DISCUSSION

Mutations in SEIPIN are associated with the most severe form of human fat loss, i.e.

CGL2/BSCL2. We and others have established an essential role of SEIPIN in adipogenesis both in vitro and in vivo (68; 16; 17). However, SEIPIN is highly expressed in mature adipocytes, where its function is completely unknown. Here, we report the generation of the ASKO mice that lack SEIPIN only in mature adipocytes.

The ASKO mice exhibit adipocyte hypertrophy, progressive loss of both WAT and

BAT, insulin resistance and hepatic steatosis. Interestingly, to our knowledge, the only other adiposespecific knockout mice via aP2Cre that exhibit adipocyte hypertrophy and progressive lipodystrophy are the FKOγ mice (18). Our results therefore uncover a critical role of seipin in the maintenance of adipose tissue, and also reveal an intimate relationship between seipin and PPARγ.

SEIPIN, adipocyte hypertrophy and progressive lipodystrophy

SEIPIN is abundantly expressed in adipose tissue, and we show here that its absence from mature adipocytes results in adipocyte hypertrophy, progressive fat loss and associated metabolic disorders. Therefore, SEIPIN is not only required for adipogenesis, but also for the normal function and survival of mature adipocytes. The hypertrophy of adipocytes is likely associated with the role of SEIPIN in LD expansion. Although aggregates of small LDs were found in SEIPIN/Fld1deficient cells (911; 27), the most striking change was the formation of giant/supersized LDs

(8; 10; 12). Upreguation of phosphatidic acid (PA), a fusogenic lipid, is believed to

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contribute to the formation of supersized LDs (21; 28), and indeed, PA is increased in

ASKO adipose tissue (Fig. 8B, E). Increased lipogenesis and reduced lipolysis can

also contribute to the formation of supersized LDs. In the ASKO adipocytes, the level

of TAG is significantly increased whereas hormonestimulated lipolysis is decreased.

A recent study showed unrestrained lipolysis in Seipin/ MEF cells which accounts for

the failure of adipogenesis (7). SEIPIN may differentially regulate lipolysis in

preadipocytes and adipocytes. It should be noted that SEIPIN overexpression in

adipocytes increased lipolysis (29), consistent with our current finding. Together,

these changes (increased PA, TAG and reduced lipolysis) may underscore the striking

enlargement of LDs in adipocytes: ASKO brown adipocytes, which usually contain

multiple small LDs, are now full of giant LDs (Fig. 2E). Therefore, we hypothesize

that the increased lipid storage in the form of supersized LDs may form the basis of

hypertrophic ASKO adipocytes.

The loss of adipose mass in ASKO mice indicates that a large number of fat cells die

with aging. Indeed, adipose tissue inflammation becomes evident as ASKO mice age

(Fig. 5). The severe hypertrophy of surviving adipocytes, which are known to be

susceptible to apoptosis, leads to further fat loss (22). Our results from lipidomic and

microarray analyses suggest that SEIPIN may directly regulate fatty

acid/sphingolipid/phospholipid metabolism in adipose tissue. Compromised SEIPIN

function leads to accumulation of toxic lipid species such as ceramides, which may

cause ER stress and eventual cell death (Fig 8). New adipocytes are continually

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formed from a preexisting stem cell or preadipocyte pool. However, cell loss eventually outpaces replenishment as ASKO mice age, causing progressive lipodystrophy (18; 30).

SEIPIN and PPARγ

The similar tissue distribution patterns of SEIPIN and PPARγ, as well as the fact that

SEIPIN and PPARγ are required for both adipogenesis and for the maintenance of adipocytes strongly suggest that they are closely connected. PPARγ function appears to be significantly impaired in ASKO adipose tissue. Indeed, although the expression of PPARγ is decreased by ~20% of 6monthold ASKO adipose tissue, the expression of PPARγ target genes is almost identical between ASKO mice and FKOγ mice (Fig.

S2).

How might SEIPIN, an ER resident protein, regulate the activity of PPARγ, a ligandactivated transcription factor? Previous results and data in this work suggest a fundamental role for SEIPIN/Fld1p in lipid metabolism (3; 21). Loss of SEIPIN function can change the quantity and/or distribution of certain lipids, such as PA (21).

PA could inhibit adipocyte differentiation by serving as highaffinity PPARγ antagonists (31). In support of this hypothesis, Rosi treatment significantly improved a number of metabolic profiles of the ASKO mice, and also rescue the adipogenic defect in Seipin/ MEFs as shown recently (Fig. 7)(8). It should also be noted that

AGPAT2 and LPIN1 are key mammalian genes linked to severe generalized

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lipodystrophy, and genetic ablation of either gene also causes accumulation of PA

which could account for the failure in adipogenesis (3234). Therefore, PA toxicity

appears to be a common theme in a few models of mammalian lipodystrophy.

In summary, our findings reveal an essential role of SEIPIN in adipocyte lipid

homeostasis and maintenance, and therefore provide important insights into the

physiological function of SEIPIN. Understanding the molecular function of SEIPIN

may lead to novel therapeutic strategies against human obesity and insulin resistance.

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Acknowledgements This work is supported in part by Major National Basic Research Program of the

People’s Republic of China (No. 2011CB503900 and 2012CB517505) to G. Liu;

National Natural Science Foundation of the People’s Republic of China to G. Liu (No.

30930037 and 81121061), H. Yang (No. 31228014) and Y. Wang (No. 30971102), and a grant from the National Health and Medical Research Council of Australia

(#1027387) to H. Yang.

L.L. Q.J. and X.W. generated the bulk of the results, conceived and designed the experiments, and drafted the manuscript. They contributed equally to this work. Y.Z. and R.C.Y.L. contributed to research data. S.M.L and G.S. performed lipidomics analysis. L.Z. and Y.W. contributed to discussion. X.C. researched data and provided advice. M.G., L.Z. and Y.L. contributed to research data. P.L. and G.X. provided advice, expertise and reagents. G.L., D.Z. and H.Y. designed the experiments, provided advice and reagents, and wrote the manuscript. G.L. and H.Y. are the guarantors of this work and, as such, have full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. H.

Yang is a Future Fellow of the Australian Research Council. We thank members of the

Liu and Yang laboratories for helpful discussions.

All authors declare that there are no competing financial interests in relation to the work described.

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References

1. Agarwal AK, Garg A: Genetic basis of lipodystrophies and management of metabolic complications. Annu Rev Med 2006;57:297-311 2. Magre J, Delepine M, Khallouf E, Gedde-Dahl T, Jr., Van Maldergem L, Sobel E, Papp J, Meier M, Megarbane A, Bachy A, Verloes A, d'Abronzo FH, Seemanova E, Assan R, Baudic N, Bourut C, Czernichow P, Huet F, Grigorescu F, de Kerdanet M, Lacombe D, Labrune P, Lanza M, Loret H, Matsuda F, Navarro J, Nivelon-Chevalier A, Polak M, Robert JJ, Tric P, Tubiana-Rufi N, Vigouroux C, Weissenbach J, Savasta S, Maassen JA, Trygstad O, Bogalho P, Freitas P, Medina JL, Bonnicci F, Joffe BI, Loyson G, Panz VR, Raal FJ, O'Rahilly S, Stephenson T, Kahn CR, Lathrop M, Capeau J: Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on 11q13. Nat Genet 2001;28:365-370 3. Fei W, Du X, Yang H: Seipin, adipogenesis and lipid droplets. Trends Endocrinol Metab 2011;22:204-210 4. Cartwright BR, Goodman JM: Seipin: from human disease to molecular mechanism. J Lipid Res 2012;53:1042-1055 5. Lundin C, Nordstrom R, Wagner K, Windpassinger C, Andersson H, von Heijne G, Nilsson I: Membrane topology of the human seipin protein. FEBS Lett 2006;580:2281-2284 6. Cui X, Wang Y, Tang Y, Liu Y, Zhao L, Deng J, Xu G, Peng X, Ju S, Liu G, Yang H: Seipin ablation in mice results in severe generalized lipodystrophy. Hum Mol Genet 2011;20:3022-3030 7. Chen W, Chang B, Saha P, Hartig SM, Li L, Reddy VT, Yang Y, Yechoor V, Mancini MA, Chan L: Berardinelli-seip congenital lipodystrophy 2/seipin is a cell-autonomous regulator of lipolysis essential for adipocyte differentiation. Mol Cell Biol 2012;32:1099-1111 8. Prieur X, Dollet L, Takahashi M, Nemani M, Pillot B, Le May C, Mounier C, Takigawa-Imamura H, Zelenika D, Matsuda F, Feve B, Capeau J, Lathrop M, Costet P, Cariou B, Magre J: Thiazolidinediones partially reverse the metabolic disturbances observed in Bscl2/seipin-deficient mice. Diabetologia 2013;56:1813-1825 9. Fei W, Li H, Shui G, Kapterian TS, Bielby C, Du X, Brown AJ, Li P, Wenk MR, Liu P, Yang H: Molecular characterization of seipin and its mutants: implications for seipin in triacylglycerol synthesis. J Lipid Res 2011;52:2136-2147 10. Fei W, Shui G, Gaeta B, Du X, Kuerschner L, Li P, Brown AJ, Wenk MR, Parton RG, Yang H: Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast. J Cell Biol 2008;180:473-482 11. Szymanski KM, Binns D, Bartz R, Grishin NV, Li WP, Agarwal AK, Garg A, Anderson RG, Goodman JM: The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology. Proc Natl Acad Sci U S A 2007;104:20890-20895 12. Tian Y, Bi J, Shui G, Liu Z, Xiang Y, Liu Y, Wenk MR, Yang H, Huang X: Tissue-autonomous function of Drosophila seipin in preventing ectopic lipid droplet formation. PLoS Genet 2011;7:e1001364 13. Tontonoz P, Hu E, Spiegelman BM: Stimulation of adipogenesis in by PPAR gamma 2, a lipid-activated transcription factor. Cell 1994;79:1147-1156 14. Tontonoz P, Spiegelman BM: Fat and beyond: the diverse biology of PPARgamma. Annu Rev Biochem 2008;77:289-312 15. Rosen ED, MacDougald OA: Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 2006;7:885-896

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16. Payne VA, Grimsey N, Tuthill A, Virtue S, Gray SL, Dalla Nora E, Semple RK, O'Rahilly S, Rochford JJ: The human lipodystrophy gene BSCL2/seipin may be essential for normal adipocyte differentiation. Diabetes 2008;57:2055-2060 17. Chen W, Yechoor VK, Chang BH, Li MV, March KL, Chan L: The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin plays a key role in adipocyte differentiation. Endocrinology 2009;150:4552-4561 18. He W, Barak Y, Hevener A, Olson P, Liao D, Le J, Nelson M, Ong E, Olefsky JM, Evans RM: Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle. Proc Natl Acad Sci U S A 2003;100:15712-15717 19. Li P, Fan W, Xu J, Lu M, Yamamoto H, Auwerx J, Sears DD, Talukdar S, Oh D, Chen A, Bandyopadhyay G, Scadeng M, Ofrecio JM, Nalbandian S, Olefsky JM: Adipocyte NCoR knockout decreases PPARgamma phosphorylation and enhances PPARgamma activity and insulin sensitivity. Cell 2011;147:815-826 20. Folch J, Lees M, Sloane Stanley GH: A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497-509 21. Fei W, Shui G, Zhang Y, Krahmer N, Ferguson C, Kapterian TS, Lin RC, Dawes IW, Brown AJ, Li P, Huang X, Parton RG, Wenk MR, Walther TC, Yang H: A role for phosphatidic Acid in the formation of "supersized" lipid droplets. PLoS Genet 2011;7:e1002201 22. Ng SF, Lin RC, Laybutt DR, Barres R, Owens JA, Morris MJ: Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Nature 2010;467:963-966 23. Zechner R, Zimmermann R, Eichmann TO, Kohlwein SD, Haemmerle G, Lass A, Madeo F: FAT SIGNALS--lipases and lipolysis in lipid metabolism and signaling. Cell Metab 2012;15:279-291 24. Brasaemle DL: Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet : stabilization of lipid droplets and control of lipolysis. J Lipid Res 2007;48:2547-2559 25. Festuccia WT, Blanchard PG, Turcotte V, Laplante M, Sariahmetoglu M, Brindley DN, Deshaies Y: Depot-specific effects of the PPARgamma agonist rosiglitazone on adipose tissue glucose uptake and metabolism. J Lipid Res 2009;50:1185-1194 26. Arioglu E, Duncan-Morin J, Sebring N, Rother KI, Gottlieb N, Lieberman J, Herion D, Kleiner DE, Reynolds J, Premkumar A, Sumner AE, Hoofnagle J, Reitman ML, Taylor SI: Efficacy and safety of troglitazone in the treatment of lipodystrophy syndromes. Ann Intern Med 2000;133:263-274 27. Boutet E, El Mourabit H, Prot M, Nemani M, Khallouf E, Colard O, Maurice M, Durand-Schneider AM, Chretien Y, Gres S, Wolf C, Saulnier-Blache JS, Capeau J, Magre J: Seipin deficiency alters fatty acid Delta9 desaturation and lipid droplet formation in Berardinelli-Seip congenital lipodystrophy. Biochimie 2009;91:796-803 28. Yang H, Galea A, Sytnyk V, Crossley M: Controlling the size of lipid droplets: lipid and protein factors. Curr Opin Cell Biol 2012;24:509-516 29. Cui X, Wang Y, Meng L, Fei W, Deng J, Xu G, Peng X, Ju S, Zhang L, Liu G, Zhao L, Yang H: Overexpression of a short human seipin/BSCL2 isoform in mouse adipose tissue results in mild lipodystrophy. Am J Physiol Endocrinol Metab 2012;302:E705-713 30. Imai T, Takakuwa R, Marchand S, Dentz E, Bornert JM, Messaddeq N, Wendling O, Mark M, Desvergne B, Wahli W, Chambon P, Metzger D: Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse. Proc Natl Acad Sci U S A 2004;101:4543-4547 31. Stapleton CM, Mashek DG, Wang S, Nagle CA, Cline GW, Thuillier P, Leesnitzer LM, Li LO, Stimmel

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JB, Shulman GI, Coleman RA: activates peroxisome proliferator activated receptor-gamma in CHO cells that over-express glycerol 3-phosphate acyltransferase-1. PLoS One 2011;6:e18932 32. Zhang P, Takeuchi K, Csaki LS, Reue K: Lipin-1 phosphatidic phosphatase activity modulates phosphatidate levels to promote peroxisome proliferator-activated receptor gamma (PPARgamma) during adipogenesis. J Biol Chem 2012;287:3485-3494 33. Nadra K, Medard JJ, Mul JD, Han GS, Gres S, Pende M, Metzger D, Chambon P, Cuppen E, Saulnier-Blache JS, Carman GM, Desvergne B, Chrast R: Cell autonomous lipin 1 function is essential for development and maintenance of white and brown adipose tissue. Mol Cell Biol 2012;32:4794-4810 34. Gale SE, Frolov A, Han X, Bickel PE, Cao L, Bowcock A, Schaffer JE, Ory DS: A regulatory role for 1-acylglycerol-3-phosphate-O-acyltransferase 2 in adipocyte differentiation. J Biol Chem 2006;281:11082-11089

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Figure Legends

Fig. 1. Generation of adipocytespecific Seipin knockout (ASKO) mice.

(A) Detection of Seipin expression in various tissues of 6monthold WT and ASKO mice by qRTPCR (top). The PCR products after 35 cycles are also resolved by agarose gel electrophoresis (bottom). (WAT), brown adipose tissue (BAT), skeletal muscle (soleus, SkM).

(B and C) Detection of Seipin mRNA (top) and protein (bottom) expression in

EpiWAT (B) and BAT (C) of 3, 6 and 10monthold WT and ASKO mice and

3monthold WT and Seipin global knockout mice (SKO). Values are fold induction of gene expression normalized to the housekeeping gene Gapdh and expressed as mean±SEM, n=6, *p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT.

Fig. 2. Seipin ablation in adipose tissue leads to progressive lipodystrophy.

(A) Weight changes of total fat (left) and gross morphology of EpiWAT

(arrows)/testis (right) from 3, 6 and 10monthold WT and ASKO mice. Fat weight is normalized to body weight. Values are the mean±SEM, n=9. ***p<0.001 for ASKO vs. WT.

(B) HE staining of EpiWAT from 3, 6 and 10monthold WT and ASKO mice.

Scale bar is 50 µm.

(C) Measurements of adipocyte area of EpiWAT from 3, 6 and 10monthold 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).

27 For Peer Review Only Page 29 of 64 Diabetes

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

10monthold WT and ASKO mice. The weight is normalized to body weight. Values

are the mean±SEM, n=9. ***p<0.001 for ASKO vs. WT.

(E) HE staining of BAT from 3, 6 and 10monthold WT and ASKO mice. Scale bar

is 50 µm.

Fig. 3. Insulin resistance and hepatic steatosis in ASKO mice.

(A, B and C) Glucose tolerance (A), plasma insulin concentrations (B) during the

glucose tolerance and insulin tolerance (C) tests in 3, 6 and 10monthold WT and

ASKO mice. Values are the mean±SEM, n=6.

(D and E) Representative Western blot images (left) for the indicated proteins in

EpiWAT (C) and liver (D) and the quantification (right) by densitometry of

phosphorylated AKT (PAKT) normalized to total AKT for 3, 6 and 10monthold

WT and ASKO mice. Values are the mean±SEM, n=4.

(F) Weight changes of the liver in 3, 6 and 10monthold WT and ASKO mice. Liver

weight is normalized to body weight. Values are the mean ±SEM, n=9.

(G) Oil red O staining of the liver from 3, 6 and 10monthold WT and ASKO mice.

Nuclei are counterstained with hematoxylin. The red color droplets represent the lipid

droplets. Scale bar is 50 µm.

(H) Liver TAG contents from 3, 6 and 10monthold WT and ASKO mice. TAG

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content is normalized to liver weight. Values are the mean ±SEM, n=45. *p<0.05,

**p<0.01, ***p<0.001 for ASKO vs. WT.

Fig. 4. Effects of high fat diet on ASKOB6 mice.

(A) Fat pad weight of 6monthold WTB6 and ASKOB6 mice. Fat weight is normalized to body weight.

(B) Body weight of WTB6 and ASKOB6 mice after 6 weeks of normal diet (ND) or high fat diet (HFD) from 6 weeks old.

(C) Total fat weight of WTB6 and ASKOB6 mice on ND or HFD.

(D) Fat pad weight of WTB6 and ASKOB6 mice on 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 WTB6 and ASKOB6 mice on ND or HFD.

Nuclei are counterstained with hematoxylin. The red color droplets represent the lipid droplets. Scale bar is 50 µm.

(H) TAG content of livers from WTB6 and ASKOB6 mice on ND or HFD. TAG content is normalized to liver weight. Values are the mean ±SEM, n=57. *p<0.05,

**p<0.01, ***p<0.001 for HFD vs. ND. #p<0.05, ##p<0.01, ###p<0.001 for

ASKOB6 vs. WTB6.

Fig. 5. Increased adipose tissue inflammation in ASKO mice.

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(A and B) Mac2 immunostaining in EpiWAT (A) and BAT (B) from 3, 6 and

10monthold WT and ASKO mice. Arrows: Crownlike structures. Scale bar is 50

µm.

(C and D) Relative mRNA levels of macrophage markers and inflammatory cytokines

in EpiWAT (C) and BAT (D) from 6 and 10monthold WT and ASKO mice.

Values are expressed as mean±SEM, n=5 in (C) and n=7 in (D). *p<0.05, **p<0.01

for ASKO vs. WT.

Fig. 6. Impaired lipolysis in ASKO mice.

(A) Circulating levels of glycerol (left) and NEFA (right) response to β3AR agonist

CL316,243 (0.1 mg/kg, i.p.) in 3and 6monthold WT and ASKO mice. Values are

expressed as mean±SEM, n=6 for 3monthold mice and n=4 for 6monthold mice.

(B and C) Basal and isoproterenol (Iso)stimulated glycerol release (B) and

intracellular cAMP content (C) in EpiWAT explants from 3monthold WT and

ASKO mice. Values are expressed as mean±SEM, n=4.

(D) Representative Western blot images for the indicated proteins and

phosphoproteins in basal and Isostimulated EpiWAT explants from 3monthold WT

and ASKO mice.

(E) Circulating levels of glycerol response to β3AR agonist CL316,243 (0.1 mg/kg,

i.p.) in 10monthold WT and ASKO mice. Values are expressed as mean±SEM, n=4.

*p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT.

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Fig. 7. Effects of rosiglitazone treatment on ASKO mice.

6monthold male WT and ASKO mice were administered rosiglitazone (Rosi, 0.03%) or vehicle 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) HE staining of subcutaneous WAT from WT and ASKO mice with or without

Rosi. Arrows indicate putative newlyformed adipocytes. Scale bar is 50 m.

(D) Expression of PPARγ and its target genes in EpiWAT.

(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.

(G and H) Plasma glucose concentrations for glucose tolerance tests (G) and insulin tolerance tests (H) in mice fasted for 16 h and 4 h, respectively. Area under curve

(AUC) is also quantified.

(I and J) Plasma levels of insulin (I), leptin and adiponectin (J) in mice fasted for 4 h.

Values are the mean±SEM, n=58, *p<0.05, **p<0.01, ***p<0.001 for Rosi diet

(Rosi) vs. normal diet (ND). # p<0.05, ## p<0.01, ### p<0.001 for ASKO vs. WT.

Fig. 8. Lipid accumulation/imbalance and ER Stress in ASKO mice.

(A) Relative mRNA levels of sphingolipid metabolism pathway genes in EpiWAT from 6monthold WT and ASKO mice. Sptlc3: serine palmitoyltransferase, long

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chain base subunit3; Sgpp1: sphingosine1phosphate phosphatase1; Acer3: alkaline

ceramidase3; Gla: galactosidasealpha. Values are expressed as mean±SEM, n=5.

(B, C, D, E, F, G and H) Lipidomic analysis of EpiWAT (B, C, D, E) and BAT ( F, G,

H) from 6monthold WT and ASKO mice using LCMS. Different lipid groups (B, F)

and different classes of Cer (C, D, G, H) and TAG (E) are compared. In D and H,

sphingolipids are the sum of all ceramide and sphingomyelin species. Values are

expressed as mean±SEM, n=4. *p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT.

Cer, ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PE,

phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM,

spingomyelin; TAG, triacylglycerol.

(I) Representative Western blot images for the indicated proteins and phosphoproteins

related to ER stress in EpiWAT from 10monthold WT and ASKO mice.

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Fig. 1. Generation of adipocyte-specific seipin knockout (ASKO) mice. (A) Detection of seipin expression in various tissues of 6-month-old WT and ASKO mice by qRT-PCR (top). The PCR products after 35 cycles are also resolved by agarose gel electrophoresis (bottom). White adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (soleus, SkM). (B and C) 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 knockout mice (SKO). Values are fold induction of gene expression normalized to the housekeeping gene Gapdh and expressed as mean±SEM, n=6, *p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT.

84x40mm (300 x 300 DPI)

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Fig. 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. Values are the mean±SEM, n=9. ***p<0.001 for ASKO vs. WT. (B) HE staining of Epi-WAT from 3-, 6- and 10-month-old WT and ASKO mice. Scale bar is 50 µm. (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±SEM, n=9. ***p<0.001 for ASKO vs. WT. (E) HE staining of BAT from 3-, 6- and 10-month-old WT and ASKO mice. Scale bar is 50 µm. 151x128mm (300 x 300 DPI)

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Fig. 3. Insulin resistance and hepatic steatosis in ASKO mice. (A, B and C) Glucose tolerance (A), plasma insulin concentrations (B) during the glucose tolerance and insulin tolerance (C) tests in 3-, 6- and 10- month-old WT and ASKO mice. Values are the mean±SEM, n=6. (D and E) Representative Western blot images (left) for the indicated proteins in Epi-WAT (C) and liver (D) 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±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 ±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 color droplets represent the lipid droplets. Scale bar is 50 µm.(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 ±SEM, n=4-5. *p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT 117x77mm (300 x 300 DPI)

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Fig. 4. Effects of high fat diet 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.(B)Body weight of WT-B6 and ASKO-B6 mice after 6 weeks of normal diet (ND) or high fat diet (HFD) from 6 weeks old.(C)Total fat weight of WT-B6 and ASKO-B6 mice on ND or HFD.(D)Fat pad weight of WT-B6 and ASKO-B6 mice on 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 on ND or HFD. Nuclei are counterstained with hematoxylin. The red color droplets represent the lipid droplets. Scale bar is 50 µm. (H)TAG content of livers from WT-B6 and ASKO-B6 mice on ND or HFD. TAG content is normalized to liver weight. Values are the mean ±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. 102x59mm (300 x 300 DPI)

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Fig. 5. Increased adipose tissue inflammation in ASKO mice. (A and B) Mac2 immuno-staining in Epi-WAT (A) and BAT (B) from 3-, 6- and 10-month-old WT and ASKO mice. Arrows: Crown-like structures. Scale bar is 50 µm.(C and D) Relative mRNA levels of macrophage markers and inflammatory cytokines in Epi-WAT (C) and BAT (D) from 6- and 10-month-old WT and ASKO mice. Values are expressed as mean±SEM, n=5 in (C) and n=7 in (D). *p<0.05, **p<0.01 for ASKO vs. WT. 100x56mm (300 x 300 DPI)

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Fig. 6. Impaired lipolysis in ASKO mice. (A)Circulating levels of glycerol (left) and NEFA (right) response to β3AR agonist CL316,243 (0.1 mg/kg, i.p.) in 3and 6monthold WT and ASKO mice. Values are expressed as mean±SEM, n=6 for 3monthold mice and n=4 for 6monthold mice. (B and C) Basal and isoproterenol (Iso)stimulated glycerol release (B) and intracellular cAMP content (C) in EpiWAT explants from 3month old WT and ASKO mice. Values are expressed as mean±SEM, n=4. (D) Representative Western blot images for the indicated proteins and phosphoproteins in basal and Isostimulated EpiWAT explants from 3month old WT and ASKO mice. (E) Circulating levels of glycerol response to β3AR agonist CL316,243 (0.1 mg/kg, i.p.) in 10monthold WT and ASKO mice. Values are expressed as mean±SEM, n=4. *p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT. 117x78mm (300 x 300 DPI)

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Fig.7. Effects of rosiglitazone treatment on ASKO mice. 6monthold male WT and ASKO mice were administered rosiglitazone (Rosi, 0.03%) or vehicle 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)HE staining of subcutaneous WAT from WT and ASKO mice with or without Rosi. Arrows indicate putative newlyformed adipocytes. Scale bar is 50 m. (D)Expression of PPARγ and its target genes in EpiWAT. (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. (G and H)Plasma glucose concentrations for glucose tolerance tests(G) and insulin tolerance tests(H) in mice fasted for 16 h and 4 h, respectively. Area under curve (AUC) is also quantified. (I and J)Plasma levels of insulin(I), leptin and adiponectin (J) in mice fasted for 4 h. Values are the mean±SEM, n=58, *p<0.05, **p<0.01, ***p<0.001 for Rosi diet(Rosi) vs. normal diet(ND). # p<0.05, ## p<0.01, ### p<0.001 for ASKO vs. WT. 204x268mm (300 x 300 DPI)

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Fig. 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 subunit3; Sgpp1: sphingosine-1-phosphate phosphatase1; Acer3: alkaline ceramidase3; Gla: galactosidase-alpha. Values are expressed as mean±SEM, n=5. (B, C, D, E, F, G and H) Lipidomic analysis of Epi-WAT (B, C, D, E) and BAT ( F, G, H) from 6-month-old WT and ASKO mice using LC-MS. Different lipid groups (B, F) and different classes of Cer (C, D, G, H) and TAG (E) are compared. In D and H, sphingolipids are the sum of all ceramide and sphingomyelin species. Values are expressed as mean±SEM, n=4. *p<0.05, **p<0.01, ***p<0.001 for ASKO vs. WT. Cer, ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, spingomyelin; TAG, triacylglycerol. (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. 186x194mm (300 x 300 DPI)

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On line supplemental materials

Table S1. List of primers used and their sequences

Seipin Forward TCAATGACCCACCAGTC F4/80 Forward TTTCCTCGCCTGCTTCTTC Reverse AAGGAGCCATAGAGGAAC Reverse CCCCGTCTCTGTATTCAACC Mac2 Forward CCTATGACCTGCCCTTGC TNFα Forward CTGTGAAGGGAATGGGTGTT Reverse CCCAGTTGGCTGATTTCC Reverse CAGGGAAGAATCTGGAAAGGTC MCP1 Forward TCCCAATGAGTAGGCTGGAG Ym1 Forward GTAATGAGTGGGTTGGTT Reverse AAGTGCTTGAGGTGGTTGTG Reverse AGTAGATGTCAGAGGGAAA IL1β Forward AGGCTCCGAGATGAACAA Mgl1 Forward TGAGAAAGGCTTTAAGAACTGGG Reverse AAGGCATTAGAAACAGTCC Reverse GACCACCTGTAGTGATGTGGG TGFβ1 Forward GGCGGTGCTCGCTTTGTA Mgl2 Forward GGATGGGACCGACTTTGA Reverse TCCCGAATGTCTGACGTATTGA Reverse GTGGGCTGAGCTGGCTTT Arg1 Forward AAGACAGCAGAGGAGGTG PPARγ Forward GACCACTCGCATTCCTTT Reverse AGTCAGTCCCTGGCTTAT Reverse CCACAGACTCGGCACTCA SCD1 Forward TGACCTGAAAGCCGAGAA LPL Forward ACTAGGTCCCACAGGACTG Reverse ATGTGCCAGCGGTACTCA Reverse GACTTCCAGAAGTAACCAACTTTG DGAT1 Forward ATCTGAGGTGCCATCGTC Adiponectin Forward CTCCTGCTTTGGTCCCTC Reverse ATGCCATACTTGATAAGGTTCT Reverse GCCAGTGCTGCCGTCATA CD36 Forward GGCAGGAGTGCTGGATTA FABP4 Forward ACACCGAGATTTCCTTCA Reverse GAGGCGGGCATAGTATCA Reverse CCTCTTCCTTTGGCTCAT HSL Forward GACTCACCGCTGACTTCC C/EBPα Forward GTTAGCCATGTGGTAGGAGACA Reverse TGTCTCGTTGCGTTTGTAG Reverse CCCAGCCGTTAGTGAAGAGT SREBP1c Forward TGGAGACATCGCAAACAAG PPARα Forward GGGCTTTCGGGATAGTTG Reverse GGTAGACAACAGCCGCATC Reverse ATTGGGCTGTTGGCTGAT FAS Forward GGGTCTATGCCACGATTC ACC Forward CCAGACCCTTTCTTCAGC Reverse GTGTCCCATGTTGGATTTG Reverse TTGTCGTAGTGGCCGTTC DGAT2 Forward TCAACCGAGACACCATAGAC Leptin Forward CACAGTCTGGAGCGAAGG Reverse CCTCAAAGATCACCTGCTT Reverse CACAATCTGGGAACAAGC Lipin Forward GGTCAAGGTTGGCAATAA Resistin Forward TCCTTGTCCCTGAACTGC Reverse AGTCAGGCACTGTTCAGG Reverse ACGAATGTCCCACGAGCC UCP1 Forward CTCTGCACTGGCACTACCT UCP2 Forward AATGTTGCCCGTAATGCC Reverse TCGGCAATCCTTCTGTTT Reverse CCCAAGCGGAGAAAGGAA AKT Forward CAGATGGTCGCCAACAGT IRS1 Forward GGATCGTCAATAGCGTAA Reverse TGCCGAGGAGTTTGAGATA Reverse GCTTGGCACAATGTAGAA GLUT4 Forward ACGGATAGGGAGCAGAAA IRS2 Forward GGGGCGAACTCTATGGGTA Reverse AAGGGTGAGTGAGGCATT Reverse GCAGGCGTGGTTAGGGAAT PEPCK1 Forward AGTCATCATCACCCAAGAGC G6P Forward AATCTCCTCTGGGTGGCA Reverse CCACCACATAGGGCGAGT Reverse GCTGTAGTAGTCGGTGTCC MTP Forward GGAAAGCAGAGCGGAGAC Col1a1 Forward CGCCATCAAGGTCTACTGC Reverse AGAGCAAGGGTCAGGCAC Reverse GAATCCATCGGTCATGCTCT Col3a1 Forward GGCAGTGATGGGCAACCT Col6a1 Forward CACTCAACGGGACACGAC Reverse TCCCTTCGCACCGTTCTT Reverse AGATACCTGGCCGACCTT Forward GCAGCCCTAACCAGAAACT CYP4A Forward TCTGCTCTAAGCCCAACC Reverse CCCACAAAGAAGAAGCACC Reverse GCAAACCATACCCAATCC CPT1A Forward CGTGACGTTGGACGAATC ACOX Forward GGAGATCACGGGCACTTA Reverse TCTGCGTTTATGCCTATC Reverse TGAGAATGAACTCTTGGGTC Sptlc3 Forward CAACCAACGCAATGAATA Sgpp1 Forward CCATTCCTCTAGCCTGTA Reverse AGACGCCCTCTACCACGA Reverse ATAGCTGGTAGCCTCCTT Acer3 Forward AATGACCCAGCCATAGAG Gla Forward AATCACTGAGCCAGCAAG Reverse TGTGATGAGAACAGAGCC Reverse GGACATCATGTAGCCAAT

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Table S2. Metabolic parameters of 3M, 6M and 10M ASKO mice

3M 6M 10M

WT ASKO WT ASKO WT ASKO

Fed State

Plasma total cholesterol, mg/dl 128.4±5.6 137.7±5.0 114.7±6.4 130.5±5.4 119.9±6.0 125.1±9.2

Plasma triacylglycerols, mg/dl 93.0±8.9 94.2±11.1 87.4±12.6 143.5±15.8* 73.7±7.8 113.1±14.3*

Plasma NEFA, mEq/L 1.39±0.15 1.40±0.16 1.37±0.11 1.87±0.14* 1.09±0.14 1.59±0.11*

Plasma glycerol, mol/L 708.8±30.2 697.4±45.4 699.2±44.9 738.0±85.7 554.0±59.4 543.8±35.6

Plasma glucose, mg/dl 115.4±12.2 128.2±7.1 121.4±7.2 119.1±7.3 131.4±21.9 111.5±11.8

Plasma leptin, ng/ml 5.90±1.02 3.37±0.87 5.57±1.12 3.66±0.50 5.91±0.74 2.93±0.80*

Plasma adiponectin, g/ml 6.62±0.63 4.69±1.40 6.50±0.29 2.19±0.35*** 6.02±0.37 1.15±0.32***

Plasma insulin, ng/ml 1.45±0.67 0.98±0.28 1.49±0.63 2.93±0.66 1.29±0.37 6.14±0.58***

4h Fast State

Plasma total cholesterol, mg/dl 157.7±7.4 161.4±10.0 121.6±7.2 139.4±5.2 112.3±6.2 137.9±9.5*

Plasma triacylglycerols, mg/dl 92.1±6.2 94.1±7.0 60.9±3.6 55.6±2.5 69.3±2.9 62.1±6.2

Plasma glucose, mg/dl 131.9±10.1 120.0±9.8 114.1±6.7 131.8±8.2 116.7±8.9 126.0±6.1

16h Fast State

Plasma total cholesterol, mg/dl 158.2±5.4 155.7±8.4 134.1±7.3 136.9±19.4 138.3±5.4 125.6±9.3

Plasma triacylglycerols, mg/dl 155.6±12.3 135.6±12.9 158.5±18.7 60.3±8.4** 160.6±15.8 85.7±7.5**

Plasma glucose, mg/dl 70.6±12.5 101.6±2.8* 71.5±6.2 95.9±5.4* 57.6±7.8 80.9±10.6*

Data represent the mean ± SEM, n=7. Blood was obtained from 3, 6 and

10monthold WT and ASKO mice in fed state and fast state. *p<0.05, **p<0.01,

***p<0.001 for ASKO vs. WT.

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Table S3. Differentially expressed genes comparing KO vs WT, P < 0.05 unadjusted and Fold Change >2 and < 2 pvalue Mean StdErr Mean StdErr FoldChange Gene Symbol RefSeq Transcript ID (KOvs.WT) (KO) (KO) (WT) (WT) (KOvs.WT) Fndc5 NM_027402 1.2E06 6.33 0.02 4.74 0.02 3.01 Renbp NM_001164704/NM_023132 3.0E05 8.79 0.04 7.82 0.03 1.96 Plxdc1 NM_001163608/NM_028199 4.8E05 7.05 0.06 5.89 0.02 2.24 Fabp3 NM_010174 6.4E05 8.56 0.06 5.70 0.15 7.25 Cxcl16 NM_023158 7.3E05 8.20 0.03 7.21 0.05 1.98 Fads6 NM_178035 1.0E04 5.01 0.04 4.07 0.04 1.92 Anxa4 NM_013471 1.9E04 9.48 0.04 8.54 0.06 1.92 Myt1l NM_001093775/NM_001093776 2.2E04 4.24 0.14 2.45 0.01 3.46 Gdpd1 NM_025638 2.6E04 7.46 0.04 6.05 0.11 2.67 Cmklr1 NM_008153 2.8E04 8.64 0.05 9.81 0.09 -2.26 Fndc5 NM_027402 2.8E04 6.29 0.04 4.75 0.12 2.90 Gm6756/Phgdh NM_016966 3.3E04 8.58 0.07 9.68 0.07 -2.14 Cxcl12 NM_001012477/NM_013655 4.8E04 11.03 0.09 9.48 0.12 2.92 Nnat NM_010923/NM_180960 4.8E04 11.02 0.09 12.30 0.08 -2.42 Hpca NM_001130419/NM_010471 4.8E04 4.88 0.08 3.64 0.09 2.36 Apcdd1 NM_133237 5.8E04 10.31 0.10 11.35 0.04 -2.06 Chchd10 NM_175329 6.6E04 11.97 0.15 10.04 0.13 3.79 Parp8 NM_001081009/NM_027272 7.9E04 6.24 0.10 5.29 0.02 1.94 Cyp51 NM_020010 8.3E04 5.91 0.12 4.83 0.03 2.12 Fgf21 NM_020013/XM_003689489 8.7E04 7.37 0.05 5.36 0.22 4.01 Cadm1 NM_001025600/NM_018770 9.4E04 8.73 0.04 6.54 0.25 4.57 Chchd10 NM_175329 9.5E04 11.41 0.18 9.39 0.14 4.06 Slc25a13 NM_001177572/NM_015829 9.6E04 6.73 0.09 5.43 0.12 2.45 Pak1 NM_011035 9.9E04 6.45 0.06 5.04 0.15 2.66 D430019H16Rik NM_001252508/NR_015481 1.0E03 7.95 0.17 9.51 0.07 -2.96 Myof NM_001099634/NM_177035 1.1E03 9.46 0.11 8.33 0.08 2.19 Osbpl3 NM_001163645/NM_027881 1.1E03 6.89 0.05 5.40 0.17 2.83 Orm3 NM_013623 1.2E03 5.49 0.14 8.39 0.33 -7.50 Ivd NM_019826 1.2E03 9.80 0.01 10.77 0.12 -1.96 Apcdd1 NM_133237 1.3E03 9.31 0.12 10.35 0.05 -2.05 Dhtkd1 NM_001081131 1.3E03 7.11 0.09 8.09 0.08 -1.97 Creld2 NM_029720 1.3E03 8.86 0.13 7.73 0.05 2.20 Phgdh NM_016966 1.4E03 9.86 0.02 10.85 0.12 -1.99 Mfap2 NM_001161799/NM_008546 1.5E03 8.62 0.10 7.51 0.11 2.16 Plxnb2 NM_001159521/NM_138749 1.5E03 9.30 0.07 8.23 0.12 2.09 Fcgr1 NM_010186 1.7E03 8.85 0.22 6.97 0.12 3.68 Prkca NM_011101 1.8E03 5.99 0.05 6.94 0.12 -1.93 Enpp1 NM_008813 1.8E03 5.04 0.05 4.09 0.12 1.93 Galnt12 NM_172693 1.9E03 6.66 0.10 5.62 0.10 2.06 Kcns3 NM_001168564/NM_173417 2.0E03 7.52 0.14 8.54 0.03 -2.03

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Klra3/Klra9 NM_010648/NM_010651 2.0E03 7.06 0.14 5.74 0.12 2.50 Mthfd2 NM_008638 2.1E03 7.91 0.09 6.50 0.18 2.65 Pak1 NM_011035 2.1E03 8.21 0.16 6.74 0.13 2.78 Glrx NM_053108 2.1E03 8.11 0.12 6.95 0.11 2.23 Cdt1 NM_026014 2.1E03 7.12 0.15 6.00 0.04 2.17 Lats1 NM_010690 2.2E03 3.75 0.11 5.37 0.21 -3.08 Rasgrp2 NM_011242 2.2E03 6.23 0.14 7.21 0.03 -1.97 Cadm1 NM_001025600/NM_018770 2.2E03 8.81 0.03 6.57 0.32 4.73 Tpd52 NM_001025261/NM_001025262 2.2E03 10.39 0.05 9.10 0.18 2.44 Srpx NM_016911 2.2E03 8.11 0.18 6.63 0.11 2.78 6330509M05Rik 2.3E03 4.30 0.16 3.16 0.05 2.21 Ak8 NM_001033874 2.3E03 5.91 0.16 4.73 0.07 2.27 Cxcl16 NM_023158 2.4E03 7.94 0.06 6.71 0.17 2.36 Cadm1 NM_001025600/NM_018770 2.4E03 6.82 0.24 5.13 0.05 3.24 Fzd4 NM_008055 2.4E03 9.60 0.10 10.54 0.09 -1.92 Glrx NM_053108 2.5E03 8.46 0.08 7.47 0.12 1.99 Kcnn4 NM_001163510/NM_008433 2.5E03 7.83 0.25 6.00 0.10 3.57 Cpxm1 NM_019696 2.5E03 9.35 0.17 7.68 0.18 3.18 Btbd11 NM_001017525/NM_028709 2.7E03 5.79 0.14 4.42 0.15 2.59 Cklf NM_001037840/NM_001037841 2.8E03 6.21 0.11 5.22 0.10 1.99 Dapp1 NM_011932 2.9E03 5.10 0.11 4.07 0.11 2.04 Thbd NM_009378 3.1E03 10.38 0.14 11.36 0.06 -1.98 Fkrp NM_173430 3.1E03 3.56 0.09 4.49 0.11 -1.91 Apobec1 NM_001134391/NM_031159 3.1E03 9.25 0.18 7.79 0.14 2.75 D17H6S56E5 NM_033075 3.2E03 8.90 0.08 7.47 0.21 2.69 Epsti1 NM_029495/NM_178825 3.2E03 6.84 0.12 5.32 0.20 2.85 Alb NM_009654 3.3E03 5.94 0.15 7.21 0.13 -2.41 Gdpd1 NM_025638 3.3E03 6.69 0.12 5.35 0.18 2.54 S100g NM_009789 3.7E03 4.99 0.27 3.32 0.07 3.19 Ankrd29 NM_001190371 3.7E03 7.11 0.21 8.59 0.13 -2.80 Cadm1 NM_001025600/NM_018770 3.8E03 9.52 0.04 7.27 0.37 4.77 6330509M05Rik 3.9E03 5.89 0.21 3.84 0.27 4.13 Gnal NM_010307/NM_177137 3.9E03 7.84 0.41 5.26 0.13 5.97 Paip1 NM_001079849/NM_145457 3.9E03 6.58 0.14 7.54 0.08 -1.94 Adcy7 NM_001037723/NM_001037724 4.0E03 6.76 0.17 5.72 0.06 2.06 Pltp NM_011125 4.1E03 10.69 0.21 9.45 0.02 2.37 Hoxd1 NM_010467 4.1E03 3.68 0.11 2.40 0.19 2.43 Dmrt2 NM_145831 4.2E03 9.00 0.12 10.27 0.18 -2.40 Cdt1 NM_026014 4.2E03 8.16 0.16 6.85 0.15 2.47 Fbn1 NM_007993 4.3E03 8.77 0.12 9.71 0.11 -1.92 Klhl2 NM_178633 4.3E03 7.45 0.03 8.75 0.22 -2.46 Tnfrsf13b NM_021349 4.4E03 6.84 0.19 5.71 0.03 2.19 Tpd52 NM_001025261/NM_001025262 4.7E03 9.45 0.14 8.06 0.20 2.61 Gnal NM_010307/NM_177137 4.9E03 8.57 0.49 5.77 0.09 6.93

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Npl NM_028749 4.9E03 8.40 0.21 7.00 0.13 2.64 Ccl24 NM_019577 5.0E03 7.41 0.17 8.53 0.10 -2.17 Got1 NM_010324 5.0E03 7.75 0.18 6.47 0.14 2.43 Amy1 NM_001110505/NM_007446 5.1E03 10.20 0.12 11.26 0.14 -2.08 Pcsk6 NM_011048 5.1E03 7.48 0.01 8.71 0.22 -2.35 Mark1 NM_145515 5.2E03 6.62 0.19 5.44 0.10 2.27 Specc1 NM_001029936 5.2E03 7.50 0.23 5.98 0.15 2.87 Glyctk NM_001039586/NM_174846 5.2E03 5.69 0.17 4.75 0.03 1.91 AI428898 5.3E03 6.17 0.12 7.20 0.14 -2.04 Car1 NM_001083957/NM_009799 5.3E03 4.82 0.16 5.80 0.07 -1.97 Cdk6 NM_009873 5.3E03 9.01 0.12 7.84 0.18 2.25 D17H6S56E5 NM_033075 5.4E03 8.28 0.09 6.75 0.26 2.90 Slc16a14 NM_027921 5.5E03 5.79 0.15 4.79 0.11 2.00 Pxmp2 NM_008993 5.5E03 9.05 0.15 10.37 0.19 -2.51 Kcnn4 NM_001163510/NM_008433 5.6E03 9.60 0.22 7.93 0.22 3.19 Mmp14 NM_008608 5.7E03 9.53 0.15 8.31 0.17 2.32 Aif1 NM_019467 5.8E03 8.16 0.15 6.98 0.16 2.26 Fen1 NM_007999 6.0E03 7.07 0.17 6.11 0.06 1.96 Mmgt2 NM_175002 6.0E03 7.12 0.07 6.00 0.20 2.17 Mmp14 NM_008608 6.3E03 7.06 0.19 5.92 0.11 2.20 Tmem176a NM_001098271/NM_025326 6.3E03 11.23 0.11 10.13 0.18 2.15 Exoc3l2 NM_028954/XM_001471750 6.3E03 6.03 0.13 5.01 0.14 2.03 Mogat1 NM_026713 6.3E03 7.97 0.16 9.20 0.17 -2.34 Rnf149 NM_001033135 6.4E03 10.78 0.01 9.66 0.21 2.17 Hmox1 NM_010442 6.6E03 6.67 0.21 5.33 0.15 2.52 Pla2g7 NM_013737 6.8E03 9.40 0.22 7.96 0.18 2.71 Pvrl3 NM_021495/NM_021496 7.0E03 7.49 0.07 6.38 0.21 2.16 Bcl2l11 NM_009754/NM_207680 7.1E03 9.05 0.15 8.07 0.12 1.97 Ms4a6b NM_027209 7.2E03 9.62 0.13 8.68 0.13 1.92 Pdk4 NM_013743 7.2E03 10.57 0.36 8.76 0.02 3.51 Msln NM_018857 7.4E03 7.55 0.12 8.56 0.16 -2.01 Tes NM_011570/NM_207176 7.5E03 8.03 0.13 7.09 0.13 1.92 Ces1d NM_053200 7.5E03 10.27 0.24 11.73 0.16 -2.76 Tgif1 NM_001164074/NM_001164075 7.6E03 7.59 0.14 6.22 0.24 2.59 Slc6a6 NM_009320 8.1E03 10.58 0.29 8.97 0.16 3.04 Vegfb NM_001185164/NM_011697 8.1E03 9.53 0.11 10.46 0.16 -1.91 Msr1 NM_001113326/NM_031195 8.1E03 4.81 0.23 3.62 0.08 2.28 Acsm3 NM_016870/NM_212441 8.1E03 4.60 0.42 6.90 0.20 -4.92 B4galt6 NM_019737 8.2E03 7.36 0.19 6.39 0.06 1.96 Clic1 NM_033444 8.2E03 10.56 0.12 9.61 0.16 1.93 Cacna1d NM_001083616/NM_028981 8.4E03 6.00 0.13 5.02 0.16 1.98 Susd3 NM_025491/NM_028340 8.4E03 7.72 0.22 6.56 0.09 2.25 Lgmn NM_011175 8.5E03 11.03 0.26 9.68 0.10 2.54 Clec10a NM_001204252/NM_010796 8.6E03 10.77 0.28 9.32 0.12 2.74

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Cox6a2 NM_009943 8.7E03 6.83 0.21 5.77 0.08 2.09 Prkag3 NM_153744/NM_153745 8.7E03 6.70 0.20 8.16 0.23 -2.75 Eif4ebp1 NM_007918 8.7E03 6.53 0.17 7.64 0.16 -2.15 Apoc2 NM_009695 8.8E03 7.41 0.23 6.08 0.16 2.52 Gsg2 NM_010353 8.9E03 4.38 0.16 3.42 0.13 1.94 Ms4a4b NM_021718 9.0E03 8.10 0.36 6.24 0.14 3.63 Lonrf3 NM_028894 9.1E03 7.79 0.01 6.71 0.23 2.12 Mucl1 NM_009268/XM_003688937 9.1E03 2.79 0.19 4.72 0.36 -3.82 Syt12 NM_134164 9.2E03 7.46 0.22 8.57 0.09 -2.16 Hpd NM_008277 9.2E03 4.60 0.13 5.53 0.15 -1.91 Galnt6 NM_001161767/NM_001161768 9.3E03 6.68 0.22 5.64 0.04 2.04 Cyp51 NM_020010 9.3E03 7.82 0.25 6.43 0.16 2.63 Ddah1 NM_026993 9.3E03 6.13 0.10 5.11 0.19 2.03 Blnk NM_008528 9.3E03 9.79 0.32 7.94 0.23 3.59 Sh3bgrl2 NM_172507 9.4E03 6.41 0.11 4.77 0.33 3.11 9630013D21Rik NM_176994/XR_104877 9.4E03 5.12 0.32 6.61 0.03 -2.80 Gm10419 XR_140732/XR_142166 9.6E03 4.80 0.19 5.76 0.07 -1.95 Stmn1 NM_019641 9.6E03 10.41 0.10 9.30 0.22 2.16 Phgdh NM_016966 9.7E03 10.71 0.05 11.67 0.20 -1.94 Cdk6 NM_009873 9.8E03 8.37 0.07 7.36 0.20 2.01 Ero1l NM_015774 9.8E03 8.40 0.28 6.91 0.16 2.81 Nceh1 NM_178772 9.8E03 9.34 0.39 7.43 0.14 3.75 A030009H04Rik NM_020591/NR_027827 9.8E03 5.54 0.07 4.45 0.23 2.14 Plod2 NM_001142916/NM_011961 9.9E03 7.65 0.18 6.48 0.18 2.26 Sox4 NM_009238 1.0E02 8.14 0.22 7.06 0.08 2.11 Ear11 NM_053113 1.0E02 3.48 0.20 6.05 0.52 -5.95 Slamf7 NM_144539 1.0E02 6.91 0.31 5.40 0.11 2.85 Atp6v1a NM_007508 1.0E02 10.54 0.18 9.61 0.10 1.91 Spp1 NM_001204201/NM_001204202 1.0E02 9.06 0.05 5.82 0.71 9.40 Tmem176a NM_001098271/NM_025326 1.0E02 9.82 0.02 8.82 0.22 2.00 Tnfrsf11b NM_008764 1.0E02 2.51 0.10 3.92 0.29 -2.66 Hpgds NM_019455 1.1E02 7.97 0.45 5.65 0.25 4.99 Enpp3 NM_134005 1.1E02 7.57 0.18 6.55 0.13 2.01 Pycard NM_023258 1.1E02 9.74 0.21 8.65 0.11 2.13 Btbd11 NM_001017525/NM_028709 1.1E02 5.55 0.13 4.52 0.19 2.05 Disp2 NM_170593 1.1E02 4.81 0.17 7.19 0.51 -5.19 Rai2 NM_001103367/NM_198409 1.1E02 5.22 0.10 6.21 0.20 -1.99 Sox4 NM_009238 1.1E02 5.57 0.15 4.58 0.17 1.98 Soat1 NM_009230 1.2E02 8.83 0.35 7.17 0.13 3.16 Cd9 NM_007657 1.2E02 10.86 0.14 9.80 0.20 2.08 Eci3 NM_026947 1.2E02 7.11 0.18 8.43 0.24 -2.50 Ankrd29 NM_001190371 1.2E02 6.04 0.23 7.35 0.19 -2.48 Mmp11 NM_008606 1.2E02 7.48 0.25 6.33 0.08 2.22 Mar01 NM_001166372/NM_001166375 1.2E02 6.90 0.20 5.95 0.08 1.93

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Osbpl3 NM_001163645/NM_027881 1.2E02 5.43 0.19 4.38 0.15 2.07 Tfec NM_031198 1.2E02 6.01 0.32 4.57 0.07 2.72 Cd28 NM_007642 1.2E02 4.39 0.18 3.38 0.15 2.01 Pitpnc1 NM_145823 1.2E02 5.48 0.16 4.31 0.22 2.25 Eepd1 NM_026189 1.2E02 9.65 0.15 10.60 0.16 -1.93 Hspb7 NM_013868 1.2E02 8.91 0.11 9.84 0.18 -1.90 Ncf4 NM_008677 1.3E02 8.43 0.24 7.39 0.05 2.06 Nceh1 NM_178772 1.3E02 7.04 0.38 5.34 0.09 3.24 4930534B04Rik NM_181815 1.3E02 5.30 0.08 4.30 0.22 2.00 Armcx4 NM_001202500 1.3E02 6.87 0.20 5.62 0.22 2.38 Klrd1 NM_010654 1.3E02 7.04 0.25 5.90 0.10 2.21 Hist1h4 NM_001195421/NM_033596 1.4E02 4.94 0.35 6.87 0.30 -3.81 Epsti1 NM_029495/NM_178825 1.4E02 8.68 0.16 7.42 0.25 2.40 Runx1 NM_001111021/NM_001111022 1.4E02 5.60 0.23 4.56 0.09 2.05 Raet1a/Raet1b NM_009016/NM_009017 1.4E02 5.42 0.22 4.49 0.06 1.91 Uap1l1 NM_001033293 1.4E02 3.80 0.26 2.68 0.08 2.19 Nceh1 NM_178772 1.4E02 8.66 0.37 6.98 0.16 3.21 Mafb NM_010658 1.4E02 10.27 0.27 9.07 0.10 2.30 Soat1 NM_009230 1.4E02 8.77 0.31 7.31 0.16 2.76 Adcy7 NM_001037723/NM_001037724 1.4E02 7.78 0.18 6.78 0.16 2.01 Slc16a7 NM_011391 1.4E02 7.95 0.22 9.01 0.14 -2.09 Cpeb2 NM_001177379/NM_175937 1.5E02 3.98 0.18 5.08 0.20 -2.15 Rhbdl1 NM_144816 1.5E02 8.08 0.15 9.19 0.22 -2.15 Ccl5 NM_013653 1.5E02 9.36 0.36 7.86 0.07 2.84 Fam26f NM_175449 1.5E02 6.97 0.24 5.86 0.12 2.16 Gm8096/Phgdh NM_016966/NR_033590 1.5E02 9.37 0.05 10.39 0.24 -2.02 Dock8 NM_028785/NM_175233 1.5E02 9.93 0.11 8.74 0.27 2.28 Mmp14 NM_008608 1.5E02 6.34 0.23 5.37 0.05 1.96 Tmem176a NM_001098271/NM_025326 1.5E02 10.68 0.17 9.66 0.19 2.02 Enpp1 NM_008813 1.5E02 6.53 0.12 5.32 0.28 2.33 Grem2 NM_011825 1.6E02 7.33 0.16 8.46 0.23 -2.20 Specc1 NM_001029936 1.6E02 8.07 0.35 6.18 0.31 3.73 Upk3b NM_175309 1.6E02 8.90 0.13 9.86 0.20 -1.95 Cdkl4 NM_001033443 1.6E02 7.37 0.09 5.53 0.45 3.57 Aldh1l2 NM_153543 1.6E02 6.97 0.43 5.20 0.12 3.40 6720475J19Rik NM_026586/XM_001474896 1.6E02 7.86 0.01 6.20 0.42 3.16 Pon1 NM_011134 1.7E02 5.01 0.33 6.40 0.11 -2.63 Soat1 NM_009230 1.7E02 9.19 0.37 7.63 0.15 2.96 Ankrd5 NM_175667/NM_177654 1.7E02 7.08 0.29 8.29 0.08 -2.30 Prkcb NM_008855 1.7E02 8.68 0.27 7.39 0.18 2.44 Trim66 NM_001170912/NM_001170913 1.7E02 3.73 0.25 2.64 0.12 2.13 Sh3bgrl2 NM_172507 1.8E02 7.31 0.10 5.78 0.38 2.89 Vsig8 NM_177723/NR_027644 1.8E02 6.05 0.50 4.10 0.04 3.87 Derl3 NM_024440 1.8E02 7.42 0.26 6.35 0.09 2.10

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Myo7a NM_001256081/NM_001256082 1.8E02 7.34 0.22 6.30 0.16 2.06 9030624J02Rik NM_027815 1.8E02 6.27 0.17 5.33 0.18 1.92 Thrsp NM_009381 1.8E02 10.85 0.21 11.85 0.15 -2.00 Rnf180 NM_027934 1.8E02 6.97 0.34 5.34 0.25 3.09 2010107G23Rik NM_027251 1.9E02 6.57 0.21 5.63 0.12 1.91 Hmga2 NM_010441 1.9E02 4.98 0.48 3.13 0.06 3.60 Tifa NM_145133 1.9E02 7.88 0.21 6.91 0.14 1.96 Rnf128 NM_001254761/NM_023270 1.9E02 9.99 0.12 6.93 0.79 8.33 Emilin1 NM_133918 1.9E02 7.90 0.21 6.89 0.17 2.03 IghVJ558/Igha NM_001024700 1.9E02 11.93 0.31 10.13 0.36 3.49 Sema4d NM_013660 1.9E02 7.25 0.29 5.76 0.26 2.81 H2Q7/H2Q8 NM_001198560/NM_001198561 1.9E02 10.20 0.07 9.21 0.25 1.99 Cd68 NM_009853 1.9E02 11.06 0.41 9.42 0.14 3.11 Klf15 NM_023184 2.0E02 8.25 0.29 9.52 0.18 -2.40 Arhgap19 NM_001163495/NM_027667 2.0E02 7.11 0.23 6.15 0.12 1.95 Ces1f NM_144930 2.0E02 9.24 0.18 10.28 0.21 -2.05 Mmp2 NM_008610 2.0E02 10.02 0.19 9.08 0.17 1.92 Klhl2 NM_178633 2.0E02 8.83 0.09 10.01 0.30 -2.26 Gabra3 NM_008067 2.0E02 7.74 0.13 6.78 0.22 1.94 Upk3b NM_175309 2.0E02 9.92 0.15 10.89 0.21 -1.96 Gm17396 XR_105326/XR_108161 2.0E02 4.89 0.12 5.83 0.22 -1.92 H2Q5 NM_010393/NR_051981 2.0E02 6.97 0.40 5.45 0.09 2.88 Gm12824 NM_001085549 2.1E02 7.70 0.23 8.65 0.11 -1.92 Ifi30 NM_023065 2.1E02 11.00 0.31 9.74 0.14 2.40 Rgs10 NM_026418 2.1E02 9.58 0.26 8.61 0.06 1.97 IghVJ558/Igha NM_001024700 2.1E02 12.20 0.29 10.53 0.34 3.19 Mmp2 NM_008610 2.1E02 10.08 0.21 9.07 0.18 2.01 Ms4a7 NM_001025610/NM_027836 2.1E02 10.01 0.45 8.34 0.01 3.17 Sgpp1 NM_030750 2.1E02 6.66 0.13 5.61 0.25 2.06 Pik3ap1 NM_031376 2.1E02 7.40 0.34 5.97 0.19 2.68 Slc25a23 NM_025877 2.1E02 7.56 0.23 8.49 0.10 -1.91 Gtpbp4 NM_027000 2.1E02 5.71 0.29 6.88 0.13 -2.25 Fbn1 NM_007993 2.1E02 3.77 0.28 4.95 0.15 -2.27 H2DMb1 NM_010387/NM_010388 2.1E02 9.43 0.27 8.31 0.13 2.16 Trhde NM_146241 2.2E02 3.94 0.30 5.80 0.41 -3.65 Fbln7 NM_024237 2.2E02 7.58 0.46 5.83 0.15 3.37 IghVJ558/Igha NM_001024700 2.2E02 12.61 0.29 11.01 0.33 3.04 Crisp2 NM_001204071/NM_009420 2.2E02 5.11 0.07 4.15 0.26 1.95 Nkg7 NM_024253 2.3E02 7.11 0.24 5.97 0.20 2.22 Mdk NM_001012335/NM_001012336 2.3E02 7.90 0.44 6.10 0.23 3.48 Laptm5 NM_010686 2.3E02 9.84 0.45 8.21 0.08 3.09 Dennd2d NM_001093754/NM_028110 2.3E02 5.62 0.21 6.71 0.21 -2.12 Myo1e NM_181072/XM_003689436 2.3E02 6.39 0.32 5.20 0.10 2.27 Sptlc3 NM_175467 2.3E02 7.87 0.68 5.42 0.08 5.48

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Timp1 NM_001044384/NM_011593 2.3E02 8.47 0.20 7.49 0.19 1.97 Ckb NM_021273 2.3E02 10.78 0.28 9.62 0.17 2.23 Hoxc5 NM_175730 2.3E02 7.86 0.20 9.07 0.27 -2.31 Cldn22 NM_029383 2.3E02 6.48 0.20 7.73 0.28 -2.37 A530016L24Rik NM_177039 2.4E02 8.27 0.18 9.29 0.23 -2.03 Ociad2 NM_026950 2.4E02 8.22 0.26 6.92 0.25 2.45 Adcy7 NM_001037723/NM_001037724 2.4E02 9.48 0.16 8.42 0.25 2.07 Cd72 NM_001110320/NM_001110321 2.4E02 7.13 0.38 5.62 0.18 2.83 Il20rb NM_001033543/NM_001037246 2.4E02 5.38 0.23 4.44 0.13 1.91 Pdia3 NM_007952 2.5E02 11.46 0.10 10.35 0.30 2.16 Dkk3 NM_015814 2.5E02 7.82 0.28 6.63 0.19 2.29 Frk NM_001159544/NM_010237 2.5E02 4.37 0.27 3.09 0.25 2.43 Sdf2l1 NM_022324 2.5E02 8.94 0.28 7.93 0.08 2.02 Hmga2 NM_010441 2.5E02 5.31 0.32 4.14 0.11 2.26 Mpeg1 NM_010821 2.5E02 11.46 0.41 9.85 0.21 3.04 Nckap1l NM_153505 2.5E02 9.54 0.34 8.30 0.11 2.36 Tnfaip8l2 NM_027206 2.6E02 7.95 0.29 6.93 0.04 2.04 Prr15 NM_030024 2.6E02 6.60 0.19 5.26 0.33 2.53 Ms4a6d NM_026835 2.6E02 8.49 0.31 7.32 0.13 2.26 Hsd17b11 NM_053262 2.6E02 8.79 0.35 7.44 0.18 2.55 Elovl3 NM_007703 2.6E02 5.22 0.44 3.68 0.05 2.90 P2rx4 NM_011026 2.6E02 8.39 0.31 7.31 0.07 2.11 Tmigd1 NM_025655 2.6E02 6.58 0.30 5.55 0.02 2.04 Gzmb NM_013542 2.6E02 6.00 0.31 4.77 0.18 2.34 Emilin2 NM_145158 2.7E02 8.98 0.07 9.92 0.27 -1.92 Ces1d NM_053200 2.7E02 10.90 0.32 12.03 0.09 -2.18 Slc6a6 NM_009320 2.7E02 12.05 0.26 10.84 0.23 2.30 Fam57b NM_001146347/NM_026884 2.7E02 6.69 0.21 7.64 0.19 -1.93 Rnf128 NM_001254761/NM_023270 2.7E02 9.98 0.08 7.10 0.84 7.36 Cyba NM_007806 2.8E02 11.18 0.28 10.15 0.13 2.04 P2ry6 NM_183168 2.8E02 9.29 0.33 8.08 0.15 2.31 B3galt2 NM_020025 2.8E02 4.73 0.25 6.00 0.29 -2.42 Tmem229b NM_001170401/NM_178745 2.8E02 8.23 0.32 7.09 0.12 2.21 Lcp1 NM_001247984/NM_008879 2.9E02 9.14 0.36 7.84 0.15 2.48 Gla NM_013463 2.9E02 7.15 0.31 6.07 0.08 2.11 Evi2a NM_001033711/NM_010161 2.9E02 9.67 0.35 8.47 0.06 2.30 Lipa NM_001111100/NM_021460 2.9E02 10.83 0.35 9.63 0.10 2.31 Esrrg NM_001243792/NM_011935 2.9E02 6.49 0.28 5.35 0.20 2.22 Lgals12 NM_019516 2.9E02 8.78 0.13 9.72 0.25 -1.92 Pyhin1 NM_175026 2.9E02 7.54 0.40 5.41 0.50 4.38 Thbs1/X99384 NM_011580/NM_013753 3.0E02 9.82 0.24 10.93 0.23 -2.15 Serpina3k NM_011458 3.0E02 6.76 0.70 4.39 0.14 5.18 Ubash3b NM_176860 3.0E02 7.46 0.31 6.43 0.06 2.04 Depdc7 NM_144804 3.0E02 6.36 0.43 4.82 0.20 2.92

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Tex9 NM_009359 3.1E02 6.84 0.25 5.53 0.32 2.47 Fcer1g NM_010185 3.1E02 10.83 0.29 9.85 0.08 1.97 Lpxn NM_134152 3.1E02 8.54 0.45 6.99 0.14 2.92 Dennd2d NM_001093754/NM_028110 3.1E02 5.67 0.35 6.97 0.20 -2.47 Btk NM_013482 3.1E02 7.29 0.35 6.14 0.05 2.21 Tcfl5 NM_178254 3.1E02 6.75 0.53 5.00 0.10 3.36 Rad51 NM_011234 3.2E02 5.70 0.17 4.46 0.34 2.35 Fcgr4 NM_144559 3.2E02 7.66 0.41 5.77 0.42 3.69 Ms4a6d NM_026835 3.2E02 7.45 0.34 6.34 0.07 2.16 Napsa NM_008437 3.2E02 8.11 0.35 6.84 0.18 2.40 Aqp11 NM_175105 3.2E02 3.99 0.25 4.94 0.15 -1.93 Padi2 NM_008812 3.3E02 6.29 0.28 5.27 0.15 2.03 Ptpn22 NM_008979 3.3E02 7.23 0.35 5.99 0.16 2.36 H2Q4/H2Q6 NM_001143689/NM_207648 3.3E02 6.80 0.12 5.56 0.37 2.36 Wif1 NM_011915 3.3E02 3.09 0.42 1.65 0.17 2.71 Cd3g NM_009850 3.3E02 6.63 0.34 5.41 0.16 2.33 Gpr18 NM_182806 3.4E02 5.69 0.33 4.58 0.11 2.15 Rgs2 NM_009061 3.4E02 9.54 0.18 8.06 0.43 2.79 Oxct1 NM_024188 3.4E02 8.11 0.26 9.21 0.23 -2.13 Ebi3 NM_015766 3.4E02 7.51 0.38 6.27 0.07 2.35 Ms4a6c NM_001166376/NM_028595 3.4E02 9.44 0.28 8.47 0.13 1.96 A530016L24Rik NM_177039 3.4E02 7.78 0.23 8.76 0.21 -1.97 Got1l1 NM_029674 3.4E02 6.57 0.33 5.43 0.15 2.21 D14Ertd449e NM_025311/NM_026679 3.5E02 7.57 0.31 6.34 0.24 2.35 1110006G14Rik XM_001478810/XM_989766 3.5E02 7.45 0.26 8.53 0.23 -2.12 Slamf8 NM_029084 3.5E02 6.69 0.51 5.02 0.17 3.18 Lypd6b NM_027990 3.5E02 4.79 0.23 6.10 0.35 -2.48 Scd3 NM_024450 3.5E02 4.64 0.29 3.71 0.08 1.90 Opn3 NM_010098 3.5E02 9.27 0.17 10.22 0.25 -1.93 Fam70a NM_172930 3.5E02 6.38 0.22 5.45 0.20 1.90 Acer3 NM_025408 3.5E02 6.28 0.32 5.19 0.13 2.13 Nrg4 NM_032002 3.6E02 7.38 0.34 8.92 0.36 -2.91 Lck NM_001162432/NM_001162433 3.6E02 6.25 0.39 5.02 0.04 2.35 Galnt7 NM_001167981/NM_144731 3.6E02 5.62 0.34 4.50 0.11 2.16 Adamts15 NM_001024139 3.6E02 7.51 0.18 6.39 0.31 2.18 Laptm5 NM_010686 3.6E02 10.13 0.30 9.11 0.14 2.03 Sirpa NM_001177646/NM_001177647 3.6E02 9.60 0.31 8.63 0.05 1.95 Adam12 NM_007400 3.6E02 5.92 0.38 4.64 0.16 2.44 Hmga1 NM_001025427/NM_001039356 3.7E02 8.18 0.27 7.19 0.17 1.98 Cd84 NM_001252472/NM_013489 3.8E02 8.76 0.55 7.07 0.05 3.24 Ccrl1 NM_145700 3.8E02 6.37 0.30 5.28 0.19 2.12 Dtna NM_010087/NM_207650 3.8E02 5.24 0.29 6.59 0.33 -2.54 Npas2 NM_008719 3.8E02 4.91 0.24 3.71 0.32 2.31 Galnt7 NM_001167981/NM_144731 3.8E02 7.42 0.30 6.44 0.11 1.97

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Reps2 NM_178256 3.9E02 8.28 0.16 7.27 0.29 2.02 Fblim1 NM_001163256/NM_133754 3.9E02 8.12 0.35 6.73 0.29 2.61 9530053H05Rik NR_002850/XR_035210 3.9E02 7.86 0.29 6.93 0.09 1.90 Ctss NM_001267695/NM_021281 3.9E02 12.39 0.28 11.27 0.24 2.18 Bnc2 NM_172870 4.0E02 5.30 0.32 6.42 0.19 -2.17 Sh3bp2 NM_001136088/NM_001145858 4.0E02 7.57 0.28 6.60 0.15 1.96 Pld3 NM_011116 4.0E02 10.20 0.38 8.99 0.14 2.32 Dkk3 NM_015814 4.0E02 7.20 0.25 6.24 0.19 1.94 Pafah1b3 NM_008776 4.0E02 7.30 0.33 6.31 0.05 1.99 Ccl8 NM_021443/XM_003085741 4.0E02 11.45 0.18 10.12 0.41 2.53 Obfc2a NM_028696 4.0E02 7.06 0.34 5.77 0.28 2.46 Prkcb NM_008855 4.1E02 6.45 0.31 5.46 0.12 1.99 Serpina3g NM_009251 4.1E02 6.86 0.43 5.51 0.15 2.55 Slc1a3 NM_148938 4.1E02 7.84 0.30 8.93 0.22 -2.13 Ptpro NM_001164401/NM_001164402 4.2E02 5.50 0.36 4.41 0.05 2.12 Id2 NM_010496 4.2E02 9.42 0.19 8.19 0.37 2.34 Pyhin1 NM_175026 4.3E02 5.56 0.32 4.46 0.19 2.14 Esm1 NM_023612 4.3E02 4.30 0.38 5.49 0.15 -2.29 Lipa NM_001111100/NM_021460 4.3E02 9.88 0.36 8.75 0.15 2.20 Prrx1 NM_001025570/NM_011127 4.3E02 8.99 0.29 7.95 0.20 2.05 Gm885 NM_001033435 4.4E02 5.98 0.36 4.86 0.13 2.17 Cd48 NM_007649 4.4E02 9.37 0.37 8.27 0.09 2.14 Sgms2 NM_028943 4.4E02 7.43 0.25 6.49 0.21 1.91 Gxylt2 NM_198612 4.4E02 6.34 0.30 5.38 0.14 1.94 Fblim1 NM_001163256/NM_133754 4.4E02 8.57 0.25 7.44 0.30 2.19 Pik3r5 NM_177320 4.5E02 6.99 0.38 5.81 0.15 2.26 Rgs2 NM_009061 4.5E02 8.69 0.23 7.17 0.47 2.86 Fgd4 NM_139232/NM_139233 4.5E02 4.29 0.36 3.23 0.10 2.09 Aoah NM_012054 4.5E02 5.83 0.35 4.77 0.10 2.08 Myo1e NM_181072/XM_003689436 4.5E02 6.94 0.39 5.80 0.06 2.20 Gpnmb NM_053110 4.6E02 5.36 0.70 3.36 0.03 4.01 C5ar1 NM_001173550/NM_007577 4.6E02 6.83 0.43 5.60 0.07 2.36 Kif5c NM_008449 4.6E02 6.67 0.30 5.65 0.19 2.03 Fdps NM_001253751/NM_134469 4.6E02 9.19 0.39 7.94 0.20 2.38 Tlr13 NM_205820 4.6E02 8.44 0.56 6.75 0.18 3.21 Marveld2 NM_001038602/NM_178410 4.6E02 5.37 0.39 4.14 0.19 2.35 Lat2 NM_020044/NM_022964 4.6E02 8.34 0.65 6.44 0.13 3.74 Cdk18 NM_008795 4.6E02 6.65 0.50 5.21 0.09 2.71 Tlr1 NM_030682 4.7E02 6.43 0.50 4.98 0.08 2.73 Rtn4 NM_024226/NM_194051 4.7E02 11.56 0.33 10.40 0.24 2.23 Fgf12 NM_010199/NM_183064 4.7E02 4.59 0.42 3.39 0.06 2.30 Pkib NM_001039050/NM_001039051 4.7E02 4.96 0.33 3.92 0.15 2.05 Brca1 NM_009764 4.8E02 5.83 0.42 4.61 0.12 2.33 Ppargc1a NM_008904/NR_027710 4.8E02 5.68 0.36 4.53 0.18 2.22

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Adcyap1r1 NM_001025372/NM_007407 4.8E02 6.84 0.02 7.84 0.35 -2.00 Oas1a NM_145211 4.8E02 8.82 0.22 7.64 0.36 2.26 Car3 NM_007606 4.8E02 8.30 0.50 9.73 0.10 -2.69 1300002K09Rik NM_028788 4.8E02 4.50 0.36 3.43 0.13 2.10 Gas2l3 NM_001033331/NM_001079876 4.9E02 7.47 0.51 5.88 0.26 3.02 Tnfrsf1b NM_011610 4.9E02 8.54 0.27 7.60 0.20 1.92 Trav9d3 4.9E02 6.95 0.66 4.84 0.37 4.31 Cd53 NM_007651 4.9E02 10.83 0.35 9.82 0.09 2.02 Pcdhb9 NM_053134 4.9E02 6.49 0.06 5.53 0.34 1.95 Acer3 NM_025408 4.9E02 8.06 0.27 7.05 0.24 2.01 Trav9d3 4.9E02 8.48 0.43 7.22 0.13 2.40 Igk 4.9E02 8.16 0.61 6.28 0.29 3.67 Syngr1 NM_009303/NM_207708 5.0E02 7.34 0.52 5.71 0.26 3.08 Slc16a2 NM_009197 5.0E02 6.99 0.29 5.99 0.21 2.00 Adam12 NM_007400 5.0E02 8.58 0.40 7.45 0.09 2.18 Note: --- denotes unannuotated genes from the Affymetrix library for this array.

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Table.S4. Pathway enrichment for fat tissue gene signature list KEGG Pathway Name number of significant genes in input list p < 0.05, fold change < 1.5 or < 1.5 1304 Enriched pathway % genes in pathway Enrichment (Enrichment P value < 0.05) that are present P value KEGG pathway ID

Natural killer cell mediated cytotoxicity 28.93 3.9E12 kegg_pathway_224 Autoimmune thyroid disease 41.82 4.3E12 kegg_pathway_55 Graftversushost disease 38.64 1.2E08 kegg_pathway_37 Allograft rejection 34.88 4.3E07 kegg_pathway_108 Systemic lupus erythematosus 20.66 5.7E06 kegg_pathway_73 Type I diabetes mellitus 28.57 1.5E05 kegg_pathway_114 Herpes simplex infection 16.40 6.1E05 kegg_pathway_11 Antigen processing and presentation 23.19 6.6E05 kegg_pathway_237 Cytosolic DNAsensing pathway 23.73 1.4E04 kegg_pathway_170 Influenza A 16.27 2.1E04 kegg_pathway_192 Phagosome 16.35 2.5E04 kegg_pathway_173 Measles 16.91 3.4E04 kegg_pathway_174 Tolllike receptor signaling pathway 18.75 4.2E04 kegg_pathway_138 RIGIlike receptor signaling pathway 21.54 4.2E04 kegg_pathway_46 Regulation of autophagy 27.27 7.6E04 kegg_pathway_9 B cell receptor signaling pathway 19.48 8.3E04 kegg_pathway_56 Viral carcinogenesis 14.15 1.1E03 kegg_pathway_194 Staphylococcus aureus infection 22.00 1.4E03 kegg_pathway_115 Intestinal immune network for IgA production 23.26 1.5E03 kegg_pathway_122 Sphingolipid metabolism 22.22 2.2E03 kegg_pathway_191 Lysosome 15.97 2.2E03 kegg_pathway_242 Viral myocarditis 17.95 2.8E03 kegg_pathway_39 Hepatitis B 14.79 3.5E03 kegg_pathway_36 Cell adhesion molecules (CAMs) 14.48 4.5E03 kegg_pathway_133 Cytokinecytokine receptor interaction 12.55 6.0E03 kegg_pathway_125 Osteoclast differentiation 14.88 6.2E03 kegg_pathway_120 Steroid biosynthesis 29.41 8.3E03 kegg_pathway_197 Tuberculosis 13.29 8.7E03 kegg_pathway_113 Fc epsilon RI signaling pathway 16.90 8.8E03 kegg_pathway_88 PI3KAkt signaling pathway 11.50 9.7E03 kegg_pathway_19 Leishmaniasis 17.19 1.1E02 kegg_pathway_152 Asthma 24.00 1.1E02 kegg_pathway_135 DNA replication 20.59 1.5E02 kegg_pathway_137 Adipocytokine signaling pathway 15.94 1.8E02 kegg_pathway_89

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Arginine and proline metabolism 17.31 1.9E02 kegg_pathway_208 Chemokine signaling pathway 12.37 2.0E02 kegg_pathway_124 T cell receptor signaling pathway 13.89 2.2E02 kegg_pathway_165 Primary immunodeficiency 18.92 2.3E02 kegg_pathway_203 Hepatitis C 13.08 2.6E02 kegg_pathway_228 Melanoma 15.07 2.7E02 kegg_pathway_198 Ether lipid metabolism 17.07 3.9E02 kegg_pathway_136 Mucin type OGlycan biosynthesis 20.00 4.2E02 kegg_pathway_48 Starch and sucrose metabolism 15.69 4.4E02 kegg_pathway_90 Riboflavin metabolism 27.27 5.0E02 kegg_pathway_76 HTLVI infection 10.74 5.2E02 kegg_pathway_168

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Table.S5. Pathway ANOVA analysis showing significant patterns between KO vs Controls across genes in a pathway (P < 0.05).

# of KEGG pvalue Ratio Pathway Name Probesets Pathway ID (KO vs.WT) (KO vs.WT) Circadian rhythm 94 241 3.88E05 0.962 Purine metabolism 347 206 2.01E04 1.035 Mucin type OGlycan biosynthesis 52 48 6.13E04 1.081 DNA replication 79 137 8.85E04 1.156 Protein export 60 22 1.59E03 1.120 Linoleic acid metabolism 61 232 1.83E03 0.983 Other glycan degradation 28 17 2.12E03 1.187 Pantothenate and CoA biosynthesis 43 251 3.85E03 1.136 Type I diabetes mellitus 89 114 4.21E03 1.185 Allograft rejection 96 108 4.93E03 1.250 Mismatch repair 45 69 5.36E03 1.112 Colorectal cancer 182 227 5.99E03 1.037 Protein processing in endoplasmic reticulum 395 27 6.45E03 1.071 Graftversushost disease 80 37 7.03E03 1.223 Sphingolipid metabolism 99 191 8.22E03 1.190 Progesteronemediated oocyte maturation 223 178 8.28E03 1.051 Aldosteroneregulated sodium reabsorption 99 240 8.43E03 1.054 Carbohydrate digestion and absorption 93 8 8.81E03 1.079 Antigen processing and presentation 146 237 9.49E03 1.182 Autoimmune thyroid disease 109 55 1.01E02 1.175 Pancreatic cancer 192 3 1.13E02 1.063 Insulin signaling pathway 357 235 1.19E02 0.972 PPAR signaling pathway 151 68 1.24E02 1.120 Glycosaminoglycan biosynthesis heparan sulfate / heparin 49 163 1.53E02 0.963 Riboflavin metabolism 34 76 1.68E02 1.098 Lysine biosynthesis 9 248 2.26E02 0.803 Pentose phosphate pathway 59 189 2.56E02 1.117 Glycerolipid metabolism 117 212 2.70E02 1.078 Collecting duct acid secretion 49 127 2.74E02 1.213 Fructose and mannose metabolism 85 255 2.74E02 1.100 Phosphatidylinositol signaling system 196 106 3.02E02 1.042 Staphylococcus aureus infection 104 115 3.18E02 1.301 Amino sugar and nucleotide sugar metabolism 102 229 3.24E02 1.066 Fatty acid elongation 54 32 3.40E02 1.180 Primary immunodeficiency 87 203 3.61E02 1.248 Transcriptional misregulation in cancer 411 13 3.72E02 1.089 Ether lipid metabolism 84 136 3.89E02 1.040 Cell adhesion molecules (CAMs) 332 133 3.95E02 1.152

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Regulation of autophagy 57 9 4.00E02 0.940 Intestinal immune network for IgA production 101 122 4.05E02 1.219 Glioma 191 188 4.05E02 1.050 Glycosaminoglycan degradation 34 21 4.67E02 1.103 Nicotinate and nicotinamide metabolism 54 43 5.10E02 1.066 Phagosome 340 173 5.24E02 1.167 GnRH signaling pathway 227 226 5.37E02 1.044 Systemic lupus erythematosus 155 73 5.40E02 1.113 Melanoma 188 198 5.47E02 1.047 Note: lipid metabolism pathways are highlighted in bold.

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Figure Legends

Fig. S1. Fat pad weight and histology of subcutaneous WAT in ASKO mice.

(A) Fat pad weight of 3, 6 and 10 monthold WT and ASKO mice. Fat weight is

normalized to body weight. Values are the mean±SEM, n=9. *p<0.05, **p<0.01,

***p<0.001 for ASKO vs. WT.

(B) Histology of subcutaneous WAT from 3, 6 and 10monthold WT and ASKO

mice. Scale bar is 50 μm.

Fig. S2. Abnormal gene expression and function in EpiWAT and BAT from ASKO

mice.

(A) Expression of adipogenesis related genes in EpiWAT of 3, 6 and 10monthold

ASKO mice.

(B) Expression of adipocytokines in EpiWAT of 3, 6 and 10monthold ASKO

mice.

(C) Expression of UCP1 and UCP2 in BAT of 3, 6 and 10monthold ASKO mice.

Values are fold induction of gene expression normalized to the housekeeping gene

Gapdh.

(D) Core body temperature of 3monthold WT and ASKO mice. Core body

temperature of mice at room temperature (25°C) was measured using a rectal probe

(Thermalet TH5). Mice were then exposed to 4°C for 4 hours and core body

temperature was examined every hour. Values are the mean±SEM, n=6. *p<0.05 for

ASKO vs. WT.

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Fig. S3. Expression of selected genes in EpiWAT and liver and lipid content in skeletal muscle.

(A) Expression of insulin signaling related genes in EpiWAT of 3, 6 and

10monthold ASKO mice.

(B) Expression of insulin signaling related genes in liver of 3, 6 and 10monthold

ASKO mice.

(C) Expression of lipid synthesis related genes in the liver of 3, 6 and 10monthold

ASKO mice. Values are fold induction of gene expression normalized to the housekeeping gene Gapdh and expressed as mean±SEM, n=6.

(D) Expression of PPARγ in the liver of 6monthold ASKO mice. Values are the mean ±SEM, n=5

(E) Representative Western blot images for the cleaved SREBP1C in the liver from

6and 10monthold WT and ASKO mice.

(F) Skeletal muscle TAG contents from 10monthold WT and ASKO mice. TAG content is normalized to weight. Values are the mean ±SEM, n=45. *p<0.05,

**p<0.01 for ASKO vs. WT.

(G) Representative Western blot images for AKT and PAKT in the skeletal muscle from 10monthold WT and ASKO mice.

Fig. S4. Increased adipose tissue cell death and fibrosis in ASKO mice and decreased, hormone stimulated lipolysis in ASKOB6 mice.

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(A) TUNEL staining of WT and ASKO adipose tissue.

(B) Sirius red staining of EpiWAT from 3, 6 and 10monthold WT and ASKO mice.

Scale bar is 50 μm.

(C) Expression of fibrosis related genes in EpiWAT of 10monthold WT and ASKO

Mice. Values are fold induction of gene expression normalized to the housekeeping

gene Gapdh and expressed as mean±SEM, n=6.

(D and E) Basal and isoproterenol (Iso)stimulated glycerol release in EpiWAT

explants from 3(D) and 6monthold(E) C57BL/6 background WTB6 and ASKOB6

mice. Values are expressed as mean±SEM, n=4. *p<0.05, **p<0.01 for ASKO vs.

WT.

Fig. S5. Improvement of hepatic steatosis in ASKO mice after rosiglitazone treatment.

(A) Oil red O staining of the liver from WT and ASKO mice with or without Rosi.

Nuclei are counterstained with hematoxylin. The red color droplets represent the lipid

droplets. Scale bar is 50 μm.

(B) Liver weight of WT and ASKO mice with or without Rosi.

(C) TAG content of the liver from WT and ASKO mice with or without Rosi. TAG

content is normalized to liver weight.

(D, E and F) Expression of lipid synthesis (D), βoxidation (E) and insulin signaling

(F) related genes in the liver of WT and ASKO mice with or without Rosi. Values are

fold induction of gene expression normalized to the housekeeping gene Gapdh.

Values are the mean±SEM, n=58, *p<0.05, **p<0.01, ***p<0.001 for Rosi diet

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(Rosi) vs. normal diet (ND). # p<0.05 for ASKO vs. WT.

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