Perilipin 5 Deletion in Hepatocytes Remodels LipidMetabolism and Causes Hepatic Insulin Resistance inMiceStacey N. Keenan,1 Ruth C. Meex,1,2 Jennifer C.Y. Lo,1 Andrew Ryan,3 Shuai Nie,4

Magdalene K. Montgomery,1,5 and Matthew J. Watt1,5

Diabetes 2019;68:543–555 | https://doi.org/10.2337/db18-0670

Defects in hepatic lipid metabolism cause nonalcoholicfatty liver disease and insulin resistance, and thesepathologies are closely linked. Regulation of lipid drop-let metabolism is central to the control of intracellularfatty acid fluxes, and perilipin 5 (PLIN5) is important inthis process. We examined the role of PLIN5 on hepaticlipid metabolism and systemic glycemic control usingliver-specific Plin5-deficient mice (Plin5LKO). Hepato-cytes isolated from Plin5LKO mice exhibited markedchanges in lipid metabolism characterized by de-creased fatty acid uptake and storage, decreasedfatty acid oxidation that was associated with reducedcontact between lipid droplets and mitochondria,and reduced triglyceride secretion. With consumptionof a high-fat diet, Plin5LKO mice accumulated intra-hepatic triglyceride, without significant changes ininflammation, ceramide or diglyceride contents, endo-plasmic reticulum stress, or autophagy. Instead, livers ofPlin5LKO mice exhibited activation of c-Jun N-terminalkinase, impaired insulin signal transduction, and insulinresistance, which impaired systemic insulin action andglycemic control. Re-expression of Plin5 in the livers ofPlin5LKOmice reversed these effects. Together, we showthat Plin5 is an important modulator of intrahepatic lipidmetabolism and suggest that the increased Plin5 expres-sion that occurs with overnutritionmay play an importantrole in preventing hepatic insulin resistance.

The liver maintains normal lipid homeostasis by regulatinga number of processes, including de novo lipogenesis; fattyuptake, oxidation, and esterification; and packaging oftriglycerides within lipoproteins for secretion (1). Dysre-gulation of one or more of these processes can lead tohepatic steatosis, which is excessive accumulation of in-tracellular lipids and is the defining component of non-alcoholic fatty liver disease (NAFLD). NAFLD is of clinicalimportance because it is closely linked to metabolic ab-normalities such as insulin resistance and dyslipidemia (2)and is a risk factor for life-threatening complications,including steatohepatitis, cirrhosis, and hepatocellularcarcinoma (3).

The excess lipids stored in hepatic steatosis are pre-dominately triglycerides, which are compartmentalizedwithin intracellular lipid droplets. The storage of trigly-cerides within lipid droplets is postulated to protecttissues from free fatty acid (FFA)–induced “lipotoxicity.”Dysregulated lipid droplet metabolism is associated withthe development of a range of cellular defects, includingactivation of stress signaling pathways, insulin resis-tance, lipoapoptosis, and organ failure (4). Lipids aremobilized from lipid droplets via the process of lipolysis,which is primarily regulated by adipose triglyceride lipase(ATGL) and hormone-sensitive lipase (HSL) (5). Proteo-mic approaches have identified many other lipid droplet–associated proteins (6) and prominent among these are

1Metabolism, Diabetes and Obesity Program, Monash Biomedicine DiscoveryInstitute, and Department of Physiology, Monash University, Clayton, Victoria,Australia2Department of Human Biology, School of Nutrition and Translational Research inMetabolism (NUTRIM), Faculty of Health, Medicine and Life Sciences, MaastrichtUniversity Medical Centre, Maastricht, the Netherlands3TissuPath, Mount Waverley, Victoria, Australia4Melbourne Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science& Biotechnology Institute, The University of Melbourne, Melbourne, Victoria,Australia5Department of Physiology, The University of Melbourne, Melbourne, Victoria,Australia

Corresponding author: Matthew J. Watt, matt.watt@unimelb.edu.au

Received 15 June 2018 and accepted 13 December 2018

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0670/-/DC1.

© 2019 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.

Diabetes Volume 68, March 2019 543

METABOLISM

the perilipin (PLIN) family of proteins, which inducetissue-specific metabolic effects, not least through theirinteractions with ATGL and HSL (7–9).

PLIN5 is actively involved in regulating lipid metab-olism in tissues with high oxidative capacity, such asheart, skeletal muscle, and brown adipose tissue(7,10–13). Studies using cell lines and transgenic micewith Plin5 deficiency or overexpression show that PLIN5interacts with ATGL and comparative geneidentification (CGI)-58 to suppress lipolysis (14–16).This limits the production of lipid intermediates andcellular stress signals and, in turn, prevents disruptionto tissue morphology and functions, including heartcontraction, skeletal muscle insulin action, and b-cellinsulin secretion (17). PLIN5 also promotes transcrip-tion of genes that mediate mitochondrial biogenesis andoxidative function in brown adipose tissue, therebycoupling protein kinase A–mediated lipolysis to trans-criptional regulation of mitochondrial fatty acid metabo-lism (13).

Studies of PLIN5 functions in the liver have yieldedequivocal results. PLIN5 levels are increased in murinemodels of obesity and hepatosteatosis (16,18), andadenovirus-mediated overexpression of PLIN5 wors-ened hepatosteatosis in high-fat diet–fed mice, butthis did not cause liver injury or adversely affect sys-temic glucose tolerance or insulin sensitivity (16,18).Two studies in whole-body Plin52/2 mice report di-vergent results. One group reported that Plin5 deletionreduces hepatosteatosis but causes hepatic inflammation,endoplasmic liver damage, and lipid peroxidation, withoutimpacting glucose metabolism (16), whereas anotherreported no evidence of steatosis or liver damage andimproved hepatic insulin sensitivity in Plin52/2 mice (7).

In light of this uncertainty, we aimed to determine theimpact of Plin5 deletion on hepatic lipid metabolism andinsulin action and whole-body glucose homeostasis.Herein, we show that mice with liver-specific Plin5 de-ficiency (Plin5LKO) have alterations in hepatic lipid metab-olism that result in mild increases in hepatic triglycerideswithout significant liver damage. These changes wereassociated with activation of c-Jun N-terminal kinase(JNK), suppression of insulin signal transduction in theliver, and the development of hepatic insulin resistanceand whole-body glucose intolerance in the context of diet-induced obesity.

RESEARCH DESIGN AND METHODS

Animal StudiesStudies were approved by the Monash University Schoolof Biomedical Science Animal Ethics Committee (MARP-2013-050) and conformed to the National Health andMedical Research Council of Australia guidelines regard-ing care and use of experimental animals. Plin5lox/+ micewere generated as described previously (19). Mice withliver-specific deletion of Plin5 were generated by crossingthese mice with mice expressing Cre recombinase under

the control of the liver-specific murine promoter(Alb-Cre; provided by T. Tiganis, Monash University) tocreate mice with liver-specific ablation of Plin5 (Plin5lox.lox.Cre+, denoted herein as Plin5LKO mice) and control wild-type (Wt) littermates (lox/lox, i.e., Plin5lox.lox.Cre– mice). Inseparate experiments, an adeno-associated virus serotype8 (AAV) driven by the albumin promotor and containingPlin5 cDNA or GFP (1 3 1012 genome copies/mouse;Vector Biolabs, Malvern, PA) was injected via the tailvein in 20-week-old high-fat–diet fed Plin5LKO. Experi-ments were conducted in mice 8 weeks later. Male micewere housed at 22°C on a 12:12-h light-dark cycle. Wt andPlin5LKO mice aged 8 weeks were fed either a regular low-fat chow (4.6% energy from fat) or high-fat diet (43%energy from fat, SF04-001; Specialty Feeds, Western Aus-tralia, Australia) for 12 weeks prior to metabolic assess-ments. Mice were fasted from 0700 h to 1100 h before allexperiments, unless stated otherwise.

Body Composition and Energy ExpenditureBody composition was measured using DEXA (LunarPixi; PIXImus, Madison, WI) (20). Oxygen consumption,carbon dioxide production, physical activity, and foodintake were measured using an Oxymax indirect calo-rimetery system (Columbus Instruments, Columbus,OH).

Glucose and Insulin Tolerance TestsMice fasted for 4 hours received an oral gavage of D-glucose(2 g/kg) or intraperitoneal injection of insulin (1 unit/kg,Actrapid) for glucose and insulin tolerance tests, respec-tively. Blood obtained from a tail nick was assessed forblood glucose (Accu-Chek) as indicated.

Hyperinsulinemic-Euglycemic ClampClamps were conducted in conscious and restrainedmice (4 mU/kg/min insulin) as described previously(7,21).

Cell CultureHepatocytes were isolated from Wt and Plin5LKO liversby collagenase digestion (22). Metabolic assessment ofhepatocytes was previously described (23). Hepatocyteswere incubated for 2 h with low-glucose DMEM Gluta-MAX (Life Technologies) containing 2% fatty acid–freeBSA, 2 mCi [1-14C]palmitate (CFA23; GE Healthcare),and 0.5 mmol/L palmitate (Sigma-Aldrich). Fatty acidoxidation was calculated as the sum of complete oxida-tion to 14CO2 and “incomplete” oxidation (i.e., acid-soluble metabolites). Fatty acid uptake was calculatedas the sum of fatty acid oxidation and fatty acids storedin complex lipids (i.e., 14C in the organic fraction of thelysed cells). Lipogenesis was measured for 2 h in DMEMcontaining 2 mCi/mL D-[2-3H]glucose (NET238C001MC;PerkinElmer). Triglyceride secretion was assessed inmedium (DMEM) after 8 h incubation and was determinedusing a colorimetric assay kit (Triglycerides GPO-PAP;

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Roche Diagnostics, Indianapolis, IN). All data are ex-pressed per milligram protein.

Mitochondria and Lipid Droplet Staining and ImagingProtocol

Hepatocytes grown in M199 medium were incubated withMitoTracker (Molecular Probes) diluted (2 mmol) in pre-warmed media for 15 min. Cells were washed with PBS.BODIPY (2 mg/mL) (Thermo Fisher Scientific, San Jose,CA) was applied to cells and incubated for 10 min. Cellswere washed with PBS and viewed using time-lapse con-focal microscopy using a TCS SP8 confocal microscope(Leica). To quantify the distance between lipid dropletsand mitochondria over several time points, a340 lens wascalibrated at 1 pixel = 0.01679mm, i.e., 100 pixels = 1.1679mm. A Euclidean pixel-distance map was derived with anyMitoTracker signal above threshold set as distance zero.The mass centers of lipid droplets were localized by theirposition on the Euclidean distance map, across three timepoints, to determine proximity time away from the mito-chondria. For example, if the lipid droplet touches a mito-chondrion, the lipid droplet is zero pixels away froma mitochondrion; thus, for that time point, that lipiddroplet will have a measurement of zero. Additionally, ifthe lipid droplet sits 10 pixels away from the nearestmitochondria, then that droplet gets assigned a value of10 for that time point. Therefore, if a droplet spends threetime points touching a mitochondria, two time pointsbeing two pixels away, and one time point being five pixelsaway, the lipid droplet will receive a value of (33 0) + (232) + (1 3 5) = 9 units of proximity time.

Liver Lipid Analysis and Blood MetabolitesLipids were extracted as previously described (24). Tri-glycerides were determined by colorimetric assay (Tri-glycerides GPO-PAP; Roche Diagnostics). Liver cytosolicand membrane fractions were isolated using a well-accepted ultracentrifugation method (25), and lipidswere extracted using a modified Folch extraction (26).Samples were spiked with internal standards and wereanalyzed using an Orbitrap Fusion Lumos mass spec-trometer coupled to a Vanquish UHPLC (ThermoFisher Scientific). For both analyses at positive andnegative ionization mode, each sample was injectedinto an Accucore C30 column (2.1 3 250 mm, 2.6mm) (Thermo Fisher Scientific). For analysis at bothpolarities, top-speed data-dependent acquisition wasperformed with a cycle time of 1 s. Diglycer-ide and ceramide species content was determined bycomparing ratios of unknowns with internal standardsand referencing to a standard curve. Plasma FFA andb-hydroxybutyrate were measured using a colorimetrickit (Wako Diagnostics, Osaka, Japan, and CaymanChemical, Ann Arbor, MI, respectively). Thiobarbituricacid–reactive substance (TBARS) and lipid hydro-peroxides were determined as previously described(27,28). AST and alanine aminotransferase (ALT)activities were determined by enzymatic colorimetricassays (29,30). Triglyceride secretion rates were as-sessed by injecting 300 mg/kg Triton WR 1339 (Tyloxapol,T8761; Sigma-Aldrich) into the tail vein of con-scious mice, and plasma triglyceride was determinedover 2 h.

Figure 1—Confirmation of liver-specific Plin5 deletion in transgenic mice. A: mRNA expression of Plin5 in liver (Wt n = 10, Plin5LKO n = 14).B: Representative immunoblot of liver PLIN5 expression. C: Immunohistochemistry analysis of PLIN5 (green) expression in liver and nucleus(blue). Imbedded squares represent negative controls. Scale bars = 20 mm. D: mRNA expression of Plin5 across various tissues (Wt n = 8,Plin5LKO n = 8). E: mRNA expression of PLIN proteins and lipases (Wt n = 8, Plin5LKO n = 8). F: Representative immunoblot of liver PLIN2 andPLIN3 expression. Representative immunoblotting shows n= 2per group, but respective quantification analysiswasdoneonn=8 per genotype.*P , 0.05 vs. Wt.

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ImmunohistochemistryLivers were fixed in 10% neutral buffered formalin andparaffin embedded, and immunohistochemistry was per-formed as described previously (7). Immunofluorescenceimages were obtained using a TCS SP8 confocal microscope(Leica).

Analysis of Gene ExpressionRNA was extracted from tissues with QIAzol lysis reagent(Qiagen, Doncaster, Victoria, Australia) and reverse tran-scribed with iScript Reverse Transcriptase (Bio-Rad Labo-ratories, Invitrogen, Mt. Waverley, Victoria, Australia), andthe gene products were determined by real-time quanti-tative RT-PCR (ep realplex4 Mastercycler; Eppendorf,Hamburg, Germany) using SYBR Green PCR Master Mix(Brilliant II SYBR Green QPCR Master Mix; Agilent Tech-nologies, Santa Clara, CA) for Plin5 and TaqMan UniversalPCR Master Mix (AmpliTaq Gold DNA Polymerase; Ap-plied Biosystems, Scoresby, Victoria, Australia) for other

genes. 18S was used as a reference gene and did not varybetween groups. The mRNA levels were determined by theDDCT method. Primer sequences are listed in Supplemen-tary Table 1.

ImmunoblottingLiver lysates were prepared in 100 mmol/L TRIS-HCl (pH7.4; for PLIN5) or RIPA buffer, proteins were resolved bySDS-PAGE electrophoresis, and immunoblot analysis wasconducted as described previously (19). Antibodies arelisted in Supplementary Table 2. Stain-free images werecollected after transfer for loading control (ChemiDoc MPand ImageLab software version 4.1; Bio-Rad Laboratories,New South Wales, Australia).

Statistical AnalysisStatistical analysis was performed using unpaired two-wayStudent t tests or two-way ANOVA with Bonferroni posthoc analysis where required. Statistical significance was

Figure 2—Lipid metabolism in isolated hepatocytes. A: Representative immunoblot of hepatocyte PLIN5 expression. B: Hepatocytetriglyceride content (Wt n = 9, Plin5LKO n = 9).C: Fatty acid uptake (Wt n = 12, Plin5LKO n = 12). D: Fatty acid oxidation (Wt n = 12, Plin5LKO n =12). E: Fatty acid storage into triglyceride (Wt n = 12, Plin5LKO n = 12). F: Fatty acid storage into diglycerides (Wt n = 12, Plin5LKO n = 12). G:mRNA expression (Wt n = 8, Plin5LKO n = 7).H: Lipid droplet (LD)–mitochondria proximity (Wt n = 10, Plin5LKO n = 10). I: Representative imagefrom live cell imaging of lipid droplets (green) andmitochondria (red) at magnification340. J: De novo lipogenesis (Wt n = 12, Plin5LKO n = 12).K: Triglyceride secretion (Wt n = 12, Plin5LKO n = 12). n represents the number of independent mouse donors per genotype per experiment,and data are normalized per milligram of cellular protein. Representative immunoblotting shows n = 2 per group, but respective quantificationanalysis was done on n = 8 per genotype. *P , 0.05 vs. Wt.

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established at P , 0.05. Data are reported as means 6SEM.

RESULTS

Generation of Plin5LKO MiceLiver Plin5 mRNA expression was reduced by 85% inPlin5LKO compared with Wt mice (Fig. 1A). The absenceof PLIN5 protein in liver was confirmed by immunoblotanalysis (Fig. 1B) and immunohistochemistry (Fig. 1C).Specificity of liver Plin5 knockdown was confirmed byqPCR, showing expression similar to that in Wt mice inskeletal muscle, heart, kidney, and white adipose tissue(Fig. 1D). There were no changes in Plin2, Plin3, orPlin4 expression in the liver of Plin5LKO mice or changesin the mRNA expression of prominent lipases includingPnpla2 and Lipe compared with Wt mice (Fig. 1E).PLIN4 was not detectable by immunoblot, whereasPLIN2 and PLIN3 protein content was not different

between Plin5LKO and Wt mice (PLIN2 1.00 6 0.06vs. 1.13 6 0.12 and PLIN3 1.00 6 0.13 vs. 1.17 6 0.18arbitrary units for Wt and Plin5LKO, respectively; n = 4 pergroup) (Fig. 1F).

Assessment of Lipid Metabolism in IsolatedHepatocytes From Wt and Plin5LKO MiceWe first investigated the metabolic effects of Plin5 de-letion in primary hepatocytes (Fig. 2A). Although tri-glyceride content was not different between genotypes(Fig. 2B), detailed analysis revealed significant remodel-ing of fatty acid metabolism in Plin5LKO hepatocytes.Fatty acid uptake was reduced (Fig. 2B) and was accom-panied by a reduction in fatty acid oxidation (Fig. 2C)and fatty acid esterification into triglycerides (Fig. 2D)and diglycerides (Fig. 2E). These changes were notaccompanied by significant changes in genes encodingkey proteins of lipid metabolism or mitochondrial ca-pacity (Fig. 2F). PLIN5 promotes a close interaction

Figure 3—Histopathology and liver biochemistry of Plin5LKO andWt mice. A: Liver cellular structure (H-E) and collagen deposition (Masson’strichrome). B: Liver triglycerides (Wt n = 19, Plin5LKO n = 20). C: Liver diglycerides (Wt n = 3, Plin5LKO n = 6). D: Liver ceramides (Wt n = 3,Plin5LKO n = 6). E: Plasma FFA (Wt n53, Plin5LKO n5 6). F: Plasma b-hydroxybutyrate (Wt n5 3, Plin5LKO n5 6). G: Plasma triglyceride (Wtn = 3, Plin5LKO n = 6). H: Plasma triglyceride secretion, after injection of Tyloxapol (Wt n = 3, Plin5LKO n = 6). I: Plasma ALT (Wt n = 7, Plin5LKO

n = 9). J: Liver 2,4-dinitrophenylhydrazine (DNPH) (Wt n = 6, Plin5LKO n = 10).K: Liver malondialdehyde and thiobarbituric acid (MDA-TBA) (Wtn = 5, Plin5LKO n = 10). For panels B–D, data are normalized to tissue mass. *P , 0.05 vs. Wt.

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between lipid droplets and mitochondria in cardiomyo-cytes, which may enhance fatty acid oxidation (31).Live cell imaging revealed a significant reduction inthe interaction and proximity distance between lipiddroplets and mitochondria in Plin5LKO hepatocytes, in-dicating reduced contact between these organelles (Fig.2G and H). De novo lipogenesis was not significantlyimpacted in Plin5LKO hepatocytes (Fig. 2I), whereastriglyceride secretion from Plin5LKO hepatocyteswas markedly reduced compared with Wt hepatocytes(Fig. 2J).

Assessment of Liver Histology, Lipids, and PlasmaMetabolites in Mice Fed a Chow DietWe next investigated the metabolic phenotype ofPlin5LKO and Wt mice fed a standard chow diet. Liverhistology showed no evidence of increased lipid deposition

or fibrosis (Fig. 3A). Consistent with this notion, livertriglyceride (Fig. 3B), diglyceride (Fig. 3C), andceramide (Fig. 3D) levels were not different betweenPlin5LKO and Wt mice. Fasting plasma FFAs (Fig. 3E),b-hydroxybutyrate (Fig. 3F), and triglyceride levels (Fig.3G), triglyceride secretion (Fig. 3H), and microsomal tri-glyceride transfer protein (MTTP) content (data notshown) were not different between genotypes, whichcontrasted with the reduction in triglyceride secretionmeasured from cultured hepatocytes of Plin5LKO mice(Fig. 2K). Plasma ALT is a clinical measure of liver damageand was not different between genotypes (Fig. 3I), norwere the liver contents of dinitrophenylhydrazine,a marker of protein carbonylation and oxidative damage(Fig. 3J). The malondialdehyde content was decreased inthe livers of Plin5LKO mice (Fig. 3K), indicating a reductionin lipid peroxidation and oxidative stress.

Figure 4—Body composition, energy homeostasis, and glucosemetabolism inWt and Plin5LKOmice fed a chow diet. A: Weekly body weight(Wt n = 10, Plin5LKO n = 15). B: DEXA analysis of lean mass, fat mass, and % body fat (Wt n = 10, Plin5LKO n = 14). C: Liver mass (Wt n = 9,Plin5LKO n = 13). D: Daily food intake (Wt n = 5, Plin5LKO n = 6). E: Indirect calorimetry assessment of VO2. F: Daily physical activity. G: Therespiratory exchange ratio (RER) (VCO2/VO2) (Wt n = 8, Plin5LKO n = 11). H: Intraperitoneal GTT (2 mg/kg; Wt n = 6, Plin5LKO n = 6). I:Intraperitoneal insulin tolerance test (0.75 units/kg;Wt n = 10,Plin5LKO n = 9). J: Fasting plasma glucose (Wt n = 12,Plin5LKO n = 16).K: Fastingplasma insulin (Wt n = 9, Plin5LKO n = 8). *P , 0.05 vs. Wt.

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Plin5LKO Mice Fed a Chow Diet Are Glucose Intolerantand Insulin ResistantWt and Plin5LKOmice had similar body weights from 5 to20 weeks of age (Fig. 4A). In addition, there was nodifference in lean or fat mass (Fig. 4B) or liver mass (Fig.4C) between genotypes. Whereas food intake was similar(Fig. 4D), energy expenditure was modestly decreased inPlin5LKO mice (Fig. 4E) and was reflective of decreasedactivity (Fig. 4F). Despite the reduction in hepatocyte fattyacid oxidation (Fig. 2B), there were no differences in therespiratory exchange ratio between genotypes (Fig. 4G),

indicating similar whole-body fat oxidation. Whole-bodyglycemic control (Fig. 4H) and insulin sensitivity (Fig. 4I)were impaired in Plin5LKO compared with Wt mice. Fastingblood glucose and plasma insulin levels were increased inthe Plin5LKO mice (Fig. 4J and K), which is consistent withthe presence of insulin resistance.

Metabolic Characterization of Plin5LKO Mice Feda High-Fat DietPLIN5 is upregulated in hepatocytes with dietary lipidoverload (16,32,33), presumably to facilitate the transfer

Figure 5—Body composition, energy homeostasis, and liver histopathology inWt andPlin5LKOmice fed a high-fat diet.A: Weekly bodymass(Wt n = 9, Plin5LKO n = 9).B: DEXA analysis of leanmass and fat mass (Wt n = 9, Plin5LKO n = 9).C: Food intake.D: Indirect calorimetry VO2 (Wtn = 6, Plin5LKO n = 9). E: Liver mass (Wt n = 13, Plin5LKO n = 9). F: Liver triglycerides (Wt n = 14, Plin5LKO n = 12).G: Histological assessment ofliver cellular structure (H-E) and collagen disposition (Masson’s trichrome).H: Plasma ALT (Wt n = 7, Plin5LKO n = 9). I: Liver malondialdehydeand thiobarbituric acid (MDA-TBA) (Wt n = 5,Plin5LKO n = 10). J: Liver 2,4-dinitrophenylhydrazine (DNPH) (Wt n = 6,Plin5LKO n = 10).K: Plasmab-hydroxybutyrate. L: FFA. M: Triglycerides (Wt n = 8, Plin5LKO n = 9). *P , 0.05 vs. Wt.

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of excess fatty acids into lipid droplets to limit lipotoxicstress (7,13). Hence, we surmised that the metabolicconsequences of Plin5 ablation would be more prominentin mice fed a high-fat diet. Plin5LKO mice were marginallyheavier than Wt mice (Fig. 5A). There was no significantdifference in lean or fat mass (Fig. 5B) or food intake (Fig.5C) between genotypes, whereas energy expenditure waslower in Plin5LKO mice (Fig. 5D). There was no differencebetween genotypes for fasting plasma triglycerides (Wt1.01 6 0.05 vs. Plin5LKO 1.04 6 0.08 mmol/L), FFA (Wt0.90 6 0.06 vs. Plin5LKO 0.91 6 0.07 mmol/L),b-hydroxybutyrate (Wt 415 6 80 vs. Plin5LKO 468 654 mmol/L), or cholesterol (Wt 67.4 6 5.4 vs. Plin5LKO

72.9 6 7.8 mg/dL).Liver mass was increased in Plin5LKO compared with Wt

mice (Fig. 5E) and coincided with a 70% increase in tri-glyceride content (Fig. 5F). Analysis of hematoxylin-eosin(H-E)–stained liver sections (Fig. 5G) confirmed the pres-ence of fatty liver, with higher liver steatosis scores inthe livers of Plin5LKO compared with Wt mice (datanot shown). Lobular inflammation and hepatocellular

ballooning were not different between genotypes (datanot shown). Fibrosis was assessed by Masson’s trichromestain and was unaffected by Plin5 deletion (Fig. 5G).Plasma ALT was increased in Plin5LKO compared withWt mice, suggesting mild damage in the livers of thesemice (Fig. 5I). In contrast, liver oxidative stress, assessed asTBARS (Fig. 5I) and protein carbonylation (Fig. 5J), wasnot different between genotypes.

Effects of Diet-InducedObesity onGlucoseMetabolismand Insulin Action in Plin5LKO MiceHepatic lipid accumulation is commonly associated withthe development of insulin resistance. Consistent with thisnotion, fasting plasma insulin, but not blood glucose, levelswere increased in Plin5LKO compared with Wt mice (Fig. 6Aand B). Plin5LKO mice had impaired glucose tolerance (Fig.6C), whereas plasma insulin levels during the oral glucosetolerance test (GTT) were not different between genotypes(Fig. 6D). In addition, Plin5LKO mice exhibited impairedinsulin sensitivity as assessed by an insulin tolerancetest (Fig. 6E). Hyperinsulinemic-euglycemic clamps were

Figure 6—Glucose metabolism and insulin sensitivity in Wt and Plin5LKO mice fed a high-fat diet. A: Fasting plasma glucose (Wt n = 10,Plin5LKO n = 16). B: Fasting plasma insulin (Wt n = 10, Plin5LKO n = 16).C: Oral GTT (Wt n = 10, Plin5LKO n = 16).D: Plasma insulin levels duringthe oral GTT (Wt n = 6, Plin5LKO n = 7). E: Insulin tolerance test (0.75 units/kg intraperitoneally) (Wt n = 10, Plin5LKO n = 10). F–L:Hyperinsulinemic-euglycemic clamps (Wt n = 6, Plin5LKO n = 7). F: Blood glucose. G: Glucose infusion rate. H: Plasma insulin. I: Glucoseinfusion rate during clamp. J: Glucose Rd.K: Endogenous glucose production (EndoRa). L: Suppression of hepatic glucose production (HGP).M: Akt Ser473 phosphorylation/Akt in response to acute intraperitoneal insulin administration; right, representative immunoblot. Represen-tative immunoblotting shows n = 2 per group, but respective quantification on n = 8 per genotype. *P , 0.05 vs. Wt.

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Figure 7—Hepatic lipids and signaling in Wt and Plin5LKO mice fed a high-fat diet. A: Diacylglycerol content in cytosolic and membranefractions of livers (Wt n = 8,Plin5LKO n = 8).B, left: Representative immunoblot showing PKCe in cytosolic andmembrane fractions of livers.B,right: Quantification of PKCe abundance (Wt n = 8, Plin5LKO n = 8). C: Representative immunoblot of liver IRE1 pSer724, elF2a pSer51, XBP1,CHOP, and ATF6. D: Liver mRNA expression of Atf4, Chop, Gadd34, Grp94, and Xbp1 (Wt n = 6, Plin5LKO n = 6). E: Representativeimmunoblot of liver autophagy proteins LC3B and p62. F: Representative immunoblot of liver IkBa.G: Expression of inflammatory genes (Wtn = 6, Plin5LKO n = 6). H: Liver mRNA expression of FGF-21 (Wt n = 8, Plin5LKO n = 8). I: Plasma FGF-21 levels (Wt n = 8, Plin5LKO n = 8).

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performed to assess whole-body and tissue-specific insulinaction. Glucose was clamped at euglycemia (Fig. 6F), anda steady-state glucose infusion rate was achieved in bothgenotypes (Fig. 6G). Plasma insulin levels were higherduring the clamp (main treatment effect) but were notdifferent between Plin5LKO and Wt mice (Fig. 6H). Whole-body insulin action was impaired in Plin5LKO mice, asreflected by a 50% reduction in the glucose infusionrate (Fig. 6I). The insulin-stimulated glucose disposalrate was not different between genotypes (Fig. 6J), in-dicating no differences in peripheral glucose uptake.Rather, Plin5LKO mice displayed marked hepatic insulinresistance compared with Wt mice, as evidenced by a fail-ure to suppress endogenous glucose production (Fig. 6Kand L) and reduced Akt Ser473 phosphorylation afterinsulin administration (Fig. 6M and N).

Molecular Changes Mediating Hepatic InsulinResistance in Plin5LKO MiceWe performed biochemical and molecular analyses inthe livers of high-fat diet–fed Plin5LKO and Wt mice todetermine the link between Plin5 ablation and hepaticinsulin resistance. We assessed diglyceride and ceramidespecies in membrane or cytosol fractions of liver.Ceramides were not different in either fraction betweengenotypes (data not shown). No diglyceride specieswere different in the membrane fractions of Plin5LKO

and Wt livers. In contrast, 16 diglyceride species were in-creased in the cytosolic fraction of Plin5LKO comparedwith Wt livers (Fig. 7A). Although cytosolic diglycerideshave been associated with hepatic PKCe activation, asreflected by PKCe translocation to the plasma membrane,we observed no differences in membrane PKCe betweengenotypes, suggesting that this mechanism is unlikely toexplain the impaired insulin signaling in Plin5LKO livers(Fig. 7B). There was no evidence for increased endoplas-mic reticulum (ER) stress in Plin5LKO mice, as indicated byimmunoblot analysis of phosphorylation in IRE1 Ser724

and eIF2a Ser51, as well as changes in total proteincontent of XBP1, CHOP, and ATF6 (Fig. 7C). Similarly,there was no difference in mRNA expression of the ERstress genes Atf4, Chop, Gadd34, Grp94, and Xbp1 (Fig.7D). There were no differences in the autophagy markersLC3B-II and p62 (Fig. 7E) or markers of inflammation,including IkBa content (Fig. 7F) and Il-6, Tnfa, andAdgre1 mRNA expression (Fig. 7G). We have previouslyshown that Plin5 deletion in muscle reduces FGF-21 (34);however, neither FGF-21 mRNA expression nor circulat-ing levels were altered in Plin5LKO compared with Wt mice(Fig. 7H and I). Of interest, livers of Plin5LKOmice showed

substantial phosphorylation (activation) of the serine/threonine kinase JNK (Fig. 7J and K). JNK activation wassupported by the finding of increased gene expressionand protein content of c-Jun, which is regulated by JNK(Fig. 7J–L). A JNK target protein is insulin receptorsubstrate 1 (IRS-1), which once phosphorylated atSer307, inhibits its activity, and contributes to hepaticinsulin resistance (35). JNK phosphorylation was in-creased by 50% in the livers of Plin5LKO compared withWt mice, and this coincided with an increase in IRS-1Ser307 phosphorylation (Fig. 7M and N). JNK phosphor-ylation was also increased in the livers of chow-fed Plin5LKO compared with Wt mice (Wt 1.00 6 0.10vs. Plin5LKO 1.52 6 0.17 arbitrary units, respectively;n = 8 per genotype, P , 0.05), demonstrating conserva-tion of this response.

Re-expression of PLIN5 in the Liver of Plin5LKO

Restores Glycemic Control in High-Fat Diet–Fed MiceAdministration of an AAV containing Plin5 restored PLIN5protein content to Wt levels (Fig. 8A). This was associatedwith no change in body mass (Fig. 8B) and restoration ofliver triglyceride content to levels of Wt littermate controlmice (Fig. 8C). Whole-body glucose tolerance (Fig. 8D) andinsulin action (Fig. 8E), which were impaired in Plin5LKO

mice, were not different between Wt mice and Plin5LKO

mice with PLIN5 re-expression. These changes in glycemiccontrol and insulin action were associated with a dampen-ing of JNK phosphorylation in Plin5LKO mice to levelsmeasured in Wt mice (Fig. 8F). As expected, re-expressionof GFP alone did not rescue the impaired glycemic control,insulin action, or defective JNK signaling in Plin5LKO mice.

DISCUSSION

Hepatic insulin resistance is often associated with dysregu-lated lipid metabolism (36), which is partly driven by theexpression levels, activity, and interactions of proteins lo-cated at the surface of lipid droplets. PLIN5 appears to berequired for the adaptation to lipid overload, as its expres-sion is increased upon exposure to fatty acids in culturedcells (33,37), with high-fat feeding (18) and prolongedfasting (37) in mice, and in humans with NAFLD (16).Here, we have shown that Plin5LKO mice exhibit systemicglucose intolerance and insulin resistance when fed a stan-dard chow diet, and that these effects were exacerbated uponhigh-fat feeding and were associated with substantial tri-glyceride accumulation, activation of JNK, inhibition ofhepatic insulin signaling, and impaired insulin-mediatedsuppression of hepatic glucose output. Altogether, thesestudies define important roles for PLIN5 in hepatic lipid

J: Representative immunoblot of liver JNK pThr183/Tyr185/JNK and c-Jun. K: Quantification of pJNK/JNK and c-Jun. L: Liver mRNAexpression of c-Fos and c-Jun (Wt n = 8, Plin5LKO n = 8). M: Representative immunoblot of liver IRS-1 pSer307/IRS-1. N: Quantification ofpIRS-1/IRS-1 protein contents (Wt n = 8, Plin5LKO n = 8). Representative immunoblotting shows n = 2 per group, but respective quantifi-cation analysis was done on n = 8 per genotype. *P , 0.05 vs. Wt.

552 PLIN5 Deletion Remodels Lipid Metabolism Diabetes Volume 68, March 2019

metabolism and insulin action without the pleiotropic effectsthat occur in global knockout mice (7,11,16).

Despite several physiological and molecular studies, therole of PLIN5 in regulating hepatic lipid metabolismremained unresolved. To better understand the roleof PLIN5 in hepatic lipid metabolism, we developedhepatocyte-specific Plin5-null mice and examined PLIN5’scell-autonomous role in hepatocytes. Plin5 deletion im-pacted lipid homeostasis by reducing uptake, storage, andoxidation of extracellular-derived fatty acids and reducedtriglyceride secretion. These findings contrast earlier workby Wang et al. (16) that reported increased fatty acidoxidation, decreased fatty acid storage, and increasedtriglyceride secretion from hepatocytes isolated fromwhole-body Plin52/2 mice. Although the molecular eventsmediating the changes in lipid metabolism remain un-resolved, the absence of marked changes in gene expres-sion of proteins controlling these key metabolic processesindicates that posttranscriptional regulation may be im-portant, and in particular, protein interactions. In thisregard, PLIN5 regulates lipolysis in nonhepatic cells byphysical interactions with ATGL, HSL, and CGI-58 (8,9)and appears to modulate lipid metabolism by facilitatingthe interaction between lipid droplets and mitochondria(31). Studies aimed at identifying PLIN5 protein-proteininteractions are ongoing and may explain the unique re-ciprocal roles of PLIN5 in hepatic lipid metabolism de-scribed here. Alternatively, the decrease in fatty acidoxidation and storage could be explained most simplyby reduced uptake of extracellular fatty acids, as fattyacid flux per se can impact distal processes and was pre-viously reported in hearts of Plin52/2 mice (38).

VLDL assembly of triglycerides occurs via a processinvolving lipolysis/re-esterification and is likely to involvetriglyceride hydrolase (39) rather than the PLIN5-interacting lipases ATGL or HSL (23). Another novelobservation observed here was the decreased triglyceridesecretion from Plin5LKO hepatocytes, which contrasts witha previous report showing that triglyceride secretion isincreased in cultured Plin5-deficient hepatocytes (16).Although triglyceride secretion was markedly impaired incultured hepatocytes, this was not associated with alteredexpression of key regulators of this process, includingtriglyceride hydrolase or MTTP (data not shown). More-over, our in vivo analysis demonstrated no effect of Plin5deletion on triglyceride secretion or plasma triglyceridelevels, the latter aligning with previous studies reportingnormal plasma triglyceride levels in three independentPlin52/2 mouse colonies (7,11,16). Taken together,these data indicate that PLIN5 appears to regulate tri-glyceride secretion from hepatocytes but that humoral orneural signals overcome this cell autonomous regulation,resulting in normal triglyceride secretion in vivo.

NAFLD and type 2 diabetes are common comorbidities(40), and hepatic steatosis often develops before insulinresistance, suggesting a causative role of hepatic lipidaccumulation in the pathogenesis of hepatic insulin re-sistance (1). We found that liver-specific Plin5 ablationreduced hepatic insulin action, without affecting insulinsensitivity of other tissues, and was sufficient to inducesystemic insulin resistance and impair glucose tolerance.Further, reintroducing PLIN5 into the livers of Plin5LKO

mice was sufficient to restore whole-body glycemic controland insulin action. We systematically evaluated the most

Figure 8—Re-expression of Plin5 in the livers of Plin5LKO mice restores glycemic control and insulin sensitivity. A: Representative PLIN5immunoblot and quantification. B: Bodymass.C: Liver triglycerides.D: Oral GTT (50mg glucose/mouse). E: Insulin tolerance test (0.75 units/kg intraperitoneally). F: Quantification of liver JNK pThr183/Tyr185/JNK (n = 7 per group for all panels). Representative immunoblotting showsn = 2 per group, but respective quantification analysis was done on n = 8 per genotype. *P , 0.05 vs. Wt.

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common mediators of lipid-induced insulin resistance andobserved no significant effect of hepatic Plin5 deletion onthe accumulation of ceramides, oxidative stress, ER stress,autophagy, or inflammation. Instead, we report activationof JNK and suppression of insulin signal transduction,which is a plausible explanation for the impaired hepaticinsulin sensitivity (41,42). By precedence, Plin5 deletionactivates JNK in aortic valve tissues of ApoE2/2 mice (43).Others have shown that adenoviral overexpression ofPLIN5 in the liver prevented the development of late-onset whole-body insulin resistance in high-fat diet–fedmice but that this was insufficient to impact whole-bodyglycemic control (18). In contrast, we have previouslyreported that whole-body Plin52/2 mice have improvedwhole-body insulin sensitivity, which was mediated byenhanced liver insulin sensitivity but was accompaniedby paradoxical reductions in muscle insulin sensitivity (7).These discordant results between mice with whole-body(7) and liver-specific Plin5 deletion could be explained byaltered intertissue communication. Supporting this prem-ise, mice with muscle-specific Plin5 deficiency have im-paired activation of the unfolded protein response, whichwas associated with reduced fibroblast growth factor21 (FGF-21) expression and secretion and FGF-21–depen-dent changes in systemic glycemic control (34). Previouswork also showed that Plin5 overexpression in muscleincreases FGF-21 production and improves glycemic con-trol (44), but this was not the case with Plin5 deletion inthe liver. Evidently, potential changes in intertissue cross-talk could extend to hormones other than FGF-21, and thiswill require further investigation. In addition, our resultsand others (45) support the notion that some PLIN5functions are highly conserved across cell types/tissues(e.g., maintenance of intracellular triglyceride levels) butthat other tissue-specific actions of PLIN5 impact localinsulin action through distinct mechanisms (e.g., alteredproduction of signaling lipids, including ceramides inskeletal muscle but not in liver [oxidative stress in theheart but not liver]).

Previous reports indicate that PLIN5 facilitates physicalcontact between lipid droplets and mitochondria withinthe heart (7,31), which is proposed to facilitate tightercoupling of triglyceride lipolysis to fatty acid oxidation. Inkeeping with this, live cell imaging studies demonstratedreduced lipid droplet–mitochondria contact in hepatocytesderived from Plin5LKO mice compared with Wt mice, andthis was associated with reduced fatty acid oxidation inPlin5LKO hepatocytes, independent of changes in mito-chondrial protein abundance. However, immunofluores-cent and electron microscopy approaches have notconfirmed this relationship in skeletal muscle (7), raisingthe distinct possibility of tissue specificity in the interact-ing/adaptor proteins that facilitate this interorganellecontact. Future studies are clearly required to elucidatethe proteins that facilitate the interaction between lipiddroplets and mitochondria under physiologically relevantconditions and determine whether the functional interplay

between these organelles is an important determinant ofmetabolic efficiency, intracellular signaling, and insulinaction.

In sum, this study affirms the importance of lipiddroplet proteins in regulating cell metabolism andexpands our knowledge of PLIN5 biology by demonstrat-ing multiple effects on hepatic lipid metabolism and therequirement of PLIN5 for normal insulin action in theliver, especially in mice fed a high-fat diet. Since PLIN5expression is increased in response to lipid oversupply inmice (18) and in steatotic livers of humans (16), andoverexpression of PLIN5 improves insulin action (18),our data suggest that PLIN5 is part of an adaptive re-sponse to protect against insulin resistance and type 2diabetes.

Acknowledgments. The authors thank Michelle Kett, Robert Lee, CamdenLo, and Maria Matzaris (Monash University) for technical assistance. The authorsthank the Melbourne Mass Spectrometry and Proteomics Facility of the Bio21Molecular Science & Biotechnology Institute at The University of Melbourne for thesupport of mass spectrometry analysis.Funding. This work was funded by the National Health and Medical ResearchCouncil of Australia (NHMRC) (1047138). S.N.K. was supported by a Bio-medicine Discovery Scholarship from Monash University, and M.J.W.(APP1077703) and M.K.M. (APP1143224) were supported by research fellowshipsfrom the NHMRC.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.N.K. and M.J.W. planned and conducted theexperiments, analyzed the data, and wrote the manuscript. R.C.M., J.C.Y.L., andM.K.M. planned and conducted the experiments and analyzed the data. A.R. andS.N. planned and conducted experiments. All authors edited the manuscript.M.J.W. is the guarantor of this work and, as such, had full access to all the datain the study and takes responsibility for the integrity of the data and the accuracyof the data analysis.

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