The Effect of the HIV Protease Inhibitor Ritonavir on Proliferation, Differentiation, Lipogenesis, Gene Expression and Apoptosis of Human Preadipocytes and Adipocytes

Abstract

HIV patients in highly active antiretroviral therapy (HAART) develop lipodystrophy and insulin resistance. Protease inhibitors have been shown to alter adipocyte metabolism in murine cell lines. In this study, biological effects of the HIV protease inhibitor, ritonavir, were investigated on human SGBS preadipocytes and adipocytes. Ritonavir dose-dependently impaired preadipocyte proliferation and adipogenic differentiation. Gene expression analysis measured by real-time PCR, showed no effect of ritonavir (up to 20 μM) on expression of mRNA of PPARγ2 and SREBP1c, but suppressed adiponectin mRNA while increasing IL-6 mRNA expression. In human adipocytes, ritonavir at therapeutic concentrations inhibited insulin-stimulated lipogenesis, reduced GLUT4 mRNA, fatty acid synthase and adiponectin expression, while increasing IL-6 mRNA expression. Finally, long-term treatment (72 and 120 h) of SGBS adipocytes but not preadipocytes with ritonavir induced apoptosis in up to 15 % of the cells. All together, these data show effects of ritonavir on human preadipocytes and adipocytes aiming at reducing adipose tissue mass and increasing insulin resistance. These in vitro findings may partly explain the clinical findings in patients under HAART. Furthermore, SGBS cells may serve as a useful tool in further investigation of the mechanism of protease inhibitor action in human adipocytes.

Key words

Lipodystrophy - HAART - Fat cell - SGBS-cells - Insulin resistance

References

• 1 Koutkia P, Grinspoon S. HIV-associated lipodystrophy: Pathogenesis, Prognosis, Treatment, and Controversies.  Annu Rev Med. 2004;  55 303-317
• 2 Kannisto K, Sutinen J, Korsheninnikova E, Fisher R M, Ehrenborg E, Gertow K, Virkamaki A, Nyman T, Vidal H, Hamsten A, Yki-Jarvinen H. Expression of adipogenic transcription factors, peroxisome proliferators-activated receptor gamma co-activator1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapy.  Aids. 2003;  17 1753-1762
• 3 Miller K D, Jones E, Yanovski J A, Shankar R, Feuerstein I, Falloon J. Visceral abdominal-fat accumulation associated with use of indinavir.  Lancet. 1998;  351 871-875
• 4 Carr A, Samaras K, Chisholm D J, Cooper D A. Abnormal fat distribution and use of protease inhibitors.  Lancet.. 1998;  351 1736
• 5 Stocker D N, Meier P J, Stoller R, Fattinger K E. “Buffalo hump” in HIV-1 infection.  Lancet.. 1998;  352 320-321
• 6 Acevedo M, Sprecher D L, Calabrese L, Pearce G L, Coyner D L, Halliburton S S, White R D, Sykora E, Kondos G T, Hoff J A. Pilot study of coronary atherosclerotic risk and plaque burden in HIV patients: ‘a call for cardiovascular prevention’.  Atherosclerosis. 2002;  163 349-354
• 7 Domingo P, Matias-Guiu X, Pujol R M, Francia E, Lagarda E, Sambeat M A, Vàzquez G. Subcutaneous adipocyte apoptosis in HIV-1 protease inhibitor-associated lipodystrophy.  Aids. 1999;  13 2261-2267
• 8 Dowell P, Flexner C, Kwiterovich P O, Lane M D. Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors.  J Biol Chem. 2000;  275 41 325-41 332
• 9 Bastard J P, Caron M, Vidal H, Jan V, Auclair M, Vigouroux C, Luboinski J, Laville M, Maachi M, Girard P M, Rozenbaum W, Levan P, Capeau J. Association between altered expression of adipogenic factor SREBP1 in lipoatrophic adipose tissue from HIV-1 infected patients and abnormal adipocyte differentiation and insulin resistance.  Lancet. 2002;  359 1026-1031
• 10 Caron M, Auclair M, Vigouroux C, Glorian M, Forest C, Capeau J. The HIV Protease inhibitor indinavir impairs stero regulatory element-binding protein-1 intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance.  Diabetes. 2001;  50 1378-1388
• 11 Zhang B, Macnaul K, Szalkowski D, Li Z, Berger J, Moller D E. Inhibition of adipocyte differentiation by HIV protease inhibitors.  J Clin Endocrinol Metab. 1999;  84 4274-4277
• 12 Lenhard J M, Furfine E S, Jain R G, Ittoop O, Orband-Miller L A, Blanchard S G, Paulik M A, Weiel J E. HIV protease inhibitors block adipogenesis and increase lipolysis in vitro.  Antiviral Res. 2000;  47 121-129
• 13 Murata H, Hruz P W, Mueckler M. The mechanism of insulin resistance caused by HIV protease inhibitor therapy.  J Biol Chem. 2000;  275 20 251-20 254
• 14 Rudich A, Vanounou S, Riesenberg K, Porat M, Tirosh A, Harman-Boehm I, Greenberg A S, Schlaeffer F, Bashan N. The HIV protease inhibitor nelfinavir induces insulin resistance and increases basal lipolysis in 3T3-L1 adipocytes.  Diabetes. 2001;  50 1425-1431
• 15 Wabitsch M, Brenner R E, Melzner I, Braun M, Möller P, Heinze E. et al . Characterization of a human preadipocyte cell strain with high capacity for adipose differentiation.  Int J Obesity. 2001;  25 8-15
• 16 Fischer-Posovszky P, Tornqvist H, Debatin K M, Wabitsch M. Inhibition of death-receptor mediated apoptosis in human adipocytes by the insulin-like growth factor I (IGF-I)/IGF-I receptor autocrine circuit.  Endocrinology. 2004;  145 1849-1859
• 17 Dalen K T, Schoonjans K, Ulven S M, Weedon-Fekjaer M S, Bentzen T G, Koutnikova H, Auwerx J, Nebb H I. Adipose tissue expression of the lipid droplet-associating proteins S3 - 12 and perilipin is controlled by peroxisome proliferator-activated receptor-gamma.  Diabetes. 2004;  53 1243-1252
• 18 Bao Y, Bing C, Hunter L, Jenkins J R, Wabitsch M, Trayhurn P. Zinc-alpha(2)-glycoprotein, a lipid mobilizing factor, is expressed and secreted by human (SGBS)adipocytes.  FEBS Lett. 2005;  579 41-47
• 19 Vidal H. Quantification of lipid-related mRNAs by reverse transcription-competitive polymerase chain reaction in human white adipose tissue biopsies.  Methods Mol Biol. 2001;  155 83-88
• 20 Flexner M D. HIV-Protease inhibitors.  N Engl J Med. 1998;  338 1281-1292
• 21 Janneh O, Hoggard P G, Tjia J F, Jones S P, Khoo S H, Maher B, Back D J, Pirmohamed M. Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes.  Antivir Ther. 2003;  8 417-426
• 22 Riddle T M, Kuhel D G, Woollett L A, Fichtenbaum C J, Hui D Y. HIV protease inhibitor induces fatty acid and sterol biosynthesis in liver and adipose tissues due to the accumulation of activated sterol regulatory element-binding proteins in the nucleus.  J Biol Chem. 2001;  276 37 514-37 519
• 23 Nguyen A T, Gagnon A, Angel J B, Sorisky A. Ritonavir increases the level of active ADD-1/SREBP-1 protein during adipogenesis.  Aids. 2000;  14 2467-2473
• 24 Vernochet C, Azoulay S, Duval D, Guedj R, Cottrez F, Vidal H, Ailhaud G, Dani C. Human Immunodeficiency virus protease inhibitors accumulate into cultured human adipocytes and alter expression of adipocytokines.  J Biol Chem. 2005;  280 2238-2243
• 25 Wentworth J M, Burris T P, Chatterjee V K. HIV protease inhibitors block human preadipocyte differentiation, but not via the PPARgamma/RXR heterodimer.  J Endocrinol. 2000;  164 R7-R10
• 26 Roche R, Poizot-Martin I, Yazidi C M, Compe E, Gastaut J A, Torresani J, Planells R. Effects of antiretroviral drug combinations on the differentiation of adipocytes.  Aids. 2002;  16 13-20
• 27 Schutt M, Zhou J, Meier M, Klein H H. Long-term effects of HIV-1 protease inhibitors on insulin secretion and insulin signaling in INS-1 beta cells.  J Endocrinol. 2004;  183 445-454
• 28 Cammalleri C, Germinario R J. The effects of protease inhibitors on basal and insulin-stimulated lipid metabolism, insulin binding, and signaling.  J Lipid Res. 2003;  44 103-108
• 29 Lagathu C, Bastard J P, Auclair M, Maachi M, Kornprobst M, Capeau J, Caron M. Antiretroviral drugs with adverse effects on adipocyte lipid metabolism and survival alter the expression and secretion of proinflammatory cytokines and adiponectin in vitro.  Antivir Ther. 2004;  9 911-920
• 30 Straub R H, Hense H W, Andus T, Scholmerich J, Riegger G A, Schunkert H. Hormone replacement therapy and interrelation between serum interleukin-6 and body mass index in postmenopausal women: a population-based study.  J Clin Endocrinol Metab. 2000;  85 1340-1344
• 31 Kado S, Nagase T, Nagata N. Circulating levels of interleukin-6, its soluble receptor and interleukin-6/interleukin-6 receptor complex in patients with type 2 diabetes mellitus.  Acta Diabetol. 1999;  36 67-72
• 32 Lagathu C, Bastard J P, Auclair M, Maachi M, Capeau J, Caron M. Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone.  Biochem Biophys Res Commun. 2003;  311 372-379
• 33 Rotter V, Nagaev I, Smith U. Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-, overexpressed in human fat cells from insulin-resistant subjects.  J Biol Chem. 2003;  278 45 777-45 784
• 34 Haque W A, Shimomura I, Matsuzawa Y, Garg A. Serum adiponectin and leptin levels in patients with lipodystrophy.  J Clin Endocrinol Metab. 2002;  87 2395-2398
• 35 Mynarcik D C, Combs T, McNurlan M A, Scherer P E, Komaroff E, Gelato M C. Adiponectin and leptin levels in HIV-infected subjects with insulin resistance and body fat redistribution.  J Acquir Immune Defic Syndr. 2002;  31 514-520
• 36 Fasshauer M, Kralisch S, Klier M, Lossner U, Bluher M, Klein J, Paschke R. Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes.  Biochem Biophys Res Commun. 2003;  301 1045-1050
• 37 Gustafson B, Jack M M, Cushman S W, Smith U. Adiponectin gene activation by thiazolidinediones requires PPAR gamma 2, but not C/EBPalpha-evidence for differential regulation of the aP2 and adiponectin genes.  Biochem Biophys Res Commun. 2003;  308 933-939
• 38 Ledru E, Christeff N, Patey O, de Truchis P, Melchior J C, Gougeon M L. Alteration of tumor necrosis factor-alpha T-cell homeostasis following potent antiretroviral therapy: contribution to the development of human immunodeficiency virus-associated lipodystrophy syndrome.  Blood. 2000;  95 3191-3198