Rosiglitazone remodels the lipid droplet and britens human visceral and subcutaneous adipocytes ex vivo

• DOI: 10.1194/jlr.m091173
• Publication Date: 2019-02-20T01:07:01Z
• Authors: Mi-Jeong Lee; Sukanta Jash; Jessica E.C. Jones; Vishwajeet Puri; Susan K. Fried
• Keywords: Ex vivo; Rosiglitazone; Lipid droplet; Subcutaneous fat

Treatment with PPARγ agonists in vivo improves human adipocyte metabolism, but the cellular mechanisms and possible depot differences responsiveness to their effects are poorly understood. To examine ex metabolic of rosiglitazone (Rosi), we cultured explants visceral (omental) abdominal subcutaneous adipose tissues for 7 days. Rosi increased mRNA levels transcriptional regulators brite/beige adipocytes (PGC1α, PRDM16), triglyceride synthesis (GPAT3, DGAT1), lipolysis (ATGL) similarly from both depots. In parallel, key modulators FA oxidation (UCP1, FABP3, PLIN5 protein), rates oxidation, protein electron transport complexes, suggesting an enhanced respiratory capacity as confirmed newly differentiated adipocytes. led formation small lipid droplets (SLDs) around central droplet; each SLD was decorated redistributed mitochondria that colocalized PLIN5. maintenance required reesterification. thus coordinated a structural remodeling depots oxidative capacity. Selective targeting these improve handling may provide new approach treat complications obesity diabetes. White specialized cells have very high esterify store FAs (TAG) within single droplet (LD) release them by variations nutritional state (1Gustafson B. Hedjazifar S. Gogg Hammarstedt A. Smith U. Insulin resistance impaired adipogenesis.Trends Endocrinol. Metab. 2015; 26: 193-200Abstract Full Text PDF PubMed Scopus (267) Google Scholar). obesity, must cope flux diet higher basal (2Koutsari C. Jensen M.D. Free fatty acid metabolism obesity.J. Lipid Res. 2006; 47: 1643-1650Abstract (167) Scholar, 3Rydén M. Arner P. Subcutaneous contributes circulating levels.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 1782-1787Crossref (54) If not efficiently esterified or oxidized, unbound cell cause lipotoxic stress deleterious consequences endocrine function (4Das S.K. Chu W.S. Mondal A.K. Sharma N.K. Kern P.A. Rasouli N. Elbein S.C. Effect pioglitazone treatment on endoplasmic reticulum response palmitate-induced liver lines.Am. J. Physiol. 2008; 295: E393-E400Crossref (48) 5Frayn K.N. Langin D. Karpe F. Fatty acid-induced mitochondrial uncoupling is promising target insulin unless increased.Diabetologia. 51: 394-397Crossref (38) 6Chitraju Mejhert Haas J.T. Diaz-Ramirez L.G. Grueter C.A. Imbriglio J.E. Pinto Koliwad Walther T.C. Farese Jr., R.V. Triglyceride DGAT1 protects lipid-induced ER during lipolysis.Cell 407-418.e3Abstract (194) As storage exceeded overflow can lead ectopic fat deposition systemic dysfunction 7Bogacka I. Xie H. Bray G.A. S.R. Pioglitazone induces biogenesis tissue vivo.Diabetes. 2005; 54: 1392-1399Crossref (404) 8Cuthbertson D.J. Steele T. Wilding J.P. Halford J.C. Harrold J.A. Hamer What experimental overfeeding studies taught us about expansion susceptibility complications?.Int. Obes. (Lond.). 41: 853-865Crossref (88) Strategies modify therapeutic potential obesity-related diseases. contrast multilocular brown "brite"/beige densely packed express UCP1, white contain relatively few mitochondria, low capacity, much lower UCP1 (9Carobbio Pellegrinelli V. Vidal-Puig Adipose expandability determinants lipotoxicity syndrome.Adv. Exp. Med. 960: 161-196Crossref (127) (WAT), however, acquire brown-like features β-adrenergic stimulation cold. Studies rodents shown be formed mature (10Barbatelli G. Murano Madsen L. Hao Q. Jimenez Kristiansen K. Giacobino De M.R. Cinti The emergence cold-induced mouse determined predominantly transdifferentiation.Am. 2010; 298: E1244-E1253Crossref (584) 11Rosenwald Perdikari Rulicke Wolfrum Bi-directional interconversion brite adipocytes.Nat. Cell 2013; 15: 659-667Crossref (593) Scholar) recruitment differentiation beige progenitors (12Seale Conroe H.M. Estall Kajimura Frontini Ishibashi Cohen Spiegelman B.M. Prdm16 determines thermogenic program mice.J. Clin. Invest. 2011; 121: 96-105Crossref (961) 13Wang Q.A. Tao Gupta R.K. Scherer P.E. Tracking adipogenesis development, regeneration.Nat. 19: 1338-1344Crossref (905) Several lines evidence indicate metabolically reprogrammed more phenotype vivo. Chronic adrenergic humans pheochromocytoma (14Frontini Vitali Perugini Romiti Ricquier Guerrieri White-to-brown transdifferentiation omental patients affected pheochromocytoma.Biochim. Biophys. Acta. 1831: 950-959Crossref (169) severe burn injury (15Sidossis L.S. Porter Saraf M.K. Borsheim E. Radhakrishnan R.S. Chao Ali Chondronikola Mlcak R. Finnerty C.C. et al.Browning after stress.Cell 22: 219-227Abstract (296) direct cold exposure several hours (16Kern Finlin B.S. Zhu McGehee R.E. Westgate P.M. Dupont-Versteegden E.E. temperature seasons humans: gene induction.J. 2014; 99: E2772-E2779Crossref (72) obese type 2 diabetes (Rosi) also tissue, including expression genes (7Bogacka 17Rasouli Das Improved sensitivity PPARgamma PPARalpha ligands mediated genetically modulated transcripts.Pharmacogenet. Genomics. 2012; 484-497Crossref (22) (18Esterson Y.B. Zhang Koppaka Kehlenbrink Kishore Raghavan Maginley Carey Hawkins sensitizing anti-inflammatory thiazolidinediones heightened patients.J. Investig. 61: 1152-1160Crossref (28) Further, activation drives culture models (19Bogacka Ukropcova McNeil Gimble J.M. Structural functional vitro.J. 90: 6650-6656Crossref (121) 20Elabd Chiellini Carmona Galitzky Cochet O. Petersen Penicaud Bouloumie Casteilla al.Human multipotent adipose-derived stem differentiate into adipocytes.Stem Cells. 2009; 27: 2753-2760Crossref (210) 21Barquissau Beuzelin Pisani D.F. Beranger G.E. Mairal Montagner Roussel Tavernier Marques M.A. Moro al.White-to-brite conversion promotes reprogramming towards anabolic catabolic pathways.Mol. 2016; 5: 352-365Crossref (96) Scholar), know no examined Rosi's Significant components, LDs occurs humans, induced chronic catecholamines exhibit mixture paucilocular 15Sidossis early ob/ob mice treated Rosi, Corvera's group observed "small, droplet-like structures" surrounded dense staining (22Wilson-Fritch Nicoloro Chouinard Lazar Chui P.C. Leszyk Straubhaar Czech M.P. Corvera Mitochondrial associated rosiglitazone.J. 2004; 114: 1281-1289Crossref (383) course carrying out actions adipocytes, similar remodeling. We therefore went investigate biochemical interaction Perilipins LD proteins expressed cell-specific manner play active roles regulation lipolysis, (re)esterification (23Sztalryd Brasaemle D.L. perilipin family proteins: Gatekeepers intracellular lipolysis.Biochim. Acta Mol. Lipids. 1862: 1221-1232Crossref (313) PLIN1 major (24Blanchette-Mackie E.J. Dwyer Barber Coxey R.A. Takeda Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos Perilipin located surface layer adipocytes.J. 1995; 36: 1211-1226Abstract while tissues, skeletal muscle (BAT), selectively (25Wolins N.E. Quaynor B.K. Skinner J.R. Tzekov Croce Gropler M.C. Varma Yao-Borengasser al.OXPAT/PAT-1 PPAR-induced utilization.Diabetes. 55: 3418-3428Crossref (259) 26Bosma Minnaard Sparks L.M. Schaart Losen de Baets M.H. Duimel Kersten Bickel Schrauwen al.The coat 5 localizes mitochondria.Histochem. 137: 205-216Crossref (136) 27Wang Sreenevasan Hu Saladino Polster Lund Gong D.W. Stanley W.C. Sztalryd 5, droplet-associated protein, provides physical linkage mitochondria.J. 52: 2159-2168Abstract (315) Both limit facilitate stimulated However, differs it physically interacts β-oxidation increases WAT (hMADs) vitro (21Barquissau so studied how its localization different anatomical capacities phenotypes. mice, (inguinal) "briten" exposure, receptor stimulation, agonist treatment, most do show browning "visceral" (epididymal) male (28Guerra Koza Yamashita Walsh Kozak L.P. Emergence under genetic control. Effects body weight adiposity.J. 1998; 102: 412-420Crossref (547) 29Ohno Shinoda induce white-to-brown through stabilization PRDM16 protein.Cell 395-404Abstract (607) 30Wu Bostrom Ye Choi J.H. Giang A.H. Khandekar Virtanen K.A. Nuutila al.Beige distinct human.Cell. 150: 366-376Abstract (2488) 31Shabalina I.G. Petrovic Jong Kalinovich A.V. Cannon Nedergaard functionally thermogenic.Cell Reports. 1196-1203Abstract (498) phenotypes were found (Om) another study detected UCP1-positive only periadrenal (32Vergnes Davies G.R. Lin J.Y. Yeh M.W. Livhits M.J. Harari Symonds M.E. Sacks H.S. Reue Adipocyte SC pheochromocytoma.J. 101: 4440-4448Crossref current study, considered possibility could increase used organ system maintains over days 3D context physiologically relevant hormone milieu (insulin glucocorticoid modeled dexamethasone) (33Lee Burkey B.F. Fried Pathways regulated glucocorticoids tissues: microarray study.Am. 300: E571-E580Crossref (80) Collectively, our results reveal toward phenotype. Om (ASATs) obtained elective surgeries total 39 subjects [BMI, 40.5 ± 1.6 kg/m2 (range 23–63 kg/m2); age, 41.4 2.0 years 25–71 years)] who free diabetes, cancers, inflammatory diseases medical history. Surgeries took place at Boston Medical Center. All provided informed consent approved Institutional Review Boards Center all abided Declaration Helsinki principles. Subject characteristics supplemental Table S1. experiments repeated samples least three donors. number experiment figure legends. placed room medium 199 (Life Technologies), transferred laboratory, minced fragments (∼5–10 mg), (0.7 nM) dexamethasone (10 without (1 μM) up serum-free 199, previously described Aliquots media final day frozen −80°C glycerol measurements. After culture, isolation stromal vascular collagenase digestion mg/ml 1; Worthington Biochemical) quick liquid nitrogen immunoblotting, RNA expression, mitochondria. assess importance reesterification, orlistat (100 triacsin C (2 added Rosi. Total extracted using Trizol (Thermo-Fisher) quantity quality assessed spectrophotometrically (Nano-Drop; Thermo-Fisher). reverse transcription (Transcriptor First Strand cDNA kit; Roche), quantitative (q)PCR performed TaqMan probes Light Cycler 480 II (Roche). Expression relative cyclophilin A presented. Human Gene 1.0 ST microarrays (Affymetrix) profile patterns control Rosi-treated two women (33 old, Hispanic, BMI = 43.0 kg/m2; 62 Caucasian, 31.4 kg/m2). Microarray analysis Resource Core Clinical Translational Science Institute University deposited NCBI's Omnibus (GSE122721). arrays normalized Robust Multi-array Average algorithm Chip definition file maps array unique Entrez identifiers. Technical duplicates first subject averaged values second prior statistical analysis. linear mixed-effects model identify changed adjusting inter-donor variability. transcripts significantly up- down-regulated (FDR q < 0.05) enrichment DAVID (https://david.ncifcrf.gov/) Significantly enriched (P 0.01) KEGG pathways reported. Tissues homogenized lysis buffer (Cell Signaling) supplemented 10% SDS (to assure extraction proteins) protease inhibitor cocktail, processed (34Lee Glucocorticoids antagonize tumor necrosis factor-alpha-stimulated antilipolytic effect adipocytes.Am. 303: E1126-E1133Crossref (37) Proteins resolved NuPAGE Bis-Tris gels MOPS-SDS MES-SDS running PVDF membranes (Thermo-Fisher). blocking 5% milk, blotted (ab10983; Abcam), porin (ab15895; (GP31; Progen Biotechnik), FABP3 (gift Dr. Judith Storch, Rutgers University), ATGL chain (ETC) (ab110411; HSP90 (sc-7947; Santa Cruz Biotechnology) followed incubation appropriate secondary antibodies conjugated HRP capture chemiluminescence images. Mitochondria isolated following procedures (35Frezza Cipolat Scorrano Organelle isolation: liver, fibroblasts.Nat. Protoc. 2007; 2: 287-295Crossref (915) slight modifications. Briefly, (0.3 M sucrose, 10 mM HEPES, 1 EGTA) 1% BSA centrifugation steps 500 g min, 700 finally 7,000 min. pellet resuspended buffer, quantified BCA assay kit immunoblotting ETC proteins. (total ∼100 mg) had been specified conditions incubated 14C-oleic μCi/ml) ml Krebs-Ringer bicarbonate (KRB) 0.2 oleic acid, glucose, 4% atmosphere 95% O2:5% CO2 3 h. Complete 14C-oleate 14CO2 measured Lipolysis accumulation culture. Glycerol concentrations fluorometrically (36Laurell Tibbling An enzymatic fluorometric micromethod determination glycerol.Clin. Chim. 1966; 13: 317-322Crossref (340) weight. directly changes lipolytic acute incubations Incubations carried KRB glucose (pH 7.4) h atrial natriuretic peptide (ANP; nM), norepinephrine (10−6 M), isoproterenol (Iso; 10−6 (37Fried Tittelbach Blumenthal Sreenivasan Robey Yi Khan Hollender Ryan Goldberg A.P. Resistance African-American compared Caucasian postmenopausal women.J. 1193-1200Abstract (21) data Dole's MS-based detection cardiolipin Michigan Nutrition Obesity Research lipids modified Bligh-Dyer method, 2:2:2 (v/v/v) water/methanol/dichloromethane. spiking internal standards, organic collected dried stream nitrogen. extract injected Waters Acquity HSS T3 column LC-MS (Waters). Cardiolipin content Isolated freshly lipid. Adipocytes MitoTracker-Green, MitoTracker-Red, LipidTOX-Deep Red washing, live mounted slides DAPI mounting (Vector Labs) confocal microscopy Zeiss LSM 710-Live Duo scan (Carl Zeiss, Germany) Nikon A1Rsi microscope (Tokyo, Japan) 100× oil immersion objective. Images Metamorph imaging software (Universal Imaging). stained (MitoTracker-Red) neutral (LipidTOX-Deep Red) fixed paraformaldehyde 20 permeabilization 0.1% Triton X-100 min 2% h, rabbit anti-human guinea pig Biotechnik) goat anti-rabbit IgG Alexa Flour 488 anti-guinea (Thermo-Fisher), respectively, Cells fluorescence microscope. c