Coordination Among Lipid Droplets, Peroxisomes, and Mitochondria Regulates Energy Expenditure Through the CIDE-ATGL-Ppara Pathway in Adipocytes

Diabetes Volume 67, October 2018 1935

Coordination Among Lipid Droplets, Peroxisomes, and Mitochondria Regulates Energy Expenditure Through the CIDE-ATGL-PPARa Pathway in Adipocytes

Linkang Zhou,1 Miao Yu,1 Muhammad Arshad,2 Wenmin Wang,1 Ye Lu,1 Jingyi Gong,1 Yangnan Gu,3 Peng Li,1 and Li Xu1

Diabetes 2018;67:1935–1948 | https://doi.org/10.2337/db17-1452

Metabolic homeostasis is maintained by an interplay The rapid increase in the global prevalence of obesity is among tissues, organs, intracellular organelles, and mol- highly deleterious, resulting in the deregulation of glucose ecules. Cidea and Cidec are lipid droplet (LD)–associated and lipid metabolism and the development of other met- proteins that promote lipid storage in brown adipose abolic diseases, including insulin resistance, hyperglycemia, tissue (BAT) and white adipose tissue (WAT). Using hepatic steatosis, dyslipidemia, and chronic inflammation 2/2 2/2 2/2 ob/ob/Cidea , ob/ob/Cidec ,andob/ob/Cidea / (1,2). A severe reduction of white adipose tissue (WAT) with Cidec2/2 CIDE fi mouse models and -de cient cells, we ectopic lipid accumulation in other tissues and organs, METABOLISM studied metabolic regulation during severe obesity which is called lipodystrophy, can also cause these metabolic to identify ways to maintain metabolic homeostasis disorders (3,4). Thus, excessive lipid storage (obesity) and and promote antiobesity effects. The phenotype of the failure to store lipids in adipose tissue (lipodystrophy) ob/ob/Cidea2/2 ob/ob mice was similar to that of mice both contribute to metabolic disorders, although these in terms of serum parameters, adipose tissues, lipid disorders are associated with opposite fat storage phe- storage, and gene expression. Typical lipodystrophy notypes. In addition to maintaining an appropriate amount accompanied by insulin resistance occurred in ob/ob/ 2/2 of adipose tissue, fat tissue types and their interactions, Cidec mice, with ectopic storage of lipids in the including WAT as an energy storage site and brown adipose BAT and liver. Interestingly, double deficiency of Cidea and Cidec activated both WAT and BAT to consume tissue (BAT) and beige adipose tissue as energy-burning more energy and to increase insulin sensitivity com- sites, are important in systemic lipid metabolism (5). At pared with their behavior in the other three mouse the cellular level, the interaction between lipid droplets models. Increased lipolysis, which occurred on the LD (LDs) and other organelles in adipocytes also contributes fl surfaces and released fatty acids, led to activated to cellular lipid homeostasis, including metabolic ux b-oxidation and oxidative phosphorylation in perox- and the availability of fatty acids (6,7). Therefore, the isomes and mitochondria in CIDE-deficient adipocytes. balance and coordination of adipocyte types and sub- The coordination among LDs, peroxisomes, and mito- cellular organelles are important in maintaining systemic chondria was regulated by adipocyte triglyceride lipase metabolic homeostasis. (ATGL)-peroxisome proliferator–activated receptor a The CIDE (cell death-inducing DNA fragmentation (PPARa). Double deficiency of Cidea and Cidec acti- factor 45-like effector) proteins, including Cidea, Cideb, vated energy consumption in both WAT and BAT, which and Cidec/Fsp27, have emerged as important regulators in provided new insights into therapeutic approaches for the maintenance of lipid metabolic homeostasis, particu- obesity and diabetes. larly in adipose tissues and the liver (8). CIDE proteins are

1State Key Laboratory of Membrane Biology and Tsinghua-Peking Center for Life Received 7 December 2017 and accepted 29 June 2018. ’ Sciences, School of Life Sciences, Tsinghua University, Beijing, People s Republic This article contains Supplementary Data online at http://diabetes of China .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1452/-/DC1. 2Department of Bioinformatics and Biotechnology, International Islamic University, L.Z. and M.Y. contributed equally to this study. Islamabad, Pakistan 3Center for Plant Biology, Tsinghua-Peking Center for Life Sciences, School of Life © 2018 by the American Diabetes Association. Readers may use this article as Sciences, Tsinghua University, Beijing, People’s Republic of China long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Peng Li, [email protected], or Li Xu, xulilulu@ .org/content/license. tsinghua.edu.cn. 1936 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018 expressed mainly on the surface of LDs and induce the of Tsinghua University (Beijing, People’sRepublicofChina). formation of large LDs by promoting lipid exchange be- All animal experiments were approved by the Institutional tween LDs that come into contact with each other in Animal Care and Use Committee of Tsinghua University. adipocytes (Cidea and Cidec) and hepatocytes (Cideb) Energy Expenditure (9–11). Cidea is abundantly expressed in BAT, and a de- O consumption, CO production, the respiratory ex- ficiency of this protein promotes energy expenditure by 2 2 change rate, and energy expenditure were determined increasing AMPK activity (12,13). Cideb is stably expressed using a PhenoMaster/LabMaster System (TSE Systems in the liver and small intestine to promote lipid storage GmbH, Bad Homburg, Germany). Three-month-old mice and VLDL maturation for secretion (14–16). Cidec is were individually monitored for 48 h, and data were highly expressed in WAT and moderately expressed in collected at intervals of 27 min after a 1-day adaptation BAT. Cidec deficiency results in multilocular LDs and period. Parameters were compared between different markedly reduced lipid storage in WAT, with increased genotypes without considering body weight differences (24). energy expenditure (17,18). However, Cidec deficiency in obese animals promotes a lipodystrophy phenotype, with Serum Biochemical Analysis fatty liver, insulin resistance, and dyslipidemia (19,20), The concentrations of serum triacylglycerol (TAG) and which is similar to the phenotype of a patient carrying glycerol were measured using a serum triglyceride deter- a point mutation in the Cidec gene (21). Both Cidea and mination kit (catalog #TR0100; Sigma-Aldrich, St. Louis, Cidec are inducible and contribute to fatty liver develop- MO) and a free glycerol reagent (catalog #F6428; Sigma- ment, although these proteins are not expressed in the Aldrich), respectively, and the concentration of serum normal liver (22,23). Based on these observations, we nonesterified fatty acids (NEFAs) was measured using investigated what changes would occur in mice with a dou- a LabAssay NEFA kit (catalog #294–63601; Wako Pure ble deficiency of Cidea and Cidec under obesity-inducing Chemical Industries, Ltd., Osaka, Japan). Serum hor- conditions and whether this double deficiency would im- mone and cytokine levels were detected using a rat insulin prove metabolic disorders. radioimmunoassay kit (catalog #RI-13K; Millipore Sigma, Hence, four genetically engineered mouse models, Burlington, MA), mouse tumor necrosis factor-a (TNF- 2/2 2/2 including ob/ob, ob/ob/Cidea , ob/ob/Cidec ,and a) and interleukin-6 (IL-6) ELISA eBioscience Ready-SET- 2/2 2/2 ob/ob/Cidea /Cidec mice, were developed and com- GO! Kits (catalog #88-7324-86 and #88-7064-88; Thermo 2/2 pared. We observed a lean phenotype in ob/ob/Cidea / Fisher Scientific, Waltham, MA), an adiponectin kit (cat- 2/2 Cidec mice, with reduced fat storage and improved alog #ab108785; Abcam, Cambridge, U.K.), and hyaluron- insulin sensitivity. Increased energy consumption occurred idase (HAase) and laminin kits (catalog #SEB217Mu and 2/2 2/2 in the WAT and BAT of ob/ob/Cidea /Cidec mice. #SEA082Mu; Cloud-Clone Corp., Wuhan, People’sRepub- Interestingly, a dramatic increase in energy consumption lic of China). Tissue or cell catalase activity was deter- was observed through the coordinated interplay of mul- mined using a catalase assay kit (Beyotime Biotechnology, tiple organelles, including LDs, peroxisomes, and mito- Shanghai, People’sRepublicofChina). chondria; this effect was mediated by the adipocyte Detection of Lipid Levels triglyceride lipase (ATGL)-peroxisome proliferator–activated The lipid contents of tissues were determined by thin-layer receptor a (PPARa)pathway. chromatography, as previously described (20). The cellular RESEARCH DESIGN AND METHODS content of TAG was measured by an enzymatic reaction, according to the manufacturer instruction manual (catalog Animal Models 2/2 2/2 #290-63701; Wako Pure Chemical Industries, Ltd.). The Double-deficient ob/ob/Cidea and ob/ob/Cidec mice 2/2 2/2 tissues were lysed, and NEFA levels were determined using were generated by crossing Cidea or Cidec mice +/2 a LabAssay NEFA kit (catalog #294-63601; Wako Pure with leptin mice on a C57BL/6 background, as pre- 2/2 Chemical Industries, Ltd.). The NEFA profile was measured viously described (20,23). Triple-deficient ob/ob/Cidea / 2/2 +/2 using a Q Exactive Mass Spectrometer with an Orbitrap Cidec mice were generated by crossing leptin 2/2 2/2 +/2 2/2 Analyzer (Thermo Fisher Scientific) in the Lipidomics Cidea mice with Cidec mice or leptin Cidec 2/2 Center at Tsinghua University. mice with Cidea mice. All mice used for studies were male. Three-month-old mice that were subjected to random Glucose Tolerance Test and Insulin Tolerance Test feeding were sacrificed during the time period 2:00–5:00 The mice were fasted for 6 h and intraperitoneally injected P.M. for sample collection for the analyses presented in Figs. with glucose (0.5 g/kg body mass) for glucose tolerance 1, 2, and 4–6. Gonadal WAT was used in this study. Two- tests (GTTs). Following a 4-h fast, an intraperitoneal in- and 4-month-old mice were used in the analyses presented jection of insulin (2 units/kg body mass) was administered in Fig. 3. For the high-fat diet (HFD) treatments presented for insulin tolerance tests (ITTs). Blood glucose concen- in Supplementary Fig. 2, 2-month-old mice were provided trations were measured using a blood glucose monitor with an HFD for 2 months. For mouse studies, biological system (ACCU-CHEK Advantage II; Roche, Basel, Switzer- replicates were from independently tested individual mice. land). To examine in vivo insulin signaling, 4-month-old Mouse experiments were performed in the animal facility mice were fasted for 2 h, anesthetized and injected with diabetes.diabetesjournals.org Zhou and Associates 1937

Figure 1—Reduced body weight and fat mass and increased energy expenditure in ob/ob/Cidea2/2/Cidec2/2 mice. Three-month-old mice fed normal chow were used for the analyses presented in panels B–O. A: Growth curves of ob/ob, ob/ob/Cidea2/2, ob/ob/Cidec2/2, and ob/ob/Cidea2/2/Cidec2/2 mice. B: Food intake. Data are shown as grams of food per day per gram body weight. C: Representative photographs of BAT and WAT. D: Whole-body OCRs (VO2, expressed in milliliters per hour per mouse). E: Carbon dioxide production rate (VCO2, expressed in milliliters per hour per mouse). F: Respiratory quotient (VCO2/VO2)(F) and energy expenditure analyses (G) of 3-month- old mice subjected to a 12-h dark/light cycle and fed a standard diet for 48 h. The average values for the ﬁrst dark period (from 8:00 P.M. to 8:00 A.M.) are shown in the right panels. Serum TAG (H), serum glycerol (I), serum NEFAs (J), serum adiponectin (K), serum TNF-a (L), serum IL-6 (M), serum HAase (N), and serum laminin (O). For panels A–G, n = 4; for panels H–O, n =8.*P , 0.05, **P , 0.01, and ***P , 0.001. 1938 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018

Figure 2—Reduced TAG content and small LDs in ob/ob/Cidea2/2/Cidec2/2 mice. Three-month-old mice were maintained on normal chow for the analyses shown in A–H. A–C: Tissue TAG content (n = 8). D: Hematoxylin-eosin staining. Scale bars, 50 mm. E: Electron microscopy. Scale bars, 5 mm. F–H: The average LD diameter from 120 LDs in BAT, 100 LDs in WAT, and 50 LDs in the liver. **P , 0.01 and ***P , 0.001. insulin (5 units/kg body mass). After 5 min, the mice were DMEM with 10% FBS supplemented with 1 mg/mL insulin sacrificed, and tissues were collected for Western blotting. and1nmol/LT3for4days.Onday6,thecellswere trypsinized and recultured for imaging or Seahorse Analyzer BAT Stromal Vascular Cell Isolation and Differentiation (Seahorse Bioscience, Billerica, MA) experiments. Primary BAT stromal vascular cell isolation and differentiation were performed as previously described (17,25). Briefly, WAT Stromal Vascular Cell Isolation and Differentiation the interscapular brown fat pad was removed from neonates Primary stromal vascular cells were isolated from the white on postnatal day 2 to obtain primary brown adipocytes. The fat tissues of 1-month-old mice (26). Briefly, the WAT was confluent cells were induced to undergo differentiation by cut into small pieces and digested in buffer (Hanks’ buffer, DMEM with 10% FBS supplemented with 1 mg/mL insulin, catalog #14025-092; Thermo Fisher Scientific) with 2% BSA 1 nmol/L triiodothyronine (T3), 0.125 mmol/L indometha- and collagenase (catalog #C6885, 2 mg/mL; Sigma- cin, 2 mg/mL dexamethasone, and 0.5 mmol/L 3-isobutyl-1- Aldrich) for 40 min at 37°C. The confluent cells were methylxanthine (IBMX) for 2 days, followed by culture with induced to differentiate for 2 days in DMEM with 10% FBS, diabetes.diabetesjournals.org Zhou and Associates 1939

Figure 3—Increased insulin sensitivity in ob/ob/Cidea2/2/Cidec2/2 mice. Mice were fed normal chow until they were 2 months old (n = 5) for panels A– E or 4 months old for panels F–J. A and F: Fasting serum insulin levels. B and G: Fasting serum glucose levels. C and H: Fed serum glucose levels. GTTs (D and I) and ITTs (E and J)(n = 5). The right panel indicates the quantiﬁcationoftheareaunderthecurve(AUC).*P , 0.05, **P , 0.01, and ***P , 0.001.

5 mg/mL insulin, 2 mg/mL dexamethasone, 0.5 mmol/L IBMX, MEFs were differentiated at high density in high-glucose and 1 mm rosiglitazone, followed by culture for an additional DMEM plus 10% FBS, 0.8% biotin, 0.4% pantothe- 4 days in DMEM with 10% FBS supplemented with 5 mg/mL nate, 100 units/mL penicillin/streptomycin, 2 mmol/L insulin. On day 6, the cells were trypsinized and recultured L-glutamine, 10 mg/mL human transferrin, 1 mg/mL insulin, for imaging or Seahorse Analyzer experiments. 0.1 mmol/L cortisol, 2 nmol/L T3, 0.25 mmol/L dexamethasone, 0.5 mmol/LIBMX,and2mmol/L rosiglitazone. Mouse Embryonic Fibroblast Cell Isolation and The cells were then cultured without dexamethasone, Differentiation IBMX, and rosiglitazone for 4 days. Small interfering For mouse embryonic ﬁbroblast (MEF) isolation, 13.5- to RNAs (siRNAs) were introduced into differentiated 14.5-day mouse embryos were used (17). The isolated MEFs by electroporation using Amaxa Nucleofector II 1940 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018

(Lonza, Basel, Switzerland) with program A-033, according Microarray Analysis, Pathway Analysis, and Real-time to the manufacturer instructions. The sequence of the PCR siRNAusedtotargetATGLwas59-GCACATTTATCCC- Microarray analysis data have been deposited in Gene GGTGTA-39. The sequences of the siRNA used to target Expression Omnibus with the number GSE100989. Briefly, PPARa were 59-GCTTCTTTCGGCGAACTAT-39 and 59- equal amounts of total RNA from three mice were com- GCTAAAGTACGGTGTGTAT-39. The cells were cultured bined to form an RNA pool, and Affymetrix gene chips for 48 h before harvesting. Triplicate samples were tested (GeneChip Mouse Gene 1.0 ST Array; Affymetrix, Santa independently with MEFs isolated from three pregnant Clara, CA) were used for hybridization and data collection. mice. Quality control and statistical analyses of the complete microarray data were conducted using R/Bioconductor. Seahorse Analyzer Experiments The genes with altered expression ($1.5-fold) were map- The oxygen consumption rates (OCRs) of tissue explants ped to biological pathways using DAVID Bioinformatics were measured using an XF24 Analyzer (Seahorse Bio- Resources 6.8 (https://david.ncifcrf.gov/summary.jsp). The science). Approximately 4 mg of WAT or BAT pieces were heat map presented in Supplementary Fig. 4 was gener- washed with Seahorse Bioscience assay buffer containing ated as a selective profile of peroxisome-related genes. The 25 mmol/L glucose and 25 mmol/L HEPES (pH 7.4) and expression of the represented genes was confirmed by real- subsequently were cultured for 1 h in the center of a Sea- time PCR or Western blotting. Related PCR primers and anti- horse Bioscience XF24 islet capture microplate contain- bodies are listed in Supplementary Tables 4 and 5, respectively. ing 500 mL of Seahorse Bioscience assay buffer with Lipolysis 25 mmol/L glucose at 37°C without CO2.Oxygencon- sumption was measured six times and normalized to tissue WAT and BAT samples were collected from mice and cut weight. For cellular Seahorse Analyzer experiments, ;1 3 into small pieces. The tissues were washed three times with 104 cells/well (96-well plates) were maintained in XF assay DMEM and then cultured in DMEM for 1 h. The medium medium supplemented with 1 mmol/L sodium pyru- was collected, and the glycerol level was determined using vate, 4 mmol/L glutamine, and 25 mmol/L glucose. The a glycerol determination kit (catalog #F6428; Sigma-Aldrich). cells were subjected to a mitochondrial stress test by Statistics the addition of oligomycin (2 mmol/L), followed by car- fl The statistical data reported include results from at least bonyl cyanide 4-(tri uoromethoxy) phenylhydrazone three biological replicates. All results are expressed as m m (FCCP; 5 mol/L), and antimycin/rotenone (1 mol/L/ the mean 6 SEM. All statistical analyses were performed m 1 mol/L). in GraphPad Prism Version 5 (GraphPad Software, San Diego, CA). Significance was established using one-way Imaging ANOVA with Tukey multiple-comparison test (Figs. 1B–O, After differentiation, adipocytes were trypsinized and 2, 3A–C and F–H,4,5D and I, and 6 and Supplementary cultured on coverslips for 12 h, followed by immunostain- Figs. 1, 2, 5A and E,and6C and D) and a two-tailed Student ing. Briefly, the cells were incubated with MitoTracker t test (Figs. 5C and H and 7 and Supplementary Figs. 3B,4, (catalog #M7512; Thermo Fisher Scientific) before fixation, 5B, C, F, and G,6E, and 7). We used two-way repeated- followed by permeabilization with 0.4% Triton X-100. A measures ANOVA to evaluate the data presented in Figs. PMP70 antibody (catalog #ab3421; Abcam), as a peroxisome 1A and 3D, E, I, and J, and Supplementary Fig. 3C and D.In marker, and a secondary antibody (catalog #A11008; Thermo all cases, differences were considered significant at P , Fisher Scientific) were used for peroxisome staining. LDs 0.05. P values are indicated in each figure as *P , 0.05, and nuclei were stained with LipidTOX (catalog #H34477; **P , 0.01, and ***P , 0.001. Thermo Fisher Scientific) and Hoechst, respectively. For tissue immunofluorescence, deparaffinization, rehydration of paraffin-embedded sections, and antigen retrieval were RESULTS required. Nonspecific antigens were blocked with 5% BSA at Improved Metabolic Status With Increased Energy 2/2 2/2 4°C overnight, and the slideswereincubatedwithPMP70 Expenditure in ob/ob/Cidea /Cidec Mice (catalog #ab3421; Abcam) and Cox4 (catalog #11967; Cell To investigate the roles of Cidea and Cidec in lipid metab- Signaling Technology, Danvers, MA) antibodies overnight at olism, we compared the metabolic phenotypes of ob/ob, ob/ 2/2 2/2 2/2 2/2 4°C. The slides were then incubated with a secondary anti- ob/Cidea , ob/ob/Cidec ,andob/ob/Cidea /Cidec body (catalog #A11008 and #A11031; Thermo Fisher mice.Growthperformance,tissueweight,andserumparam- 2/2 Scientific). Nuclei were stained with DAPI for 10 min. eters were similar between ob/ob and ob/ob/Cidea mice Images of immunostained cells were collected on a Nikon (Fig. 1 and Supplementary Fig. 1). As a lipodystrophy model, 2/2 (Tokyo, Japan) A1R laser-scanning confocal microscope ob/ob/Cidec mice showed increased liver and BAT weights, with a CFI Plan Apo 1003 oil immersion objective (nu- withhigherserumTAG,HAase,andlamininlevels(Fig.1H, merical aperture 1.45) and 405/488/561/640-nm lasers. N,andO and Supplementary Fig. 1D). Interestingly, 2/2 2/2 Images of the cells after oil-red staining were collected on ob/ob/Cidea /Cidec micedidnotdeveloplipodystro- 2/2 a Nikon Eclipse 90i system. phy, which occurred in ob/ob/Cidec mice. Compared with diabetes.diabetesjournals.org Zhou and Associates 1941

Figure 4—Increased mitochondrial and peroxisome activity in the BAT and WAT of ob/ob/Cidea2/2/Cidec2/2 mice. Three-month-old mice that were fed a normal diet were used. Protein expression in the BAT (A) and WAT (B). C: Cox4 and PMP70 immunoﬂuorescence in the BAT and WAT. Scale bars, 10 mm. Basal OCR of BAT (D) and WAT (E) using Seahorse Bioscience equipment (n = 4). Catalase activity of BAT (F) and WAT (G) from the indicated mice (n = 4). WT, wild type. **P , 0.01 and ***P , 0.001.

2/2 2/2 2/2 2/2 2/2 ob/ob and ob/ob/Cidea mice, ob/ob/Cidea /Cidec ob/ob/Cidea /Cidec mice had the lowest BAT and mice exhibited an improved metabolic status, including WAT weights, whereas the liver weights of these mice were 2/2 a low body weight, high oxygen and energy consumption, low similar to those of ob/ob and ob/ob/Cidea mice (Fig. 1C levels of serum NEFAs and inﬂammatory cytokines (TNF-a and Supplementary Fig. 1C and D). No signiﬁcant differ- and IL-6), and high adiponectin levels (Fig. 1). In addition, ences in food intake per mouse were observed among these 1942 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018

A F

B G

C H

DE IJ

Figure 5—Increased mitochondrial and peroxisome pathway activity in Cidea and Cidec double-deﬁcient adipocytes. The BAT from newborn mice was used to isolate stromal vascular cells. The cells were then induced to differentiate to generate the data presented in A–E. The WAT from 1-month-old mice was used to isolate stromal vascular cells. The cells were then induced to differentiate to generate the data presented in F–J. A and F: Adipocytes were stained with BODIPY (boron-dipyrromethene) (white, indicates LDs), MitoTracker (red, indicates mitochondria), PMP70 (green, indicates peroxisomes), and Hoechst (blue, indicates nuclei). Scale bars, 10 mm. B, C, G, and H: OCR in the indicated adipocytes. Oligomycin, FCCP, and rotenone/antimycin A (RA) were added at the indicated time points. Cells from ﬁve mice were used. D and I: Catalase activity of the cells (n = 4). E and J: Western blot analysis showing the levels of various proteins in primary brown adipocytes. WT, wild type. *P , 0.05, **P , 0.01, and ***P , 0.001. diabetes.diabetesjournals.org Zhou and Associates 1943

Figure 6—Increased ATGL and PPARa expression in Cidea and Cidec double-deﬁcient mice. Three-month-old mice that were fed normal chow were used to generate the data presented in panels A–F. A and B: Expression of the indicated proteins in BAT and WAT. C and D: Lipolysis rates of BAT and WAT. The fat tissues were cultured for 1 h, and the culture media were collected for glycerol detection (n = 4). E and F: NEFA levels in tissues (n = 5). CGI58, abhydrolase domain containing 5; HSL, hormone-sensitive lipase; PGC1, peroxisome proliferator– activated receptor g coactivator; WT, wild type. **P , 0.01 and ***P , 0.001.

2/2 2/2 four types of genetically engineered mice, but food Cidea /Cidec mice fed an HFD were also examined, intake per gram of body weight was significantly increased and the observed tendency was similar to that of mice 2/2 2/2 2/2 in ob/ob/Cidec and ob/ob/Cidea /Cidec mice (Fig. with an ob/ob genetic background, including WAT and 1B and Supplementary Fig. 1B). BAT performance (Supplementary Fig. 2). Increases in liver 2/2 weight and hepatic TAG levels were observed in Cidea / 2/2 Decreased Triacylglycerol Storage and Small LDs in the Cidec mice compared with those of wild-type mice. Adipose Tissues of Cidea/Cidec Double-Deficient Mice As the key metabolic organs and CIDE-expressing tissues, Increased Insulin Sensitivity in ob/ob/Cidea2/2/ adipose tissues and the liver were selected for further Cidec2/2 Mice 2/2 study. The ob/ob/Cidea mice had moderately lower Next, we assessed the blood glucose levels and insulin 2/2 2/2 TAG levels in BAT and liver than did the ob/ob mice, sensitivity status of ob/ob/Cidea /Cidec mice com- 2/2 but no differences were observed in WAT (Fig. 2A–C). pared with those of ob/ob, ob/ob/Cidea , and ob/ob/ 2/2 2/2 The ob/ob/Cidec mice failed to store TAG in WAT, with Cidec mice by GTTs and ITTs when the mice were 2, a much lower TAG content in WAT, but higher TAG levels 4, and 8.5 months old. The fasting serum glucose and 2/2 2/2 2/2 were observed in the BAT and liver of ob/ob/Cidec insulin levels of ob/ob/Cidea /Cidec mice were lower mice than in the other three types of mice (Fig. 2A–C). In than those of the other three types of mice at 2 and 2/2 2/2 contrast, ob/ob/Cidea Cidec mice had obviously re- 4 months of age (Fig. 3A, B, F, and G). The fed serum duced TAG levels in BAT and WAT and maintained liver glucose levels exhibited no differences among ob/ob, 2/2 2/2 2/2 TAG levels similar to those of ob/ob mice (Fig. 2A–C). ob/ob/Cidea ,andob/ob/Cidea /Cidec mice, but the Consistent with these findings, compared with the LD fed serum glucose level was slightly higher in 2-month-old 2/2 2/2 size of ob/ob mice, Cidea deficiency (ob/ob/Cidea ) ob/ob/Cidec mice and was significantly higher at age resulted in slightly smaller LDs in BAT and liver (Fig. 4months(Fig.3C and H). Insulin sensitivity became 2D–H).ComparedwiththeLDsizeoftheob/ob and complex at different ages in these four types of genetically 2/2 2/2 ob/ob/Cidea mice, Cidec deficiency (ob/ob/Cidec ) engineered mice (Fig. 3D, E, I, and J, Supplementary Fig. caused dramatically smaller LDs in WAT but larger LDs 3C and D, and Supplementary Table 6). No difference in in BAT and liver (Fig. 2D–H). Compared with the LD size insulin sensitivity was observed between ob/ob and ob/ob/ 2/2 2/2 2/2 of ob/ob and ob/ob/Cidea mice, double deficiency of Cidea mice from 2 to 8.5 months of age. ob/ob/Cidec 2/2 2/2 Cidea and Cidec (ob/ob/Cidea Cidec mice) caused mice were more sensitive to insulin than ob/ob and ob/ 2/2 strikingly smaller LDs in both BAT and WAT (Fig. 2D– ob/Cidea mice, according to GTT curves, at 2 months 2/2 2/2 H). The performance of wild-type, Cidea , Cidec , and of age (Fig. 3D). However, compared with ob/ob and 1944 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018

Figure 7—ATGL and PPARa mediate increased mitochondrial and peroxisome pathway activity in Cidea and Cidec double-deficient adipocytes. Cidea and Cidec double-deficient MEFs were differentiated. siRNAs against PPARa (A–C) or ATGL (D–F) were electroporated into cells. A and D: Protein expression profiles. B and E: Expression profile of mitochondria-related genes. C and F: Expression profile of peroxisome-related genes. G: Proposed model for the roles of Cidea and Cidec in the regulation of lipid metabolism and energy expenditure. Cidea and Cidec deficiency led to smaller LDs and increased expression of ATGL, which led to increased lipolysis. Free fatty acid release increased the protein levels of PPARa, which induced the expression of genes related to the mitochondrial and peroxisome pathway, consequently increasing fatty acid oxidation and oxidative phosphorylation. NC, negative control; PGC1, peroxisome proliferator–activated receptor g coactivator; si, small interfering; WT, wild type. *P , 0.05, **P , 0.01, and ***P , 0.001. diabetes.diabetesjournals.org Zhou and Associates 1945

2/2 ob/ob/Cidea mice, these mice exhibited similar insulin activate mitochondria and peroxisomes. It is worth noting sensitivity when they were 4 months old (Fig. 3I and J) and that Cidec deficiency was sufficient to activate mitochondria developed severe insulin resistance when they were and peroxisomes in white adipocytes, but double deficiency of 8.5 months old, according to ITT curves (Supplementary Cidea and Cidec was required for the activation of mitochon- Fig. 3D). Interestingly, compared with that of the other dria and peroxisomes in brown adipocytes. three types of genetically modified mice, the insulin sen- 2/2 2/2 a sitivity of ob/ob/Cidea /Cidec mice was significantly ATGL-PPAR Is Required for Increased Mitochondrial and Peroxisome Activity improved at 2, 4, and 8.5 months of age. Consistent with fi In addition to activated mitochondria and peroxisomes, this nding, the phosphorylation status of AKT was dra- Cidea2/2 Cidec2/2 matically increased in the BAT and WAT of ob/ob/ smaller LDs were always observed in / 2/2 2/2 adipocytes in vivo and in vitro (Figs. 2E and 5A and F). Cidea /Cidec mice compared with those of ob/ob 2/2 Consistent with this finding, protein levels of ATGL and and ob/ob/Cidea mice (Supplementary Fig. 3A and B). CGI58 (a key lipolysis enzyme and regulator, respectively) fi fi were signi cantly increased in brown and white adipocytes Signi cantly Increased Mitochondrial and Peroxisome Cidea Cidec fi E J Activity in Cidea2/2/Cidec2/2 Adipocytes when both and were de cient (Figs. 5 and and 6A and B). The levels of the lipolysis products glycerol A microarray analysis was performed to observe transcrip- and NEFAs were also enhanced in the BAT and WAT of tional changes in the BAT, WAT, and liver of ob/ob and ob/ 2/2 2/2 2/2 2/2 ob/ob/Cidea /Cidec C–F ob/Cidea /Cidec mice. The enriched pathways of the mice (Fig. 6 ), demonstrating increased lipolysis. Lipidomics analysis showed that nearly altered genes were analyzed to identify the key upregulated all species of free fatty acids were significantly increased, pathways, focusing mainly on mitochondria, peroxisomes, fatty including fatty acids with different chain lengths and acid oxidation, and oxidative phosphorylation in both the BAT 2/2 2/2 saturation degrees (Supplementary Fig. 7A and B). PPARa and WAT of ob/ob/Cidea /Cidec mice compared with and peroxisome proliferator–activated receptor g coacti- those of their ob/ob counterparts (Supplementary Tables 1–3). vator 1a, which are known to regulate mitochondrial and Further, a significant induction of mitochondrial and peroxisome biogenesis, were also dramatically induced in peroxisome-related proteins and genes, together with an ob/ob/Cidea2/2 Cidec2/2 increased number of mitochondria and peroxisomes, was the BAT and WAT of / mice (Fig. 2/2 2/2 6A and B and Supplementary Fig. 7C and D). To determine observed in the BAT of ob/ob/Cidea /Cidec mice and 2/2 2/2 2/2 whether PPARa andATGLwereinvolvedinthisincreased in the WAT of ob/ob/Cidec and ob/ob/Cidea /Cidec b-oxidation, PPARa and ATGL were efficiently knocked mice (Fig. 4 and Supplementary Fig. 4). Consistent with this Cidea2/2/Cidec2/2 A–F fi down in MEFs from mice (Fig. 7 ). nding, dramatically increased oxygen consumption and ob/ob/Cidea2/2/Cidec2/2 ob/ob/Cidea2/2/ Consistent with those of mice, catalase activity were observed in the BAT of Cidea2/2/ Cidec2/2 ob/ob/Cidec2/2 PPARa and ATGL levels were increased in mice and in the WAT of and Cidec2/2 ob/ob/Cidea2/2/Cidec2/2 D–G MEFs compared with those of wild-type mice mice (Fig. 4 ), demonstrat- A D ing increased activity of mitochondria and peroxisomes. (Fig. 7 and ). Genes related to mitochondria and peroxisomes, including Cpt1, Acox1, the Acad family, and the Primary stromal vascular cells from BAT or WAT were 2/2 Pex family, were significantly induced in Cidea / induced and differentiated into brown or white adipocytes, 2/2 Cidec MEFs (Fig. 7B and C). When PPARa or ATGL respectively, to observe changes in LDs, mitochondria, and 2/2 2/2 was knocked down in Cidea /Cidec MEFs, this in- peroxisomes (Fig. 5). Interestingly, compared with that of creased gene expression was blocked, indicating that the their wild-type counterparts, an increased number of mito- increased activity of mitochondria and peroxisomes was chondria and peroxisomes was observed in brown and white 2/2 2/2 regulated by PPARa or ATGL (Fig. 7B, C, E, and F). adipocytes from Cidea /Cidec mice; this change was a accompanied by smaller LDs and lower cellular TAG levels Interestingly, the knockdown of PPAR did not affect the expression of ATGL (Fig. 7A). However, the knock- (Fig. 5A and F and Supplementary Fig. 5A, D,andE). down of ATGL suppressed the expression of PPARa (Fig. Consistent with this finding, genes related to the activity 7D), suggesting that increased expression of PPARa in and biogenesis of mitochondria and peroxisomes were 2/2 2/2 2/2 2/2 Cidea /Cidec MEFs was regulated by induced ATGL. significantly induced in Cidea /Cidec adipocytes (Sup- plementary Fig. 5B, C, F,andG). Similar to those of ob/ob/ 2/2 2/2 2/2 2/2 Cidea /Cidec mice, Cidea /Cidec adipocytes ex- DISCUSSION hibited increased basal and maximal oxygen consumption The key novel finding in the current study was the obser- (Fig. 5B, C, G,andH) and catalase activity (Fig. 5D and I). vation that a coordinated interplay among different organ- 2/2 2/2 In addition, differentiated Cidea /Cidec MEF cells elles, including LDs, peroxisomes, and mitochondria, is exhibited low cellular TAG contents and increased expression mediated by the CIDE-ATGL-PPARa pathway in adipose of genes related to mitochondria and peroxisomes (Supple- tissues. Similar to our previous reports (12,17,20), the mentary Fig. 6). Overall, the three sets of data from ob/ob/ current study demonstrates that increased lipolysis occurs 2/2 2/2 Cidea /Cidec mice, differentiated stromal vascular cells in CIDE-deficient adipocytes; this lipolysis occurred on the 2/2 2/2 from BAT or WAT, and MEF cells from Cidea /Cidec surface of LDs and released fatty acids. The coordination of embryos all demonstrated that CIDE protein deficiency could peroxisomes and mitochondria is required to complete the 1946 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018 oxidation of long-chain acyl-CoA, the citric acid cycle, and ATGL activity and lipolysis (43). Cidec was also found to interact oxidative phosphorylation and ultimately to produce CO2 with early growth response protein 1 to inhibit ATGL pro- CIDE and H2O (27), which was demonstrated in the current moter activity (44). Therefore, increased lipolysis in - study. Increased energy expenditure and increased mito- deficient adipocytes was potentially regulated by the 2/2 chondrial activity were previously reported in Cidea , decreased size of LDs and increased ATGL enzyme activity 2/2 2/2 2/2 Cideb , Cidec , and ob/ob/Cidec mice (8,28). The or protein expression levels, although the mechanism by active regulation of peroxisomes, in addition to mitochon- which ATGL activity was regulated remains unclear. dria, by CIDE proteins is proposed in the current study. The current study helps to elucidate the functions of Peroxisomes and mitochondria both contain fatty acid Cidea and Cidec in adipocytes. Both Cidea and Cidec are b-oxidation machinery; however, the enzymes and reac- expressed in brown adipocytes, but white adipocytes spe- tion processes used for b-oxidation in peroxisomes and cifically and abundantly express Cidec. Our data demon- 2/2 mitochondria vary significantly. Peroxisomes are respon- strated that significant changes occurred in Cidec white sible for the oxidation of very-long-chain fatty acids to adipocytes, even when Cidea was induced to some degree, produce shortened acyl-CoAs, acetyl-CoA, and propionyl- indicating the predominant role of Cidec in white adipo- CoA. We observed that the key enzymes Acadvl and cytes. However, in the mouse model and in primary brown ACOX1, which are specific to the peroxisome for the adipocytes, the double deficiency of Cidea and Cidec strik- oxidation of very-long-chain fatty acids (29), were upregu- ingly decreased cellular TAG levels and activated mitochon- lated when CIDE proteins were deficient. In addition, CIDE dria and peroxisomes in brown adipocytes, demonstrating deficiency caused increased expression of several peroxins, the redundant roles of Cidea and Cidec in brown adipocytes. which are involved in peroxisome biogenesis. For example, It is widely regarded that inducing WAT browning/beiging Pex3 and Pex16 play multiple roles in the direct targeting is an effective way to alleviate obesity or prevent obe- of peroxisomal membrane proteins (PMPs) and in the sity development in mice and humans (45–48). Significant synthesis of the peroxisome membrane (30,31). Pex5 and browning and increased energy consumption occurred in 2/2 Pex7 are responsible for the targeting of matrix proteins the WAT of ob/ob/Cidec mice, but these mice still and the import machinery. In addition to the increased exhibited metabolic disorder due to severe fatty liver and mRNA and protein expression levels of these molecular inactive BAT. This finding shows that BAT function should markers, immunofluorescence analysis demonstrated in- be considered even when browning is induced in WAT. The creased contents of mitochondria and peroxisomes in current study demonstrates the simultaneous activation of BAT CIDE-deficient adipose tissues or cells. Moreover, increased and WAT in Cidea and Cidec double-deficient mice contributed OCRs, ATP products, and catalase activity directly supported to dramatically increased energy consumption, providing some the enhanced activity of mitochondria and peroxisomes. additional strategies when considering obesity therapy. The current study has demonstrated that the ATGL- Recently, a new isoform of Cidec, designated Cidec2/ PPARa pathway is necessary to activate peroxisomes and Fsp27b, was identified in fatty liver; this isoform contains mitochondria in CIDE-deficient adipocytes. Several pre- 10 additional amino acids at the N terminus of the original vious studies reported ATGL activated PPARa/d and mi- Cidec/Fsp27a (49). Both Cidec/Fsp27a and Cidec2/ tochondrial metabolism in the liver, heart, small intestine, Fsp27b are localized on the surface of LDs and suppress pancreas, and skeletal muscle (32–37). The current study lipolysis (49). More recently, Cidec2/Fsp27b was reported has revealed the involvement of the ATGL-PPARa pathway to be expressed in BAT and to prevent LD fusion by in the metabolic activity of both BAT and WAT. ATGL- inhibiting Cidea function (50). It is well known that deficient mice showed reduced gene expression related to CIDE proteins promote LD fusion and lipid storage oxidative phosphorylation (38) and defective lipolysis, and (51). In the current study, we also observed two bands these mice accumulated large amounts of lipids in the for Cidec in wild-type BAT; the upper band (designated heart, causing cardiac dysfunction and premature death Cidec2/Fsp27b) was more abundant than the lower (39,40). Similar defects in lipid and energy disorders were band (designated Cidec/Fsp27a), which appeared as the reported in humans with ATGL mutations (41,42). This only form in WAT (Supplementary Fig. 8). However, in the evidence suggested that lipolysis induced by ATGL is ob/ob BAT, Cidec/Fsp27a was more abundant than necessary to promote the expression of PPARa/d and Cidec2/Fsp27b. The current study did not provide any its target genes. The induction of increased lipolysis by direct evidence to show different functions of Cidec iso- ATGL in CIDE-deficient cells could be explained from two forms in BAT, which has been proposed previously (50). aspects. First, CIDE proteins play a predominant role in LD In conclusion, the current study demonstrates that the fusion (8,11), and double deficiency of Cidea and Cidec led coupled functional interplay among LDs, peroxisomes, and to a failure to form large LDs in adipocytes. Small LDs have mitochondria is mediated by the ATGL-PPARa pathway in an enlarged total surface area, which makes the LDs Cidea and Cidec double-deficient adipocytes. accessible to lipase attack and lipolysis. Second, the double deficiency of Cidea and Cidec significantly increased the protein level or activity of ATGL in adipocytes. Cidec was Acknowledgments. The authors thank the members of P.L.’s laboratory at reported to physically interact with ATGL and inhibit Tsinghua University for their helpful discussion. The authors also thank Libing Mu diabetes.diabetesjournals.org Zhou and Associates 1947

(Tsinghua University) for assistance with drawing the model. In addition, the 16. Zhang LJ, Wang C, Yuan Y, et al. Cideb facilitates the lipidation of chylo- authors thank Pengcheng Jiao for assistance with the Seahorse Analyzer experi- microns in the small intestine. J Lipid Res 2014;55:1279–1287 ments and Jinyu Wang and Huizhen Cao (all from Tsinghua University) for 17. Toh SY, Gong J, Du G, et al. Up-regulation of mitochondrial activity and assistance with image processing. acquirement of brown adipose tissue-like property in the white adipose tissue of Funding. This work was supported by grants from the National Natural fsp27 deficient mice. PLoS One 2008;3:e2890 Science Foundation of China (31430040, 31690103, and 31621063 to P.L. 18. Nishino N, Tamori Y, Tateya S, et al. 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