Perilipin-2 deletion promotes carbohydrate-mediated browning of white adipose tissue at ambient temperature
2018; Elsevier BV; Volume: 59; Issue: 8 Linguagem: Inglês
10.1194/jlr.m086249
ISSN1539-7262
AutoresAndrew E. Libby, Elise S. Bales, Jenifer Monks, David J. Orlicky, James L. McManaman,
Tópico(s)Cardiovascular Disease and Adiposity
ResumoMice lacking perilipin-2 (Plin2-null) are resistant to obesity, insulin resistance, and fatty liver induced by Western or high-fat diets. In the current study, we found that, compared with WT mice on Western diet, Plin2-null adipose tissue was more insulin sensitive and inguinal subcutaneous white adipose tissue (iWAT) exhibited profound browning and robust induction of thermogenic and carbohydrate-responsive genetic programs at room temperature. Surprisingly, these Plin2-null responses correlated with the content of simple carbohydrates, rather than fat, in the diet, and were independent of adipose Plin2 expression. To define Plin2 and sugar effects on adipose browning, WT and Plin2-null mice were placed on chow diets containing 20% sucrose in their drinking water for 6 weeks. Compared with WT mice, iWAT of Plin2-null mice exhibited pronounced browning and striking increases in the expression of thermogenic and insulin-responsive genes on this diet. Significantly, Plin2-null iWAT browning was associated with reduced sucrose intake and elevated serum fibroblast growth factor (FGF)21 levels, which correlated with greatly enhanced hepatic FGF21 production. These data identify Plin2 actions as novel mediators of sugar-induced adipose browning through indirect effects of hepatic FGF21 expression, and suggest that adipose browning mechanisms may contribute to Plin2-null resistance to obesity. Mice lacking perilipin-2 (Plin2-null) are resistant to obesity, insulin resistance, and fatty liver induced by Western or high-fat diets. In the current study, we found that, compared with WT mice on Western diet, Plin2-null adipose tissue was more insulin sensitive and inguinal subcutaneous white adipose tissue (iWAT) exhibited profound browning and robust induction of thermogenic and carbohydrate-responsive genetic programs at room temperature. Surprisingly, these Plin2-null responses correlated with the content of simple carbohydrates, rather than fat, in the diet, and were independent of adipose Plin2 expression. To define Plin2 and sugar effects on adipose browning, WT and Plin2-null mice were placed on chow diets containing 20% sucrose in their drinking water for 6 weeks. Compared with WT mice, iWAT of Plin2-null mice exhibited pronounced browning and striking increases in the expression of thermogenic and insulin-responsive genes on this diet. Significantly, Plin2-null iWAT browning was associated with reduced sucrose intake and elevated serum fibroblast growth factor (FGF)21 levels, which correlated with greatly enhanced hepatic FGF21 production. These data identify Plin2 actions as novel mediators of sugar-induced adipose browning through indirect effects of hepatic FGF21 expression, and suggest that adipose browning mechanisms may contribute to Plin2-null resistance to obesity. The obesity epidemic is recognized as one of the major public health challenges facing modern society, with 66% of adults in the United States being either overweight or obese (1.Nguyen D.M. El-Serag H.B. The epidemiology of obesity.Gastroenterol. Clin. North Am. 2010; 39: 1-7Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Health problems associated with obesity include insulin resistance and risk of diabetes development, fatty liver formation, alterations in circulating lipid profiles, cardiovascular disease, hypertension, and increased risk of certain cancers (2.Catenacci V.A. Hill J.O. Wyatt H.R. The obesity epidemic.Clin. Chest Med. 2009; 30: 415-444Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 3.Polsky S. Ellis S.L. Obesity, insulin resistance, and type 1 diabetes mellitus.Curr. Opin. Endocrinol. Diabetes Obes. 2015; 22: 277-282Crossref PubMed Scopus (137) Google Scholar, 4.Hubert H.B. Feinleib M. McNamara P.M. Castelli W.P. 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In the last decade, the lipid droplet coat protein family of perilipins (PLINs) has been recognized as the physiological regulator of lipid accumulation in many tissues under both healthy and pathophysiological states (10.Ducharme N.A. Bickel P.E. Lipid droplets in lipogenesis and lipolysis.Endocrinology. 2008; 149: 942-949Crossref PubMed Scopus (387) Google Scholar). Five mammalian PLIN family members have been identified (PLIN1 through PLIN5), with differential tissue expression of the members under both healthy and disease states. PLIN2 is a constitutively associated intracellular lipid droplet coat protein expressed in many organs, including the liver, adipose tissue, and skeletal muscle (11.Greenberg A.S. Coleman R.A. Kraemer F.B. McManaman J.L. Obin M.S. Puri V. Yan Q.W. Miyoshi H. Mashek D.G. The role of lipid droplets in metabolic disease in rodents and humans.J. Clin. Invest. 2011; 121: 2102-2110Crossref PubMed Scopus (456) Google Scholar). Our laboratory has previously generated mice with whole-body knockout of Plin2 (Plin2-null). These animals are resistant to obesity, adipose tissue inflammation, insulin resistance, and liver steatosis (nonalcoholic fatty liver disease) when chronically exposed to a Western diet (WD) or a high-fat (60%) diet (HFD) (12.McManaman J.L. Bales E.S. Orlicky D.J. Jackman M. MacLean P.S. Cain S. Crunk A.E. Mansur A. Graham C.E. Bowman T.A. et al.Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.J. Lipid Res. 2013; 54: 1346-1359Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 13.Libby A.E. Bales E. Orlicky D.J. McManaman J.L. Perilipin-2 deletion impairs hepatic lipid accumulation by Interfering with sterol regulatory element-binding protein (SREBP) activation and altering the hepatic lipidome.J. Biol. Chem. 2016; 291: 24231-24246Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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To date, a variety of mechanisms have been shown to induce white-to-brown conversion of WAT. The first fully defined mechanism involved browning of WAT in response to cold exposure (25.Wu J. Cohen P. Spiegelman B.M. Adaptive thermogenesis in adipocytes: is beige the new brown?.Genes Dev. 2013; 27: 234-250Crossref PubMed Scopus (629) Google Scholar). Similar to activation of BAT, cold exposure leads to a sympathetic nervous system release of norepinephrine into WAT depots, which causes a white-to-brown phenotypic switch. Mammals are able to use this mechanism to increase nonshivering thermogenesis and heat output during cold stress. Additionally, other mechanisms for browning of WAT have since been described, including a variety of endogenous hormones. Irisin, a hormone primarily secreted by skeletal muscle, has powerful effects on WAT browning and is currently the subject of therapeutic interest (26.Boström P. Wu J. Jedrychowski M.P. Korde A. Ye L. Lo J.C. Rasbach K.A. Boström E.A. 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ChREBP is a protein that is responsive to carbohydrates and controls transcription of a suite of lipogenic genes, including FASN and acetyl-CoA carboxylase (ACC). Transgenic increase of ChREBP in adipose tissue greatly enhances insulin sensitivity, PPAR activity, and transcription of the thermogenic program. Here, we identify PLIN2 as a novel determinant of inguinal subcutaneous WAT (iWAT) browning at ambient temperature. Mice lacking whole-body Plin2 exhibit drastic differences in subcutaneous iWAT histology and thermogenic program gene/protein expression compared with WT animals. Remarkably, the degree of browning in subcutaneous iWAT in Plin2-null mice is strongly influenced by the diet these animals are fed, with diets high in simple carbohydrates evoking the strongest iWAT browning responses. Significantly, our data implicate liver-derived FGF21 in the mechanism of Plin2 regulation of these diet-induced browning effects and possibly effects of Plin2 on caloric intake. Taken together, our data provide critical insights into the molecular mechanisms of obesity resistance in Plin2-null animals. All animal protocols were approved by the IACUC of the University of Colorado Denver. Plin2-null mice were generated as previously described (12.McManaman J.L. Bales E.S. Orlicky D.J. Jackman M. MacLean P.S. Cain S. Crunk A.E. Mansur A. Graham C.E. Bowman T.A. et al.Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.J. Lipid Res. 2013; 54: 1346-1359Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Adipose-specific Plin2 knockout mice were generated by crossing the Plin2-floxed mouse with one expressing Cre recombinase driven by the FABP4 promoter [B6.Cg-Tg(Fabp4-cre)1Rev/J]. Mice were housed in the University of Colorado Anschutz Medical Campus animal facility at ambient temperatures (22–24°C) on a 10:14 h light-dark cycle. Feeding was ad libitum with free access to food and water. For the 30 week feeding studies, mice were fed standard chow from weaning until 8 weeks, after which they were fed either control diet (CD) (Teklad TD.08485), WD (Teklad TD.88137), HFD (Research Diets D12492), low-fat diet (LFD) (Research Diets D12450B), or standard chow (Teklad 2920X) for 30 weeks. For the sucrose supplementation study, animals were raised on standard chow until 8 weeks of age. Mice were then split into two groups and given either water or 20% sucrose in water bottles for 6 weeks with free access to chow. Liquid intake, food intake, and body weights (BWs) were monitored weekly. Upon completion of the feeding experiments, mice were fasted for 4 h and euthanized by CO2 exposure and cardiac puncture. Samples of freshly excised adipose tissue were fixed for 24–36 h in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E as described previously (12.McManaman J.L. Bales E.S. Orlicky D.J. Jackman M. MacLean P.S. Cain S. Crunk A.E. Mansur A. Graham C.E. Bowman T.A. et al.Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.J. Lipid Res. 2013; 54: 1346-1359Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Processing of histological samples was performed by the Pathology Core at the University of Colorado Denver Anschutz Medical Campus. RNA was extracted from snap-frozen tissue using a modified phenol-chloroform method. Approximately 50–75 mg of tissue were homogenized in 1 ml of ice-cold QIAzol® (Qiagen) using bead homogenization with two rounds of shaking at 30 Hz for 2 min each. To maximize RNA yield, homogenates were quantitatively transferred to Phase Lock Gel Heavy tubes (Quantabio Inc.), 400 μl of chloroform were added, and samples were shaken for 30 s. Samples were then centrifuged at 21,000 g for 15 min at 4°C. The RNA-containing aqueous layer above the gel was transferred to a new tube, mixed 1:1 with Buffer RLT Plus (Qiagen), mixed with 1 ml 70% ethanol, and subjected to RNA cleanup using the RNeasy Plus Mini kit (Qiagen). Genomic DNA was removed by on-column DNase digestion. RNA quality was assessed by the Genomics and Microarray Core Facility at the University of Colorado Denver Anschutz Medical Campus. cDNA was synthesized from 900 ng RNA using the iScript cDNA synthesis kit (Bio-Rad). Quantitative (q)RT-PCR was performed on a Bio-Rad CFX96 instrument using SYBR green probes and melting curve analysis to ensure single-product amplification. The qRT-PCR master mix was 2× SYBR Green qPCR Mastermix (BioTool). All gene expression was normalized to 18S rRNA, and relative expression levels were calculated using the ΔΔCt method. All primer sequences used for qRT-PCR can be found in the supplemental materials (supplemental Table S1). Protein extraction was performed by homogenizing ∼100 mg of frozen tissue directly into 2:1 chloroform:methanol in a bead homogenizer for two cycles at 30 Hz for 2 min each. The insoluble protein pellet was spun down, washed with an additional 2 ml of 2:1 chloroform:methanol to remove residual lipids, washed twice with ice-cold methanol, briefly dried, and resuspended in 10% SDS containing protease and phosphatase inhibitors (Thermo Pierce). Protein was quantified with the BCA assay (Thermo Pierce) with standards prepared in 10% SDS. For Western blotting, 37.5 μg of protein were separated on 4–20% Tris-glycine gels (Bio-Rad) and transferred to 0.2 μM nitrocellulose membranes (Bio-Rad) at 100 V for 1 h and 10 min. All membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20, pH 7.3. All antibodies and dilutions used for Western blotting can be found in the supplemental materials (supplemental Table S2). Immunoreactive bands were detected with corresponding labeled secondary antibodies (Li-COR) and quantified by imaging on a Li-COR CLx instrument. Protein intensities were normalized to α-tubulin for quantitative analysis. Approximately 10 mg of frozen adipose tissue were homogenized in 1 ml 2:1 chloroform:methanol containing 300 μg of tritridecanoin reference standard (Nu-Check Prep Inc.) using bead homogenization with two cycles at 30 Hz for 2 min each cycle. Homogenates were diluted into a total of 4 ml 2:1 chloroform:methanol in glass tubes, mixed with 800 μl of 0.9% sodium chloride, vortexed, and centrifuged at 3,250 g for 5 min. The lower phase was removed and dried under N2 gas. Total lipids were resuspended in 330 ul 100% chloroform and applied to HyperSep SI SPE columns (Thermo Scientific) preequilibrated with 15 column volumes of chloroform. Neutral lipid (NL) was eluted with 3 ml of chloroform followed by 5 ml of acetone:methanol (9:1) to elute glycolipids. Phospholipids (PLs) were then eluted with 3 ml of 100% methanol. All lipid fractions were dried under N2 gas and resuspended in 1 ml of methanol containing 2.5% H2SO4. Fatty acid methyl ester (FAME) production was initiated by heating at 80°C for 1.5 h with occasional vortexing. One milliliter of HPLC-grade water was added to quench the reactions, and FAMEs were extracted in 400 μl of hexane by vortexing. A Trace 1310 gas chromatograph with a TG-5MS column (Thermo Scientific) was used to separate FAMEs chromatographically, and lipids were analyzed with an ISQ single quadrupole mass spectrometer (Thermo Scientific). For each lipid species analyzed, serial dilutions were performed to ensure that peak areas were within linear range. Xcalibur software (Thermo Scientific) was used to calculate peak areas. Areas were normalized to the reference standard and then to total protein. Mice were fasted for 4 h starting at 8:00 AM and then split into two groups, with one receiving saline and the other receiving insulin (0.5 U/kg BW) via intraperitoneal injection. The insulin used was Humulin N® (Eli Lilly). Blood was taken 20 min postinjection, and FFA was measured calorimetrically in serum using the Serum/Plasma FFA Detection kit (ZenBio Inc., product number SFA-1). Serum FGF21 was assessed with the mouse FGF21 ELISA kit (Abcam, product number ab212160). Pilot dilutions were performed to ensure that samples were within the range of standards, and development of TMB was monitored kinetically at 600 nM in 20 s intervals for 20 min on a spectrophotometer equipped with shaking capabilities (Molecular Devices). Data were analyzed using a four-parameter curve fit with SoftMax Pro software (Molecular Devices). All data are presented as mean ± SEM and were analyzed by two-way ANOVA unless otherwise indicated. Two-way ANOVA with post hoc Tukey's multiple comparison testing was performed with GraphPad Prism software. Three-way ANOVA was performed with STATA v15 software. For lipidomic analysis under individual dietary conditions, Student's t-test was performed with Bonferroni correction using GraphPad Prism. P < 0.05 was considered to be statistically significant. WT and Plin2-null mice were placed on either a WD containing 42% kcal from fat, 42.7% kcal from carbohydrate, 15.2% kcal from protein, and 0.2% cholesterol or a matched CD containing 13% kcal from fat, 67.9% kcal from carbohydrate, 19.1% kcal from protein, and no added cholesterol for 30 weeks at ambient temperature. We previously reported that WD-fed Plin2-null mice are completely protected from the obesity, glucose intolerance, and nonalcoholic fatty liver formation found in WT mice on this diet (13.Libby A.E. Bales E. Orlicky D.J. McManaman J.L. Perilipin-2 deletion impairs hepatic lipid accumulation by Interfering with sterol regulatory element-binding protein (SREBP) activation and altering the hepatic lipidome.J. Biol. Chem. 2016; 291: 24231-24246Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In this study, we investigated the molecular differences that underlie the resistance of Plin2-null animals to obesity. We first performed histological examination of various adipose tissues of WT and Plin2-null mice, including interscapular BAT (iBAT), iWAT, and epididymal (perigonadal) WAT (eWAT). As expected, iBAT from both WT and Plin2-null mice exhibited classic widespread and homogenous cytoplasmic lipid droplet multilocularity on both diets (Fig. 1A), although WT mice appeared to have larger lipid droplets in their iBAT on WD compared with CD. No major differences were observed in the iBAT of Plin2-null mice on either diet. Next, we examined the iWAT of WT and Plin2-null mice on these diets (Fig. 1B). WT mice demonstrated classic iWAT, with unilocular adipocytes that were slightly larger on WD compared with CD. Remarkably, iWAT from Plin2-null mice fed either WD or CD very closely resembled the characteristics of iBAT, with primarily small multilocular adipocytes dominating the specimens. These multilocular droplets were homogenous throughout the bulk of the tissue, suggesting that the response was widespread. Interestingly, lipid droplets in iWAT from WD-fed Plin2-null mice appeared to be slightly smaller than those from CD-fed animals. Overall, the histological appearance and multilocularity of iWAT from Plin2-null mice at ambient temperatures was reminiscent of the browning response in iWAT normally observed in mice exposed to cold temperatures (37.Barbatelli G. Murano I. Madsen L. Hao Q. Jimenez M. Kristiansen K. Giacobino J.P. De Matteis R. Cinti S. The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E1244-E1253Crossref PubMed Scopus (556) Google Scholar). Finally, we examined eWAT from WT and Plin2-null mice on WD and CD (Fig. 1C). Compared with CD-fed mice, eWAT from WT mice fed the WD exhibited larger adipocytes and the presence of crown-like structures indicative of inflammation (38.Lumeng C.N. Bodzin J.L. Saltiel A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization.J. Clin. Invest. 2007; 117: 175-184Crossref PubMed Scopus (3335) Google Scholar). Conversely, Plin2-null mice showed much smaller adipocytes on both diets compared with WT, and no crown-like structures were observed in these mice. Importantly, we did not observe multilocular adipocytes or browning in eWAT from either genotype. We also performed mass analysis of iBAT, iWAT, and eWAT fat pads from Plin2-null and WT mice on long-term WD and CD. We previously reported quantitative MRI data from these animals (13.Libby A.E. Bales E. Orlicky D.J. McManaman J.L. Perilipin-2 deletion impairs hepatic lipid accumulation by Interfering with sterol regulatory element-binding protein (SREBP) activation and altering the hepatic lipidome.J. Biol. Chem. 2016; 291: 24231-24246Abstract Full Text Full Text PDF PubMed Scopus (51) Google Sch
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