Coordination Among Lipid Droplets, Peroxisomes, andMitochondria Regulates Energy Expenditure Through theCIDE-ATGL-PPARa Pathway in AdipocytesLinkang 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 interplayamong tissues, organs, intracellular organelles, andmol-ecules. Cidea andCidec are lipid droplet (LD)–associatedproteins that promote lipid storage in brown adiposetissue (BAT) and white adipose tissue (WAT). Usingob/ob/Cidea2/2, ob/ob/Cidec2/2, and ob/ob/Cidea2/2/Cidec2/2 mouse models and CIDE-deficient cells, westudied metabolic regulation during severe obesityto identify ways to maintain metabolic homeostasisand promote antiobesity effects. The phenotype ofob/ob/Cidea2/2mice was similar to that of ob/obmicein terms of serum parameters, adipose tissues, lipidstorage, and gene expression. Typical lipodystrophyaccompanied by insulin resistance occurred in ob/ob/Cidec2/2 mice, with ectopic storage of lipids in theBAT and liver. Interestingly, double deficiency of Cideaand Cidec activated both WAT and BAT to consumemore energy and to increase insulin sensitivity com-pared with their behavior in the other three mousemodels. Increased lipolysis, which occurred on the LDsurfaces and released fatty acids, led to activatedb-oxidation and oxidative phosphorylation in perox-isomes and mitochondria in CIDE-deficient adipocytes.The coordination among LDs, peroxisomes, and mito-chondria was regulated by adipocyte triglyceride lipase(ATGL)-peroxisome proliferator–activated receptor a

(PPARa). Double deficiency of Cidea and Cidec acti-vated energy consumption in both WAT and BAT, whichprovided new insights into therapeutic approaches forobesity and diabetes.

The rapid increase in the global prevalence of obesity ishighly deleterious, resulting in the deregulation of glucoseand lipid metabolism and the development of other met-abolic diseases, including insulin resistance, hyperglycemia,hepatic steatosis, dyslipidemia, and chronic inflammation(1,2). A severe reduction of white adipose tissue (WAT) withectopic lipid accumulation in other tissues and organs,which is called lipodystrophy, can also cause these metabolicdisorders (3,4). Thus, excessive lipid storage (obesity) andthe failure to store lipids in adipose tissue (lipodystrophy)both contribute to metabolic disorders, although thesedisorders are associated with opposite fat storage phe-notypes. In addition to maintaining an appropriate amountof adipose tissue, fat tissue types and their interactions,including WAT as an energy storage site and brown adiposetissue (BAT) and beige adipose tissue as energy-burningsites, are important in systemic lipid metabolism (5). Atthe cellular level, the interaction between lipid droplets(LDs) and other organelles in adipocytes also contributesto cellular lipid homeostasis, including metabolic fluxand the availability of fatty acids (6,7). Therefore, thebalance and coordination of adipocyte types and sub-cellular organelles are important in maintaining systemicmetabolic homeostasis.

The CIDE (cell death-inducing DNA fragmentationfactor 45-like effector) proteins, including Cidea, Cideb,and Cidec/Fsp27, have emerged as important regulators inthe maintenance of lipid metabolic homeostasis, particu-larly in adipose tissues and the liver (8). CIDE proteins are

1State Key Laboratory of Membrane Biology and Tsinghua-Peking Center for LifeSciences, School of Life Sciences, Tsinghua University, Beijing, People’s Republicof China2Department of Bioinformatics and Biotechnology, International Islamic University,Islamabad, Pakistan3Center for Plant Biology, Tsinghua-Peking Center for Life Sciences, School of LifeSciences, Tsinghua University, Beijing, People’s Republic of China

Corresponding author: Peng Li, li-peng@mail.tsinghua.edu.cn, or Li Xu, xulilulu@tsinghua.edu.cn.

Received 7 December 2017 and accepted 29 June 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1452/-/DC1.

L.Z. and M.Y. contributed equally to this study.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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METABOLISM

expressed mainly on the surface of LDs and induce theformation of large LDs by promoting lipid exchange be-tween LDs that come into contact with each other inadipocytes (Cidea and Cidec) and hepatocytes (Cideb)(9–11). Cidea is abundantly expressed in BAT, and a de-ficiency of this protein promotes energy expenditure byincreasing AMPK activity (12,13). Cideb is stably expressedin the liver and small intestine to promote lipid storageand VLDL maturation for secretion (14–16). Cidec ishighly expressed in WAT and moderately expressed inBAT. Cidec deficiency results in multilocular LDs andmarkedly reduced lipid storage in WAT, with increasedenergy expenditure (17,18). However, Cidec deficiency inobese animals promotes a lipodystrophy phenotype, withfatty liver, insulin resistance, and dyslipidemia (19,20),which is similar to the phenotype of a patient carryinga point mutation in the Cidec gene (21). Both Cidea andCidec are inducible and contribute to fatty liver develop-ment, although these proteins are not expressed in thenormal liver (22,23). Based on these observations, weinvestigated what changes would occur in mice with a dou-ble deficiency of Cidea and Cidec under obesity-inducingconditions and whether this double deficiency would im-prove metabolic disorders.

Hence, four genetically engineered mouse models,including ob/ob, ob/ob/Cidea2/2, ob/ob/Cidec2/2, andob/ob/Cidea2/2/Cidec2/2mice, were developed and com-pared. We observed a lean phenotype in ob/ob/Cidea2/2/Cidec2/2 mice, with reduced fat storage and improvedinsulin sensitivity. Increased energy consumption occurredin the WAT and BAT of ob/ob/Cidea2/2/Cidec2/2mice.Interestingly, a dramatic increase in energy consumptionwas observed through the coordinated interplay of mul-tiple organelles, including LDs, peroxisomes, and mito-chondria; this effect was mediated by the adipocytetriglyceride lipase (ATGL)-peroxisome proliferator–activatedreceptor a (PPARa) pathway.

RESEARCH DESIGN AND METHODS

Animal ModelsDouble-deficient ob/ob/Cidea2/2 and ob/ob/Cidec2/2 micewere generated by crossing Cidea2/2 or Cidec2/2 micewith leptin+/2 mice on a C57BL/6 background, as pre-viously described (20,23). Triple-deficient ob/ob/Cidea2/2/Cidec2/2 mice were generated by crossing leptin+/2

Cidea2/2 mice with Cidec2/2 mice or leptin+/2Cidec2/2

mice with Cidea2/2 mice. All mice used for studies weremale. Three-month-old mice that were subjected to randomfeeding were sacrificed during the time period 2:00–5:00P.M. for sample collection for the analyses presented in Figs.1, 2, and 4–6. Gonadal WAT was used in this study. Two-and 4-month-old mice were used in the analyses presentedin Fig. 3. For the high-fat diet (HFD) treatments presentedin Supplementary Fig. 2, 2-month-old mice were providedwith an HFD for 2 months. For mouse studies, biologicalreplicates were from independently tested individual mice.Mouse experiments were performed in the animal facility

of Tsinghua University (Beijing, People’s Republic of China).All animal experiments were approved by the InstitutionalAnimal Care and Use Committee of Tsinghua University.

Energy ExpenditureO2 consumption, CO2 production, the respiratory ex-change rate, and energy expenditure were determinedusing a PhenoMaster/LabMaster System (TSE SystemsGmbH, Bad Homburg, Germany). Three-month-old micewere individually monitored for 48 h, and data werecollected at intervals of 27 min after a 1-day adaptationperiod. Parameters were compared between differentgenotypes without considering body weight differences (24).

Serum Biochemical AnalysisThe concentrations of serum triacylglycerol (TAG) andglycerol were measured using a serum triglyceride deter-mination kit (catalog #TR0100; Sigma-Aldrich, St. Louis,MO) and a free glycerol reagent (catalog #F6428; Sigma-Aldrich), respectively, and the concentration of serumnonesterified fatty acids (NEFAs) was measured usinga LabAssay NEFA kit (catalog #294–63601; Wako PureChemical Industries, Ltd., Osaka, Japan). Serum hor-mone and cytokine levels were detected using a rat insulinradioimmunoassay kit (catalog #RI-13K; Millipore Sigma,Burlington, MA), mouse tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) ELISA eBioscience Ready-SET-GO! Kits (catalog #88-7324-86 and #88-7064-88; ThermoFisher Scientific, Waltham, MA), an adiponectin kit (cat-alog #ab108785; Abcam, Cambridge, U.K.), and hyaluron-idase (HAase) and laminin kits (catalog #SEB217Mu and#SEA082Mu; Cloud-Clone Corp., Wuhan, People’s Repub-lic of China). Tissue or cell catalase activity was deter-mined using a catalase assay kit (Beyotime Biotechnology,Shanghai, People’s Republic of China).

Detection of Lipid LevelsThe lipid contents of tissues were determined by thin-layerchromatography, as previously described (20). The cellularcontent of TAG was measured by an enzymatic reaction,according to the manufacturer instruction manual (catalog#290-63701; Wako Pure Chemical Industries, Ltd.). Thetissues were lysed, and NEFA levels were determined usinga LabAssay NEFA kit (catalog #294-63601; Wako PureChemical Industries, Ltd.). The NEFA profile was measuredusing a Q Exactive Mass Spectrometer with an OrbitrapAnalyzer (Thermo Fisher Scientific) in the LipidomicsCenter at Tsinghua University.

Glucose Tolerance Test and Insulin Tolerance TestThe mice were fasted for 6 h and intraperitoneally injectedwith glucose (0.5 g/kg body mass) for glucose tolerancetests (GTTs). Following a 4-h fast, an intraperitoneal in-jection of insulin (2 units/kg body mass) was administeredfor insulin tolerance tests (ITTs). Blood glucose concen-trations were measured using a blood glucose monitorsystem (ACCU-CHEK Advantage II; Roche, Basel, Switzer-land). To examine in vivo insulin signaling, 4-month-oldmice were fasted for 2 h, anesthetized and injected with

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Figure 1—Reduced body weight and fat mass and increased energy expenditure in ob/ob/Cidea2/2/Cidec2/2 mice. Three-month-old micefed 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, andob/ob/Cidea2/2/Cidec2/2 mice. B: Food intake. Data are shown as grams of food per day per gram body weight. C: Representativephotographs 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-oldmice 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 8:00A.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.

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insulin (5 units/kg body mass). After 5 min, the mice weresacrificed, and tissues were collected for Western blotting.

BAT Stromal Vascular Cell Isolation and DifferentiationPrimary BAT stromal vascular cell isolation and differenti-ationwere performed as previously described (17,25). Briefly,the interscapular brown fat pad was removed from neonateson postnatal day 2 to obtain primary brown adipocytes. Theconfluent cells were induced to undergo differentiation byDMEM with 10% FBS supplemented with 1 mg/mL insulin,1 nmol/L triiodothyronine (T3), 0.125 mmol/L indometha-cin, 2 mg/mL dexamethasone, and 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX) for 2 days, followed by culture with

DMEM with 10% FBS supplemented with 1 mg/mL insulinand 1 nmol/L T3 for 4 days. On day 6, the cells weretrypsinized and recultured for imaging or Seahorse Analyzer(Seahorse Bioscience, Billerica, MA) experiments.

WAT Stromal Vascular Cell Isolation and DifferentiationPrimary stromal vascular cells were isolated from the whitefat tissues of 1-month-old mice (26). Briefly, the WAT wascut into small pieces and digested in buffer (Hanks’ buffer,catalog #14025-092; Thermo Fisher Scientific) with 2% BSAand collagenase (catalog #C6885, 2 mg/mL; Sigma-Aldrich) for 40 min at 37°C. The confluent cells wereinduced to differentiate for 2 days in DMEMwith 10% FBS,

Figure 2—Reduced TAG content and small LDs in ob/ob/Cidea2/2/Cidec2/2 mice. Three-month-old mice were maintained on normal chowfor 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.

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5mg/mL insulin, 2mg/mL dexamethasone, 0.5mmol/L IBMX,and 1 mm rosiglitazone, followed by culture for an additional4 days in DMEMwith 10% FBS supplemented with 5 mg/mLinsulin. On day 6, the cells were trypsinized and reculturedfor imaging or Seahorse Analyzer experiments.

Mouse Embryonic Fibroblast Cell Isolation andDifferentiationFor mouse embryonic fibroblast (MEF) isolation, 13.5- to14.5-day mouse embryos were used (17). The isolated

MEFs were differentiated at high density in high-glucoseDMEM plus 10% FBS, 0.8% biotin, 0.4% pantothe-nate, 100 units/mL penicillin/streptomycin, 2 mmol/LL-glutamine, 10mg/mL human transferrin, 1mg/mL insulin,0.1 mmol/L cortisol, 2 nmol/L T3, 0.25 mmol/L dexameth-asone, 0.5 mmol/L IBMX, and 2 mmol/L rosiglitazone.The cells were then cultured without dexamethasone,IBMX, and rosiglitazone for 4 days. Small interferingRNAs (siRNAs) were introduced into differentiatedMEFs by electroporation using Amaxa Nucleofector II

Figure 3—Increased insulin sensitivity inob/ob/Cidea2/2/Cidec2/2mice.Micewere fednormal chowuntil theywere2monthsold (n=5) for panelsA–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 quantification of the area under the curve (AUC). *P , 0.05, **P , 0.01, and***P , 0.001.

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(Lonza, Basel, Switzerland) with program A-033, accordingto the manufacturer instructions. The sequence of thesiRNA used to target ATGL was 59-GCACATTTATCCC-GGTGTA-39. The sequences of the siRNA used to targetPPARa were 59-GCTTCTTTCGGCGAACTAT-39 and 59-GCTAAAGTACGGTGTGTAT-39. The cells were culturedfor 48 h before harvesting. Triplicate samples were testedindependently with MEFs isolated from three pregnantmice.

Seahorse Analyzer ExperimentsThe oxygen consumption rates (OCRs) of tissue explantswere measured using an XF24 Analyzer (Seahorse Bio-science). Approximately 4 mg of WAT or BAT pieces werewashed with Seahorse Bioscience assay buffer containing25 mmol/L glucose and 25 mmol/L HEPES (pH 7.4) andsubsequently were cultured for 1 h in the center of a Sea-horse Bioscience XF24 islet capture microplate contain-ing 500 mL of Seahorse Bioscience assay buffer with25 mmol/L glucose at 37°C without CO2. Oxygen con-sumption was measured six times and normalized to tissueweight. For cellular Seahorse Analyzer experiments, ;13104 cells/well (96-well plates) were maintained in XF assaymedium supplemented with 1 mmol/L sodium pyru-vate, 4 mmol/L glutamine, and 25 mmol/L glucose. Thecells were subjected to a mitochondrial stress test bythe addition of oligomycin (2 mmol/L), followed by car-bonyl cyanide 4-(trifluoromethoxy) phenylhydrazone(FCCP; 5 mmol/L), and antimycin/rotenone (1 mmol/L/1 mmol/L).

ImagingAfter differentiation, adipocytes were trypsinized andcultured on coverslips for 12 h, followed by immunostain-ing. Briefly, the cells were incubated with MitoTracker(catalog #M7512; Thermo Fisher Scientific) before fixation,followed by permeabilization with 0.4% Triton X-100. APMP70 antibody (catalog #ab3421; Abcam), as a peroxisomemarker, and a secondary antibody (catalog #A11008; ThermoFisher Scientific) were used for peroxisome staining. LDsand nuclei were stained with LipidTOX (catalog #H34477;Thermo Fisher Scientific) and Hoechst, respectively. Fortissue immunofluorescence, deparaffinization, rehydrationof paraffin-embedded sections, and antigen retrieval wererequired. Nonspecific antigens were blocked with 5% BSA at4°C overnight, and the slides were incubated with PMP70(catalog #ab3421; Abcam) and Cox4 (catalog #11967; CellSignaling Technology, Danvers, MA) antibodies overnight at4°C. The slides were then incubated with a secondary anti-body (catalog #A11008 and #A11031; Thermo FisherScientific). Nuclei were stained with DAPI for 10 min.Images of immunostained cells were collected on a Nikon(Tokyo, Japan) A1R laser-scanning confocal microscopewith a CFI Plan Apo 1003 oil immersion objective (nu-merical aperture 1.45) and 405/488/561/640-nm lasers.Images of the cells after oil-red staining were collected ona Nikon Eclipse 90i system.

Microarray Analysis, Pathway Analysis, and Real-timePCRMicroarray analysis data have been deposited in GeneExpression Omnibus with the number GSE100989. Briefly,equal amounts of total RNA from three mice were com-bined to form an RNA pool, and Affymetrix gene chips(GeneChip Mouse Gene 1.0 ST Array; Affymetrix, SantaClara, CA) were used for hybridization and data collection.Quality control and statistical analyses of the completemicroarray data were conducted using R/Bioconductor.The genes with altered expression ($1.5-fold) were map-ped to biological pathways using DAVID BioinformaticsResources 6.8 (https://david.ncifcrf.gov/summary.jsp). Theheat map presented in Supplementary Fig. 4 was gener-ated as a selective profile of peroxisome-related genes. Theexpression of the represented genes was confirmed by real-time PCR or Western blotting. Related PCR primers and anti-bodies are listed in Supplementary Tables 4 and 5, respectively.

LipolysisWAT and BAT samples were collected from mice and cutinto small pieces. The tissues were washed three times withDMEM and then cultured in DMEM for 1 h. The mediumwas collected, and the glycerol level was determined usinga glycerol determination kit (catalog #F6428; Sigma-Aldrich).

StatisticsThe statistical data reported include results from at leastthree biological replicates. All results are expressed asthe mean 6 SEM. All statistical analyses were performedin GraphPad Prism Version 5 (GraphPad Software, SanDiego, CA). Significance was established using one-wayANOVA with Tukey multiple-comparison test (Figs. 1B–O,2, 3A–C and F–H, 4, 5D and I, and 6 and SupplementaryFigs. 1, 2, 5A and E, and 6C andD) and a two-tailed Studentt test (Figs. 5C and H and 7 and Supplementary Figs. 3B, 4,5B, C, F, and G, 6E, and 7). We used two-way repeated-measures ANOVA to evaluate the data presented in Figs.1A and 3D, E, I, and J, and Supplementary Fig. 3C andD. Inall cases, differences were considered significant at P ,0.05. P values are indicated in each figure as *P , 0.05,**P , 0.01, and ***P , 0.001.

RESULTS

Improved Metabolic Status With Increased EnergyExpenditure in ob/ob/Cidea2/2/Cidec2/2 MiceTo investigate the roles of Cidea and Cidec in lipid metab-olism, we compared the metabolic phenotypes of ob/ob, ob/ob/Cidea2/2, ob/ob/Cidec2/2, and ob/ob/Cidea2/2/Cidec2/2

mice. Growth performance, tissue weight, and serum param-eters were similar between ob/ob and ob/ob/Cidea2/2 mice(Fig. 1 and Supplementary Fig. 1). As a lipodystrophy model,ob/ob/Cidec2/2mice showed increased liver and BATweights,with higher serum TAG, HAase, and laminin levels (Fig. 1H,N, and O and Supplementary Fig. 1D). Interestingly,ob/ob/Cidea2/2/Cidec2/2 mice did not develop lipodystro-phy, which occurred in ob/ob/Cidec2/2 mice. Compared with

1940 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018

ob/ob and ob/ob/Cidea2/2 mice, ob/ob/Cidea2/2/Cidec2/2

mice exhibited an improved metabolic status, includinga low body weight, high oxygen and energy consumption, lowlevels of serumNEFAs and inflammatory cytokines (TNF-aand IL-6), and high adiponectin levels (Fig. 1). In addition,

ob/ob/Cidea2/2/Cidec2/2 mice had the lowest BAT andWAT weights, whereas the liver weights of these mice weresimilar to those of ob/ob and ob/ob/Cidea2/2 mice (Fig. 1Cand Supplementary Fig. 1C and D). No significant differ-ences in food intake per mouse were observed among these

Figure 4—Increased mitochondrial and peroxisome activity in the BAT and WAT of ob/ob/Cidea2/2/Cidec2/2 mice. Three-month-old micethat were fed a normal diet were used. Protein expression in the BAT (A) and WAT (B). C: Cox4 and PMP70 immunofluorescence in the BATand 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.

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A

B

C

D E

G

H

I J

F

Figure 5—Increased mitochondrial and peroxisome pathway activity in Cidea and Cidec double-deficient adipocytes. The BAT fromnewborn 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 datapresented in F–J. A and F: Adipocytes were stained with BODIPY (boron-dipyrromethene) (white, indicates LDs), MitoTracker (red, indicatesmitochondria), PMP70 (green, indicates peroxisomes), and Hoechst (blue, indicates nuclei). Scale bars, 10 mm. B, C, G, and H: OCR in theindicated adipocytes. Oligomycin, FCCP, and rotenone/antimycin A (RA) were added at the indicated time points. Cells from five mice wereused. D and I: Catalase activity of the cells (n = 4). E and J: Western blot analysis showing the levels of various proteins in primary brownadipocytes. WT, wild type. *P , 0.05, **P , 0.01, and ***P , 0.001.

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four types of genetically engineered mice, but foodintake per gram of body weight was significantly increasedin ob/ob/Cidec2/2 and ob/ob/Cidea2/2/Cidec2/2 mice (Fig.1B and Supplementary Fig. 1B).

Decreased Triacylglycerol Storage and Small LDs in theAdipose Tissues of Cidea/Cidec Double-Deficient MiceAs the key metabolic organs and CIDE-expressing tissues,adipose tissues and the liver were selected for furtherstudy. The ob/ob/Cidea2/2 mice had moderately lowerTAG levels in BAT and liver than did the ob/ob mice,but no differences were observed in WAT (Fig. 2A–C).The ob/ob/Cidec2/2 mice failed to store TAG in WAT, witha much lower TAG content in WAT, but higher TAG levelswere observed in the BAT and liver of ob/ob/Cidec2/2

mice than in the other three types of mice (Fig. 2A–C). Incontrast, ob/ob/Cidea2/2Cidec2/2 mice had obviously re-duced TAG levels in BAT and WAT and maintained liverTAG levels similar to those of ob/ob mice (Fig. 2A–C).Consistent with these findings, compared with the LDsize of ob/ob mice, Cidea deficiency (ob/ob/Cidea2/2)resulted in slightly smaller LDs in BAT and liver (Fig.2D–H). Compared with the LD size of the ob/ob andob/ob/Cidea2/2 mice, Cidec deficiency (ob/ob/Cidec2/2)caused dramatically smaller LDs in WAT but larger LDsin BAT and liver (Fig. 2D–H). Compared with the LD sizeof ob/ob and ob/ob/Cidea2/2 mice, double deficiency ofCidea and Cidec (ob/ob/Cidea2/2Cidec2/2 mice) causedstrikingly smaller LDs in both BAT and WAT (Fig. 2D–H). The performance of wild-type, Cidea2/2, Cidec2/2, and

Cidea2/2/Cidec2/2 mice fed an HFD were also examined,and the observed tendency was similar to that of micewith an ob/ob genetic background, including WAT andBAT performance (Supplementary Fig. 2). Increases in liverweight and hepatic TAG levels were observed in Cidea2/2/Cidec2/2 mice compared with those of wild-type mice.

Increased Insulin Sensitivity in ob/ob/Cidea2/2/Cidec2/2 MiceNext, we assessed the blood glucose levels and insulinsensitivity status of ob/ob/Cidea2/2/Cidec2/2 mice com-pared with those of ob/ob, ob/ob/Cidea2/2, and ob/ob/Cidec2/2 mice by GTTs and ITTs when the mice were 2,4, and 8.5 months old. The fasting serum glucose andinsulin levels of ob/ob/Cidea2/2/Cidec2/2 mice were lowerthan those of the other three types of mice at 2 and4 months of age (Fig. 3A, B, F, and G). The fed serumglucose levels exhibited no differences among ob/ob,ob/ob/Cidea2/2, and ob/ob/Cidea2/2/Cidec2/2 mice, but thefed serum glucose level was slightly higher in 2-month-oldob/ob/Cidec2/2 mice and was significantly higher at age4 months (Fig. 3C and H). Insulin sensitivity becamecomplex at different ages in these four types of geneticallyengineered mice (Fig. 3D, E, I, and J, Supplementary Fig.3C and D, and Supplementary Table 6). No difference ininsulin sensitivity was observed between ob/ob and ob/ob/Cidea2/2mice from 2 to 8.5 months of age. ob/ob/Cidec2/2

mice were more sensitive to insulin than ob/ob and ob/ob/Cidea2/2 mice, according to GTT curves, at 2 monthsof age (Fig. 3D). However, compared with ob/ob and

Figure 6—Increased ATGL and PPARa expression in Cidea and Cidec double-deficient mice. Three-month-old mice that were fed normalchow 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 andWAT. The fat tissues were cultured for 1 h, and the culturemedia were collected for glycerol detection (n = 4). E andF: 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.

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Figure 7—ATGL and PPARa mediate increased mitochondrial and peroxisome pathway activity in Cidea and Cidec double-deficientadipocytes. Cidea and Cidec double-deficient MEFs were differentiated. siRNAs against PPARa (A–C ) or ATGL (D–F ) were electroporatedinto cells. A and D: Protein expression profiles. B and E: Expression profile of mitochondria-related genes. C and F: Expression profile ofperoxisome-related genes.G: Proposedmodel 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 releaseincreased 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–activatedreceptor g coactivator; si, small interfering; WT, wild type. *P , 0.05, **P , 0.01, and ***P , 0.001.

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ob/ob/Cidea2/2 mice, these mice exhibited similar insulinsensitivity when they were 4 months old (Fig. 3I and J) anddeveloped severe insulin resistance when they were8.5 months old, according to ITT curves (SupplementaryFig. 3D). Interestingly, compared with that of the otherthree types of genetically modified mice, the insulin sen-sitivity of ob/ob/Cidea2/2/Cidec2/2 mice was significantlyimproved at 2, 4, and 8.5 months of age. Consistent withthis finding, the phosphorylation status of AKT was dra-matically increased in the BAT and WAT of ob/ob/Cidea2/2/Cidec2/2 mice compared with those of ob/oband ob/ob/Cidea2/2mice (Supplementary Fig. 3A and B).

Significantly Increased Mitochondrial and PeroxisomeActivity in Cidea2/2/Cidec2/2 AdipocytesA microarray analysis was performed to observe transcrip-tional changes in the BAT, WAT, and liver of ob/ob and ob/ob/Cidea2/2/Cidec2/2 mice. The enriched pathways of thealtered genes were analyzed to identify the key upregulatedpathways, focusingmainly onmitochondria, peroxisomes, fattyacid oxidation, and oxidative phosphorylation in both the BATand WAT of ob/ob/Cidea2/2/Cidec2/2 mice compared withthose of their ob/ob counterparts (Supplementary Tables 1–3).

Further, a significant induction of mitochondrial andperoxisome-related proteins and genes, together with anincreased number of mitochondria and peroxisomes, wasobserved in the BAT of ob/ob/Cidea2/2/Cidec2/2 mice andin the WAT of ob/ob/Cidec2/2 and ob/ob/Cidea2/2/Cidec2/2

mice (Fig. 4 and Supplementary Fig. 4). Consistent with thisfinding, dramatically increased oxygen consumption andcatalase activity were observed in the BAT of ob/ob/Cidea2/2/Cidec2/2 mice and in the WAT of ob/ob/Cidec2/2 andob/ob/Cidea2/2/Cidec2/2 mice (Fig. 4D–G), demonstrat-ing increased activity of mitochondria and peroxisomes.

Primary stromal vascular cells from BAT or WAT wereinduced and differentiated into brown or white adipocytes,respectively, to observe changes in LDs, mitochondria, andperoxisomes (Fig. 5). Interestingly, compared with that oftheir wild-type counterparts, an increased number of mito-chondria and peroxisomes was observed in brown and whiteadipocytes from Cidea2/2/Cidec2/2 mice; this change wasaccompanied by smaller LDs and lower cellular TAG levels(Fig. 5A and F and Supplementary Fig. 5A, D, and E).Consistent with this finding, genes related to the activityand biogenesis of mitochondria and peroxisomes weresignificantly induced in Cidea2/2/Cidec2/2 adipocytes (Sup-plementary Fig. 5B, C, F, and G). Similar to those of ob/ob/Cidea2/2/Cidec2/2 mice, Cidea2/2/Cidec2/2 adipocytes ex-hibited increased basal and maximal oxygen consumption(Fig. 5B, C, G, and H) and catalase activity (Fig. 5D and I).In addition, differentiated Cidea2/2/Cidec2/2 MEF cellsexhibited low cellular TAG contents and increased expressionof genes related to mitochondria and peroxisomes (Supple-mentary Fig. 6). Overall, the three sets of data from ob/ob/Cidea2/2/Cidec2/2 mice, differentiated stromal vascular cellsfrom BAT or WAT, and MEF cells from Cidea2/2/Cidec2/2

embryos all demonstrated that CIDE protein deficiency could

activate mitochondria and peroxisomes. It is worth notingthat Cidec deficiency was sufficient to activate mitochondriaand peroxisomes inwhite adipocytes, but double deficiency ofCidea and Cidec was required for the activation of mitochon-dria and peroxisomes in brown adipocytes.

ATGL-PPARa Is Required for Increased Mitochondrialand Peroxisome ActivityIn addition to activated mitochondria and peroxisomes,smaller LDs were always observed in Cidea2/2/Cidec2/2

adipocytes in vivo and in vitro (Figs. 2E and 5A and F).Consistent with this finding, protein levels of ATGL andCGI58 (a key lipolysis enzyme and regulator, respectively)were significantly increased in brown and white adipocyteswhen both Cidea and Cidec were deficient (Figs. 5E and Jand 6A and B). The levels of the lipolysis products glyceroland NEFAs were also enhanced in the BAT and WAT ofob/ob/Cidea2/2/Cidec2/2 mice (Fig. 6C–F), demonstratingincreased lipolysis. Lipidomics analysis showed that nearlyall species of free fatty acids were significantly increased,including fatty acids with different chain lengths andsaturation degrees (Supplementary Fig. 7A and B). PPARaand peroxisome proliferator–activated receptor g coacti-vator 1a, which are known to regulate mitochondrial andperoxisome biogenesis, were also dramatically induced inthe BAT and WAT of ob/ob/Cidea2/2/Cidec2/2 mice (Fig.6A and B and Supplementary Fig. 7C and D). To determinewhether PPARa and ATGL were involved in this increasedb-oxidation, PPARa and ATGL were efficiently knockeddown in MEFs from Cidea2/2/Cidec2/2 mice (Fig. 7A–F).Consistent with those of ob/ob/Cidea2/2/Cidec2/2 mice,PPARa and ATGL levels were increased in Cidea2/2/Cidec2/2 MEFs compared with those of wild-type mice(Fig. 7A and D). Genes related to mitochondria and per-oxisomes, including Cpt1, Acox1, the Acad family, and thePex family, were significantly induced in Cidea2/2/Cidec2/2 MEFs (Fig. 7B and C). When PPARa or ATGLwas knocked down in Cidea2/2/Cidec2/2 MEFs, this in-creased gene expression was blocked, indicating that theincreased activity of mitochondria and peroxisomes wasregulated by PPARa or ATGL (Fig. 7B, C, E, and F).Interestingly, the knockdown of PPARa did not affectthe expression of ATGL (Fig. 7A). However, the knock-down of ATGL suppressed the expression of PPARa (Fig.7D), suggesting that increased expression of PPARa inCidea2/2/Cidec2/2 MEFs was regulated by induced ATGL.

DISCUSSION

The key novel finding in the current study was the obser-vation that a coordinated interplay among different organ-elles, including LDs, peroxisomes, and mitochondria, ismediated by the CIDE-ATGL-PPARa pathway in adiposetissues. Similar to our previous reports (12,17,20), thecurrent study demonstrates that increased lipolysis occursin CIDE-deficient adipocytes; this lipolysis occurred on thesurface of LDs and released fatty acids. The coordination ofperoxisomes and mitochondria is required to complete the

diabetes.diabetesjournals.org Zhou and Associates 1945

oxidation of long-chain acyl-CoA, the citric acid cycle, andoxidative phosphorylation and ultimately to produce CO2

and H2O (27), which was demonstrated in the currentstudy. Increased energy expenditure and increased mito-chondrial activity were previously reported in Cidea2/2,Cideb2/2, Cidec2/2, and ob/ob/Cidec2/2 mice (8,28). Theactive regulation of peroxisomes, in addition to mitochon-dria, by CIDE proteins is proposed in the current study.Peroxisomes and mitochondria both contain fatty acidb-oxidation machinery; however, the enzymes and reac-tion processes used for b-oxidation in peroxisomes andmitochondria vary significantly. Peroxisomes are respon-sible for the oxidation of very-long-chain fatty acids toproduce shortened acyl-CoAs, acetyl-CoA, and propionyl-CoA. We observed that the key enzymes Acadvl andACOX1, which are specific to the peroxisome for theoxidation of very-long-chain fatty acids (29), were upregu-lated when CIDE proteins were deficient. In addition, CIDEdeficiency caused increased expression of several peroxins,which are involved in peroxisome biogenesis. For example,Pex3 and Pex16 play multiple roles in the direct targetingof peroxisomal membrane proteins (PMPs) and in thesynthesis of the peroxisome membrane (30,31). Pex5 andPex7 are responsible for the targeting of matrix proteinsand the import machinery. In addition to the increasedmRNA and protein expression levels of these molecularmarkers, immunofluorescence analysis demonstrated in-creased contents of mitochondria and peroxisomes inCIDE-deficient adipose tissues or cells. Moreover, increasedOCRs, ATP products, and catalase activity directly supportedthe enhanced activity of mitochondria and peroxisomes.

The current study has demonstrated that the ATGL-PPARa pathway is necessary to activate peroxisomes andmitochondria in CIDE-deficient adipocytes. Several pre-vious studies reported ATGL activated PPARa/d and mi-tochondrial metabolism in the liver, heart, small intestine,pancreas, and skeletal muscle (32–37). The current studyhas revealed the involvement of the ATGL-PPARa pathwayin the metabolic activity of both BAT and WAT. ATGL-deficient mice showed reduced gene expression related tooxidative phosphorylation (38) and defective lipolysis, andthese mice accumulated large amounts of lipids in theheart, causing cardiac dysfunction and premature death(39,40). Similar defects in lipid and energy disorders werereported in humans with ATGL mutations (41,42). Thisevidence suggested that lipolysis induced by ATGL isnecessary to promote the expression of PPARa/d andits target genes. The induction of increased lipolysis byATGL in CIDE-deficient cells could be explained from twoaspects. First, CIDE proteins play a predominant role in LDfusion (8,11), and double deficiency of Cidea and Cidec ledto a failure to form large LDs in adipocytes. Small LDs havean enlarged total surface area, which makes the LDsaccessible to lipase attack and lipolysis. Second, the doubledeficiency of Cidea and Cidec significantly increased theprotein level or activity of ATGL in adipocytes. Cidec wasreported to physically interact with ATGL and inhibit

ATGL activity and lipolysis (43). Cidecwas also found to interactwith early growth response protein 1 to inhibit ATGL pro-moter activity (44). Therefore, increased lipolysis in CIDE-deficient adipocytes was potentially regulated by thedecreased size of LDs and increased ATGL enzyme activityor protein expression levels, although the mechanism bywhich ATGL activity was regulated remains unclear.

The current study helps to elucidate the functions ofCidea and Cidec in adipocytes. Both Cidea and Cidec areexpressed in brown adipocytes, but white adipocytes spe-cifically and abundantly express Cidec. Our data demon-strated that significant changes occurred in Cidec2/2 whiteadipocytes, even when Cidea was induced to some degree,indicating the predominant role of Cidec in white adipo-cytes. However, in the mouse model and in primary brownadipocytes, the double deficiency of Cidea and Cidec strik-ingly decreased cellular TAG levels and activated mitochon-dria and peroxisomes in brown adipocytes, demonstratingthe redundant roles of Cidea and Cidec in brown adipocytes.It is widely regarded that inducing WAT browning/beigingis an effective way to alleviate obesity or prevent obe-sity development in mice and humans (45–48). Significantbrowning and increased energy consumption occurred inthe WAT of ob/ob/Cidec2/2 mice, but these mice stillexhibited metabolic disorder due to severe fatty liver andinactive BAT. This finding shows that BAT function shouldbe considered even when browning is induced in WAT. Thecurrent study demonstrates the simultaneous activation of BATand WAT in Cidea and Cidec double-deficient mice contributedto dramatically increased energy consumption, providing someadditional strategies when considering obesity therapy.

Recently, a new isoform of Cidec, designated Cidec2/Fsp27b, was identified in fatty liver; this isoform contains10 additional amino acids at the N terminus of the originalCidec/Fsp27a (49). Both Cidec/Fsp27a and Cidec2/Fsp27b are localized on the surface of LDs and suppresslipolysis (49). More recently, Cidec2/Fsp27b was reportedto be expressed in BAT and to prevent LD fusion byinhibiting Cidea function (50). It is well known thatCIDE proteins promote LD fusion and lipid storage(51). In the current study, we also observed two bandsfor Cidec in wild-type BAT; the upper band (designatedCidec2/Fsp27b) was more abundant than the lowerband (designated Cidec/Fsp27a), which appeared as theonly form in WAT (Supplementary Fig. 8). However, in theob/ob BAT, Cidec/Fsp27a was more abundant thanCidec2/Fsp27b. The current study did not provide anydirect evidence to show different functions of Cidec iso-forms in BAT, which has been proposed previously (50).

In conclusion, the current study demonstrates that thecoupled functional interplay among LDs, peroxisomes, andmitochondria is mediated by the ATGL-PPARa pathway inCidea and Cidec double-deficient adipocytes.

Acknowledgments. The authors thank the members of P.L.’s laboratory atTsinghua University for their helpful discussion. The authors also thank Libing Mu

1946 CIDE-ATGL-PPARa Pathway and Energy Expenditure Diabetes Volume 67, October 2018

(Tsinghua University) for assistance with drawing the model. In addition, theauthors thank Pengcheng Jiao for assistance with the Seahorse Analyzer experi-ments and Jinyu Wang and Huizhen Cao (all from Tsinghua University) forassistance with image processing.Funding. This work was supported by grants from the National NaturalScience Foundation of China (31430040, 31690103, and 31621063 to P.L.and 31771303 to L.Z.), from the National Basic Research Program(2018YFA0506901 and 2016YFA0502002), and from the China PostdoctoralScience Foundation (2012M520249 and 2013T60103 to L.Z.). L.Z. was supportedby the Young Elite Scientists Sponsorship Program of the China Association forScience and Technology (CAST).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. L.Z. designed the study, performed most of theexperiments, analyzed the data, and wrote the manuscript. M.Y. performed mostof the experiments and analyzed the data. M.A. and W.W. performed some of theimmunoblotting and analyzed the data. Y.L. performed the HFD experiment. J.G.performed the MEF isolation. Y.G. performed the microarray data analyses. P.L.and L.X. designed the study, analyzed the data, and wrote the manuscript. L.X. isthe guarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of the dataanalysis.

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