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

2019; Elsevier BV; Volume: 60; Issue: 4 Linguagem: Inglês

10.1194/jlr.m091173

ISSN

1539-7262

Autores

Mi‐Jeong Lee, Sukanta Jash, Jessica Jones, Vishwajeet Puri, Susan K. Fried,

Tópico(s)

Adipokines, Inflammation, and Metabolic Diseases

Resumo

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

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