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• W2053701857 abstract "Many human immunodeficiency virus (HIV)-infected patients taking combination antiretroviral therapy that includes HIV protease inhibitors experience atrophy of peripheral subcutaneous adipose tissue. We investigated the effects of HIV protease inhibitors on adipogenesis and adipocyte survival using the 3T3-L1 preadipocyte cell line. Several HIV protease inhibitors were found either to inhibit preadipocyte differentiation or to promote adipocyte cell death. One protease inhibitor, nelfinavir, elicited both of these effects strongly. When induced to differentiate in the presence of nelfinavir, 3T3-L1 preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the adipogenic transcription factors CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ. The level of the proteolytically processed, active 68-kDa form of sterol regulatory element-binding protein-1, a transcription factor known to promote lipogenic gene expression, also was reduced markedly in nelfinavir-treated cells, whereas the level of the 125-kDa precursor form of this protein was unaffected. The inhibitory effect of nelfinavir occurred subsequent to critical early events in preadipocyte differentiation, expression of CCAAT/enhancer-binding protein β and completion of the mitotic clonal expansion phase, because these events were unaffected by nelfinavir treatment. In addition, nelfinavir treatment of fully differentiated 3T3-L1 adipocytes resulted in DNA strand cleavage and severe loss of cell viability. In contrast, cell proliferation and viability of preadipocytes were unaffected by nelfinavir treatment. Thus, molecular or cellular changes that occur during acquisition of the adipocyte phenotype promote susceptibility to nelfinavir-induced cell death. When considered together, these results suggest that nelfinavir may promote adipose tissue atrophy by compromising adipocyte viability and preventing replacement of lost adipocytes by inhibiting preadipocyte differentiation. Many human immunodeficiency virus (HIV)-infected patients taking combination antiretroviral therapy that includes HIV protease inhibitors experience atrophy of peripheral subcutaneous adipose tissue. We investigated the effects of HIV protease inhibitors on adipogenesis and adipocyte survival using the 3T3-L1 preadipocyte cell line. Several HIV protease inhibitors were found either to inhibit preadipocyte differentiation or to promote adipocyte cell death. One protease inhibitor, nelfinavir, elicited both of these effects strongly. When induced to differentiate in the presence of nelfinavir, 3T3-L1 preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the adipogenic transcription factors CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ. The level of the proteolytically processed, active 68-kDa form of sterol regulatory element-binding protein-1, a transcription factor known to promote lipogenic gene expression, also was reduced markedly in nelfinavir-treated cells, whereas the level of the 125-kDa precursor form of this protein was unaffected. The inhibitory effect of nelfinavir occurred subsequent to critical early events in preadipocyte differentiation, expression of CCAAT/enhancer-binding protein β and completion of the mitotic clonal expansion phase, because these events were unaffected by nelfinavir treatment. In addition, nelfinavir treatment of fully differentiated 3T3-L1 adipocytes resulted in DNA strand cleavage and severe loss of cell viability. In contrast, cell proliferation and viability of preadipocytes were unaffected by nelfinavir treatment. Thus, molecular or cellular changes that occur during acquisition of the adipocyte phenotype promote susceptibility to nelfinavir-induced cell death. When considered together, these results suggest that nelfinavir may promote adipose tissue atrophy by compromising adipocyte viability and preventing replacement of lost adipocytes by inhibiting preadipocyte differentiation. highly active antiretroviral therapy HIV protease inhibitor reverse transcriptase inhibitor HIV/HAART-associated syndrome CCAAT/enhancer-binding protein peroxisome proliferator-activated receptor retinoid X receptor sterol regulatory element-binding protein terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling human immunodeficiency virus Dulbecco's modified Eagle's medium phosphate-buffered saline 4′,6′-diamidino-2-phenylindole Highly active antiretroviral therapy (HAART)1 has proven effective at reducing morbidity and mortality in HIV-infected individuals displaying symptoms of disease progression (1Hammer S.M. Squires K.E. Hughes M.D. Grimes J.M. Demeter L.M. Currier J.S. Eron J.J.J. Feinberg J.E. Balfour H.H.J. Deyton L.R. Chodakewitz J.A. Fischl M.A. N. Engl. J. Med. 1997; 337: 725-733Crossref PubMed Scopus (2381) Google Scholar). Currently, the recommended therapy for such patients includes the use of one or two HIV protease inhibitors (PIs) combined with two nucleoside reverse transcriptase inhibitors (RTIs) or two nucleoside RTIs combined with one nonnucleoside reverse transcriptase inhibitor (2Carpenter C.C. Cooper D.A. Fischl M.A. Gatell J.M. Gazzard B.G. Hammer S.M. Hirsch M.S. Jacobsen D.M. Katzenstein D.A. Montaner J.S. Richman D.D. Saag M.S. Schechter M. Schooley R.T. Thompson M.A. Vella S. Yeni P.G. Volberding P.A. J. Am. Med. Assoc. 2000; 283: 381-390Crossref PubMed Scopus (1034) Google Scholar). Inhibition of the HIV protease prevents cleavage and maturation of the viral polyprotein precursor leading to production of noninfectious viral particles (reviewed in Ref. 3Flexner C. N. Engl. J. Med. 1998; 338: 1281-1291Crossref PubMed Scopus (763) Google Scholar). The HIV reverse transcriptase is required to copy the viral RNA genome and inhibitors used to target this enzyme consist of nonnucleoside, noncompetitive inhibitors or chain-terminating nucleoside analogues (reviewed in Ref. 4Beach J.W. Clin. Therapeutics. 1998; 20: 2-24Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Despite the clinical benefits of HIV suppression by HAART, a serious metabolic syndrome has arisen in treated patients. The syndrome often includes atrophy of subcutaneous adipose tissue, thus giving rise to the widely used term “lipodystrophy syndrome,” and increased visceral and dorsocervical adipose tissue (5Hengel R.L. Watts N.B. Lennox J.L. Lancet. 1997; 350: 1596Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Carr A. Samaras K. Burton S. Law M. Freund J. Chisholm D.J. Cooper D.A. AIDS. 1998; 12: F51-F58Crossref PubMed Scopus (2192) Google Scholar, 7Roth V.R. Kravcik S. Angel J.B. Clin. Infect. Dis. 1998; 27: 65-67Crossref PubMed Scopus (91) Google Scholar, 8Miller K.D. Jones E. Yanovski J.A. Shankar R. Feuerstein I. Falloon J. Lancet. 1998; 351: 871-875Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, 9Yanovski J.A. Miller K.D. Kino T. Friedman T.C. Chrousos G.P. Tsigos C. Fallon J. J. Clin. Endocrinol. Metab. 1999; 84: 1925-1931Crossref PubMed Scopus (0) Google Scholar, 10Stocker D.N. Meier P.J. Stroller R. Fattinger K.E. Lancet. 1998; 352: 320-321Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Other symptoms include dyslipidemia (6Carr A. Samaras K. Burton S. Law M. Freund J. Chisholm D.J. Cooper D.A. AIDS. 1998; 12: F51-F58Crossref PubMed Scopus (2192) Google Scholar, 8Miller K.D. Jones E. Yanovski J.A. Shankar R. Feuerstein I. Falloon J. Lancet. 1998; 351: 871-875Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar, 9Yanovski J.A. Miller K.D. Kino T. Friedman T.C. Chrousos G.P. Tsigos C. Fallon J. J. Clin. Endocrinol. Metab. 1999; 84: 1925-1931Crossref PubMed Scopus (0) Google Scholar, 11Periard D. Telenti A. Sudre P. Cheseaux J.-J. Halfon P. Reymond M.J. Marcovina S.M. Glauser M.P. Nicod P. Darioli R. Mooser V. Circulation. 1999; 100: 700-705Crossref PubMed Scopus (582) Google Scholar), hyperglycemia (12Dube M.P. Johnson D.L. Currier J.S. Leedom J.M. Lancet. 1997; 350: 713-714Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 13Eastone J.A. Decker C.F. Ann. Intern. Med. 1997; 127: 948Crossref PubMed Scopus (104) Google Scholar), and insulin resistance (6Carr A. Samaras K. Burton S. Law M. Freund J. Chisholm D.J. Cooper D.A. AIDS. 1998; 12: F51-F58Crossref PubMed Scopus (2192) Google Scholar, 14Walli R. Herfort O. Michl G.M. Demant T. Jager H. Dieterle C. Bogner J.R. Landgraf R Goebel F.D. AIDS. 1998; 12: F167-F173Crossref PubMed Scopus (504) Google Scholar). Emergence of the syndrome has been correlated temporally with the widespread use of PIs. However, similar symptoms have been reported in therapy naive HIV-infected patients (15Kotler D.P. Rosenbaum K. Wang J. Pierson R.N. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 1999; 20: 228-237Crossref PubMed Scopus (185) Google Scholar) and in patients receiving non-PI containing antiviral regimens (16Lo J.S. Mulligan K. Tai V.W. Algren H. Schambelan M. Lancet. 1998; 351: 867-870Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 17Gervasoni C. Ridolfo A.L. Trifiro G. Santambrogio S. Norbiato G. Musicco M. Clerici M. Galli M. Moroni M. AIDS. 1999; 13: 465-471Crossref PubMed Scopus (276) Google Scholar, 18Madge S. Kinloch de Loes S. Mercey D. Johnson M.A. Weller I.V.D. AIDS. 1999; 13: 735-737Crossref PubMed Scopus (167) Google Scholar, 19Saint-Marc T. Partisani M. Poizot-Martin I. Bruno F. Rouviere O. Lang J.M. Gastaut J.A. Touraine J. AIDS. 1999; 13: 1659-1667Crossref PubMed Scopus (547) Google Scholar). The underlying cause of the syndrome may be a complex physiological response to multiple factors including one or more components of combination drug regimens, viral infection, or effective viral suppression. Currently, the cause of this syndrome, referred to hereafter as HIV/HAART-associated syndrome (HAS), is unknown. A commonly reported symptom of HAS appears to be alteration of adipose tissue depots. Considerable progress has been made in understanding the molecular mechanisms of adipocyte biology using the 3T3-L1 preadipocyte cell line (20Green H. Kehinde O. Cell. 1974; 1: 113-116Abstract Full Text PDF Scopus (744) Google Scholar) as a model. 3T3-L1 preadipocytes, when growth-arrested at confluence, can be induced to differentiate into adipocytes in the presence of fetal bovine serum and a hormonal mixture that includes insulin, dexamethasone, and isobutylmethylxanthine (reviewed in Refs. 21Cornelius P. MacDougald O.A. Lane M.D. Annu. Rev. Nutr. 1994; 14: 99-129Crossref PubMed Scopus (573) Google Scholar, 22MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (942) Google Scholar, 23Hwang C.-S. Loftus T.M. Mandrup S. Lane M.D. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (209) Google Scholar). At least two classes of transcription factors serve important roles in regulating adipogenesis, CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs) belonging, respectively, to the basic leucine zipper class of transcription factors and to the nuclear hormone receptor superfamily (reviewed in Refs. 22MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (942) Google Scholar and 24Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). After the onset of differentiation, a cascade of gene expression begins with the rapid induction of C/EBP β and δ (25Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1345) Google Scholar, 26Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (812) Google Scholar). Concomitantly, synchronous re-entry into the cell cycle occurs, and cells proceed through a mitotic clonal expansion phase that consists of approximately two rounds of mitosis (27Bernlohr D.A. Bolanowski M.A. Kelly T.J. Lane M.D. J. Biol. Chem. 1985; 260: 5563-5567Abstract Full Text PDF PubMed Google Scholar, 28Tang Q.-Q. Lane M.D. Genes Dev. 1999; 13: 2231-2241Crossref PubMed Scopus (310) Google Scholar). Near to or upon completion of mitotic clonal expansion, expression of C/EBPα (25Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1345) Google Scholar) and PPARγ (29Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2000) Google Scholar) is induced, and expression of C/EBPβ and δ begins to decline (25Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1345) Google Scholar, 26Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (812) Google Scholar). C/EBPα and PPARγ then promote sustained expression of numerous adipocyte genes, including that which encodes the fatty acid binding protein 422/aP2 (reviewed in Refs. 22MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (942) Google Scholar, 24Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). The cells then become rounded and engorged with cytoplasmic triacylglycerol droplets. Both C/EBPα (30Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar, 31Herrera R. Ro H.S. Robinson G.S. Xanthopoulos K.G. Spiegelman B.M. Mol. Cell. Biol. 1989; 9: 5331-5339Crossref PubMed Scopus (134) Google Scholar) and PPARγ (29Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2000) Google Scholar, 32Tontonoz P. Graves R.A. Budavari A.I. Erdjument-Bromage H. Lui M. Hu E. Tempst P. Spiegelman B.M. Nucleic Acids Res. 1994; 22: 5628-5634Crossref PubMed Scopus (331) Google Scholar), the latter in combination with the obligate heterodimeric partner and nuclear hormone receptor superfamily member, retinoid X receptor (RXR) α, have been shown to bind regulatory elements within the promoter of the 422/aP2 gene. Similar regulatory elements have been identified within the promoters of numerous other adipocyte genes through which transcriptional activation is achieved (reviewed in Refs. 22MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (942) Google Scholar, 24Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). In addition, sterol regulatory element-binding protein-1 (SREBP-1, also referred to as ADD1) is expressed during 3T3-L1 differentiation (33Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar, 34Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (846) Google Scholar) and, like C/EBPα and PPARγ, is classified as a proadipogenic transcription factor. SREBP-1/ADD1, a member of the basic helix-loop-helix-leucine zipper transcription factor class, promotes lipogenic gene expression (34Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (846) Google Scholar) and stimulates production of an unidentified PPARγ ligand (35Kim J.B. Wright H.M. Wright M. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4333-4337Crossref PubMed Scopus (558) Google Scholar). Thus, C/EBPα, PPARγ, and SREBP-1/ADD1 cooperatively promote adipogenesis and subsequent maintenance of the adipocyte phenotype. Inhibition of preadipocyte differentiation by PIs has been reported recently. Several PIs, including amprenavir, indinavir, nelfinavir, and ritonavir, have been shown to inhibit triacylglycerol accumulation and expression of 422/aP2 mRNA in 3T3-L1 preadipocytes (36Zhang B. Macnaul K. Szalkowski D. Li Z. Berger J. Moller D.E. J. Clin. Endocrinol. Metabol. 1999; 84: 4274-4277Crossref PubMed Google Scholar). Indinavir and saquinavir were demonstrated to inhibit glycerol-3-phosphate dehydrogenase activity, a late lipogenic marker of the adipogenic process, in primary human preadipocytes (37Wentworth J.M. Burris T.P. Chatterjee V.K.K. J. Endocrinol. 2000; 164: R7-R10Crossref PubMed Google Scholar). Indinavir also has been shown to augment the inhibitory action of all-trans-retinoic acid on lipogenesis in the pluripotent mesenchymal stem cell line, C3H10T1/2 (38Lenhard J.M. Weiel J.E. Paulik M.A. Furfine E.S. Biochem. Pharmacol. 2000; 59: 1063-1068Crossref PubMed Scopus (66) Google Scholar). A poorly understood mechanism whereby indinavir enhances retinoic acid receptor signaling has been proposed (38Lenhard J.M. Weiel J.E. Paulik M.A. Furfine E.S. Biochem. Pharmacol. 2000; 59: 1063-1068Crossref PubMed Scopus (66) Google Scholar). Collectively, these findings demonstrate that PIs inhibit preadipocyte differentiation, although the precise molecular mechanisms involved remain unknown. We conducted a detailed analysis of the effects of nelfinavir on both preadipocyte differentiation and adipocyte survival. Results from our investigation indicate that nelfinavir inhibits differentiation of 3T3-L1 preadipocytes at a point following two early, differentiation-associated events, C/EBPβ expression and mitotic clonal expansion, because these events were not affected by nelfinavir. Nelfinavir-dependent inhibition of adipogenesis was manifest by severe reductions in both triacylglycerol accumulation and expression of C/EBPα, PPARγ, SREBP-1/ADD1, and 422/aP2 protein. Furthermore, nelfinavir inhibited expression of the same adipogenic transcription factors and promoted cell death in fully differentiated 3T3-L1 adipocytes. Thus, nelfinavir and other PIs may promote adipose tissue atrophy by promoting adipocyte loss and/or preventing replacement of lost adipocytes by inhibiting preadipocyte differentiation. 3T3-L1 preadipocytes (20Green H. Kehinde O. Cell. 1974; 1: 113-116Abstract Full Text PDF Scopus (744) Google Scholar) were maintained in DMEM containing 10% calf serum (Intergen Co., Purchase, NY). Differentiation was induced as described (39Student A.K. Hsu R.Y. Lane M.D. J. Biol. Chem. 1980; 255: 4745-4750Abstract Full Text PDF PubMed Google Scholar) by incubating 2-day postconfluent cells (designated day 0) in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and a hormonal mixture composed of 520 μm 3-isobutyl-1-methylxanthine, 1 μm dexamethasone and 167 nm insulin (termed MDI) for 48 h. Cells were then incubated in DMEM containing 10% fetal bovine serum and 167 nm insulin for another 48 h, after which they were maintained in DMEM containing 10% fetal bovine serum with a medium change every 48 h. All cell culture medium was supplemented throughout with 62.5 μg/ml penicillin, 100 μg/ml streptomycin, and 8 μg/ml biotin. All test agents were diluted into culture medium, and in all experiments cells were exposed to an identical concentration of vehicle (0.1% v/v). Oil Red O staining was performed by fixing cell monolayers in 3.7% formaldehyde, washing in water and staining with a 0.6% (w/v) Oil Red O solution (60% isopropanol, 40% water) for 1 h at room temperature. Cell monolayers were then washed extensively with water to remove unbound dye. Trypan blue staining was performed by incubating cell monolayers in a 0.2% (w/v) trypan blue solution (0.15 m NaCl) for 10 min at room temperature. Cell number data shown in Fig. 3 Cwere determined by trypsinizing cell monolayers from 6-cm culture dishes followed by counting with a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL).Figure 4Effects of anti-HIV drugs on fully differentiated adipocytes. 3T3-L1 preadipocytes were induced to differentiate with MDI and cultured for 6 days, a time period sufficient for complete differentiation into adipocytes. Adipocytes at day 6 were then treated with vehicle, stavudine (20 μm), or the indicated protease inhibitor (20 μm) for a period of 6 days after which cells were stained with Oil Red O. Preadipocytes not induced to differentiate (NO MDI), simultaneously cultured in the presence of vehicle for an identical period of time and stained with Oil Red O are shown for comparison. A, images of cells treated as described above. B, microscopic view (200×) of selected dishes shown in A. STVD, stavudine;IDV, indinavir; NFV, nelfinavir; RTV, ritonavir; SQV, saquinavir.View Large Image Figure ViewerDownload (PPT)Figure 3Nelfinavir does not influence early events in preadipocyte differentiation. Nuclear extracts were prepared from preadipocytes before (0 h after MDI) and during differentiation (33 and 46 h after MDI). Extracts were obtained at all time points from preadipocytes cultured in the presence of vehicle or 20 μm nelfinavir. A, nuclear extracts were examined for C/EBP DNA binding activity. Supershift analysis was performed by addition of preimmune serum (PRE,lanes 4–7) or serum that specifically recognizes C/EBPβ (ANTI-β, lanes 8–11). Lane 1 represents recombinant C/EBPβ (38-kDa form only) shown for comparative purposes. B, nuclear extracts examined for binding activity in A were subjected to immunoblot analysis using antiserum that recognizes C/EBPβ. Lanes 1–7of B correspond to samples taken from those represented inlanes 1–7 of A. C, preadipocytes were induced to differentiate (MDI) in the presence of vehicle or 20 μm nelfinavir and cultured for a period of 3 days. At the same time, additional dishes of preadipocytes not induced to differentiate (NO MDI) were grown under otherwise identical conditions. Cell number was then determined using a Coulter Counter. Two independent experiments were conducted in triplicate with similar results. Data shown are the means ± S.E. of two experiments.View Large Image Figure ViewerDownload (PPT) Whole cell extracts were prepared at the indicated times by washing cell monolayers from 6-cm plates once in phosphate-buffered saline (PBS, pH 7.5) followed by lysis in 1% SDS, 60 mm Tris-HCl (pH 8.0). Nuclear extracts were prepared according to the method of Dignam et al. (40Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). Protein concentrations of all samples were determined using the bicinchoninic acid assay (41Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18647) Google Scholar). Data shown in Figs. 2 and 4 were obtained by subjecting 100 μg of total protein from each experimental extract to SDS-polyacrylamide gel electrophoresis. Two identical sets of protein extracts were run individually on 8% and 12.5% acrylamide gels to achieve maximum resolution for each series of immunoblots. After transferring to polyvinylidene fluoride membranes (0.45-micron pore size; Immobilon-P, Millipore), blots were probed with antibodies (see below) recognizing the proteins indicated. Blots were sequentially probed, stripped in 2% SDS, 0.1 m 2-mercaptoethanol, 62.5 mm Tris-HCl (pH 6.7) at 65 °C for 1 h, equilibrated in Tris-buffered saline (137 mm NaCl, 25 mmTris-HCl, pH 7.6) + 0.1% Tween-20 (TTBS), and reprobed after blocking nonspecific protein binding by incubation for 30 min in 5% (w/v) nonfat dried milk dissolved in TTBS. In Fig. 2, extracts separated on the 12.5% gel were probed for C/EBPβ, C/EBPα, and 422/aP2, and those on the 8% gel were probed for PPARγ, RXRα, and SREBP-1. In Fig. 4, extracts separated on the 12.5% gel were probed for C/EBPα and 422/aP2, and those on the 8% gel were probed for PPARγ, RXRα, and SREBP-1. Commercially available primary antibodies recognizing the following proteins were used: PPARγ (Santa Cruz, catalogue number sc-7273), RXRα (Santa Cruz, catalogue number sc-553), and SREBP-1 (Santa Cruz, catalogue number sc-367; note this antibody recognizes both SREBP-1a and -1c (also known as ADD1)). Rabbit polyclonal antisera specific to C/EBPβ, C/EBPα, and 422/aP2 were generated in this laboratory. Protein detection was performed by ECL using commercially available reagents as per the manufacturer's instructions (Amersham Pharmacia Biotech). Identical procedures were used to generate immunoblot data shown in Fig. 3 B except 10 μg of nuclear extract was loaded per lane. Assays were performed as described (28Tang Q.-Q. Lane M.D. Genes Dev. 1999; 13: 2231-2241Crossref PubMed Scopus (310) Google Scholar) with 10-μg nuclear extract except extracts were prepared according to the method of Dignam et al. (40Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). Recombinant C/EBPβ (38-kDa isoform) was generated using a TNT-coupled reticulocyte lysate system (Promega, Madison, WI). Cells grown on No. 1 coverslips (22 × 22 mm) were washed twice in ice-cold PBS and fixed in freshly prepared 1% paraformaldehyde on ice for 15 min. Coverslips were washed once with PBS and incubated in 70% methanol at −20 °C for 60 min to permeabilize cells. Coverslips were then washed three times in PBS, and 50 μl of TUNEL assay reaction mixture was pipetted gently onto coverslips. The mixture included 200 mm potassium cacodylate, 0.2 mm dithiothreitol, 0.25 mm cobalt chloride, 25 mm Tris-HCl (pH 6.6) supplemented with 0.5 nmol ChromaTide Alexa Fluor 488–5-dUTP (Molecular Probes, Eugene, OR), and 10 units terminal deoxynucleotidyl transferase (Roche Molecular Biochemicals). Reactions were allowed to proceed for 90 min at 37 °C followed by one wash in 2× SSC to terminate reactions. Coverslips were washed twice in PBS, incubated in PBS + 1 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI, Sigma) for 15 min at room temperature to stain nuclei and washed three times before mounting on microscope slides in Prolong Antifade solution (Molecular Probes). Samples were viewed at 630× magnification using a Zeiss Axioskop microscope, and images were obtained using IP Lab software (Scanalytics, Inc., Fairfax, VA). Percentage of cells exhibiting TUNEL reactivity (TUNEL index in Fig. 6) was determined by the number of TUNEL staining cells divided by the total number of cells (DAPI-stained cells). Five different fields were scored for each treatment group in three independent experiments with similar results. Each field included approximately 10 and 40 cells for undifferentiated preadipocytes and differentiated adipocytes, respectively. Statistical analysis was performed using a two-tailed Student's t test. Indinavir (Merck, West Point, PA), nelfinavir (Agouron Pharmaceuticals, Torrey Pines, CA), ritonavir (Abbott Laboratories, Abbott Park, IL), and saquinavir (Roche Molecular Biochemicals) were supplied as powders and were kind gifts from the indicated sources. All protease inhibitors were dissolved in Me2SO at a concentration of 20 mm. Stavudine (2′,3′-didehydro-3′-deoxythymidine; Sigma) was dissolved in PBS at a concentration of 20 mm and filter sterilized before use. Under appropriate culture conditions, including incubation with a hormonal mixture (see the Introduction and “Experimental Procedures”), 3T3-L1 preadipocytes undergo differentiation and assume adipocyte characteristics that include a specific pattern of gene expression and accumulation of cytoplasmic, triacylglycerol-rich lipid droplets. Experiments were conducted to determine whether HIV PIs affect this process. Preadipocytes were induced to differentiate in the presence or absence of the PIs indinavir, ritonavir, nelfinavir, and saquinavir (20 μm). Six days after the onset of differentiation, when cytoplasmic lipid droplets are normally abundant, cells were stained with the lipophilic dye Oil Red O to determine the extent of triacylglycerol accumulation. Exposure to nelfinavir throughout the course of differentiation severely inhibited lipid accumulation, but this effect was not observed readily with 20 μmindinavir, ritonavir, or saquinavir under identical conditions (Fig. 1, A, lower panels, and B). The inhibitory effect of nelfinavir was not observed when preadipocytes were exposed to this drug only during the first 2 days of the differentiation program (Fig. 1 A, upper panels). Similarly, exposure to nelfinavir from days 2–6 of the program did not inhibit lipid accumulation (data not shown). Thus, the inhibitory effect of nelfinavir was observed only when preadipocytes were exposed to this drug throughout the course of differentiation. Adipose tissue abnormalities independent of PI-containing therapy regimens have been reported in some HIV-infected patients (15Kotler D.P. Rosenbaum K. Wang J. Pierson R.N. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 1999; 20: 228-237Crossref PubMed Scopus (185) Google Scholar, 16Lo J.S. Mulligan K. Tai V.W. Algren H. Schambelan M. Lancet. 1998; 351: 867-870Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 17Gervasoni C. Ridolfo A.L. Trifiro G. Santambrogio S. Norbiato G. Musicco M. Clerici M. Galli M. Moroni M. AIDS. 1999; 13: 465-471Crossref PubMed Scopus (276) Google Scholar, 18Madge S. Kinloch de Loes S. Mercey D. Johnson M.A. Weller I.V.D. AIDS. 1999; 13: 735-737Crossref PubMed Scopus (167) Google Scholar, 19Saint-Marc T. Partisani M. Poizot-Martin I. Bruno F. Rouviere O. Lang J.M. Gastaut J.A. Touraine J. AIDS. 1999; 13: 1659-1667Crossref PubMed Scopus (547) Google Scholar). The HIV reverse transcriptase inhibitor, stavudine, has been associated with such abnormalities in one group of patients (19Saint-Marc T. Partisani M. Poizot-Martin I. Bruno F. Rouviere O. Lang J.M. Gastaut J.A. Touraine J. AIDS. 1999; 13: 1659-1667Crossref PubMed Scopus (547) Google Scholar). Therefore, th" @default.
• W2053701857 created "2016-06-24" @default.
• W2053701857 creator A5012475457 @default.
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• W2053701857 date "2000-12-01" @default.
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• W2053701857 title "Suppression of Preadipocyte Differentiation and Promotion of Adipocyte Death by HIV Protease Inhibitors" @default.
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• W2053701857 doi "https://doi.org/10.1074/jbc.m006474200" @default.
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