摘要
目的:1、研究司他夫定(D4T)、齐多夫定(AZT)和去羟肌苷(DDI)对3T3-L1前脂肪细胞增殖和分化的影响。
2、观察D4T、AZT和DDI对处于增殖和分化过程中的3T3-L1前脂肪细胞分泌胰岛素样生长因子—1(IGF-1),以及成熟脂肪细胞分泌脂联素(APN)的影响。
通过上述研究,探讨HIV/AIDS患者接受高效抗逆转录病毒治疗(HAART)后出现脂肪萎缩的发生机制。
方法:1、将不同浓度的D4T、AZT和DDI(0.1μg/ml、1μg/ml、10μg/ml)作用于CFSE标记的3T3-L1前脂肪细胞,应用流式细胞仪测定细胞增殖情况。
2、选择D4T(1μg/ml、4μg/ml)、AZT(0.1μg/ml、1μg/ml)或DDI(1μg/ml、10μg/ml)作用于3T3-L1前脂肪细胞,使用MTT比色法测定细胞增殖情况。
3、将不同浓度的D4T、AZT和DDI(0.1μg/ml、1μg/ml、10μg/ml)作用于3T3-L1前脂肪细胞,诱导分化至第8天,使用油红0脂肪染色法观察细胞分化情况。
4、收集3T3-L1前脂肪细胞增殖和分化过程中的培养上清液,应用ELISA方法测定APN和IGF-1水平。
结果:1、流式细胞仪CFSE标记法检测结果显示,与对照组相比,不同浓度D4T干预组(0.1μg/ml、1μg/ml、10μg/ml)的增殖指数降低程度分别为19.1%、39.8%和50.8%;AZT干预组(0.1μg/ml、1μg/ml、10μg/ml)的降低程度分别为6.7%、18.1%和30.9%。MTT比色法也证实D4T和AZT能够减弱3T3-L1前脂肪细胞的增殖能力。
2、高浓度D4T(10μg/ml)和AZT(10μg/ml)能够抑制3T3-L1前脂肪细胞分化能力,造成成熟脂肪细胞数量减少。
3、ELISA检测结果发现,在细胞增殖过程中,与对照组相比,不同浓度D4T干预组(0.1μg/ml、1μg/ml、10μg/ml)中的IGF-1含量下降程度分别为21.5%、31.7%和48.7%;D4T还能降低分化第8天的成熟脂肪细胞分泌IGF-1的能力,呈浓度依赖性,下降程度分别为10.5%、30.7%和42.6%;而AZT(0.1μg/ml、1μg/ml、10μg/ml)仅能降低增殖过程中前脂肪细胞分泌IGF-1的能力,与对照组相比下降程度分别为8.6%、21.7%和29.5%。
4、与对照组相比,在10μg/ml的D4T干预组和AZT干预组中,成熟脂肪细胞分泌的APN水平下降。
5、DDI对3T3-L1前脂肪细胞增殖、分化和IGF-1、APN的分泌未见明显影响。
结论:1、本研究首次发现D4T能够降低3T3-L1前脂肪细胞的增殖能力,为浓度依赖性;同时证实AZT能够降低3T3-L1前脂肪细胞的增殖能力。
2、本研究证实了D4T和AZT能够抑制3T3-L1前脂肪细胞分化能力,使成熟脂肪细胞数量下降。
3、本研究首次在体外实验中发现,D4T和AZT均能够减弱增殖过程中的前脂肪细胞分泌IGF-1的能力,呈浓度依赖性;D4T还能够减弱成熟脂肪细胞分泌IGF-1的能力。
4、本研究证实了D4T和AZT能够使成熟脂肪细胞分泌的APN含量下降。
由此,D4T和AZT可能通过影响前脂肪细胞的增殖、分化及脂肪因子分泌等途径,导致脂肪萎缩的出现。
Objective:
1、To study the effects of D4T,AZT and DDI on proliferation and differentiation in 3T3-L1 preadipocytes.
2、To study the effects of D4T,AZT and DDI on IGF-1 production by cultured proliferating preadipocytes and differentiating adipocytes.To study the effects of D4T,AZT and DDI on adiponectin secretion by differentiated adipocytes.
To investigate the pathogenesis of highly active antiretroviral therapy(HAART) associated lipoatrophy based on above studies.
Methods:
1、3T3-L1 preadipocytes dying with CFSE were cultured in DMEM/F12 growth medium containing D4T,AZT or DDI at different concentrations(0.1μg/ml、1μg/ml、10μg/ml).Flow cytometry was used to measure the proliferation index of preadipocytes in treated and control groups.
2、3T3-L1 preadipocytes were cultured with D4T(1μg/ml、4μg/ml), AZT(0.1μg/ml、1μg/ml) or DDI(1μg/ml、10μg/ml).MTT assay was applied to evaluate growth of preadipocytes.
3、3T3-L1 preadipocytes were induced to differentiate in both treated groups containing D4T、AZT or DDI at different concentrations(0.1μg/ml、1μg/ml、10μg/ml) and control groups.Oilred O staining was used to assess lipid accumulation.
4、ELISA was designed to measure IGF-1 and adiponectin levels in cell culture supernatants during the course of proliferation and differentiation.
Results:
1、It was shown by CFSE with flow cytometry that D4T in different concentrations(0.1μg/ml、1μg/ml、10μg/ml) could make proliferation index decrease 19.1%,39.8%,50.8%respectively compared with control group.The decreased levels by AZT interfering groups(0.1μg/ml、1μg/ml、10μg/ml) were 6.7%、18.1%,30.9%respectively.The fact that D4T and AZT were able to attenuate the proliferation ability of 3T3-L1 preadipocyte was also confirmed by MTT method.
2、D4T and AZT in high concentration could also inhibit the differentiation ability of 3T3-L1 preadipocyte,then make less mature adipocyte.
3、It was also discovered by ELISA testing that D4T in different concentrations(0.1μg/ml、1μg/ml、10μg/ml) could make the concentration of IGF-1 decrease 21.5%,31.7%,48.7%respectively in proliferation phrase compared with control group.D4T could also low down the IGF-1 secretion by mature adipocyte in the 8~(th) day of differentiation.The secretion decrease, which was concentration-dependent,were 10.5%,30.7%,42.6%respectively. AZT in different concentrations(0.1μg/ml、1μg/ml、10μg/ml) could only make the concentration of IGF-1 decrease 8.6%,21.7%,29.5%respectively in proliferation phrase compared with control group.
4、Adiponectin secreted by mature adipocyte was also decreased in D4T in 10μg/ml and AZT in 10μg/ml interfering groups compared with control group.
5、DDI had no effect on proliferation,differentiation and production of IGF-1 and APN in 3T3-L1 preadipocytes.
Conclusion:
1、Our study discovers that D4T and AZT impair the proliferative activity of 3T3-L1 preadipocytes dose-dependently by flow cytometry and MTT assay.
2、We confirm that D4T and AZT induce the less number of mature adipocytes by impairing the differentiation of 3T3-L1 preadipocytes.
3、To our knowledge,in vitro experiments our study is the first to discover that D4T and AZT impair the production of IGF-1 in proliferating preadipocytes,and D4T impairs the production of IGF-1 in mature adipocytes.
4、It is confirmed that D4T and AZT can lead to the reduction of adiponectin in supernanants of differentiated adipocytes.
So,D4T and AZT might induce lipoatrophy by bothering the proliferation, differentiation of preadipocytes and the secretion of adipokines.
引文
1.Safrin S, Grunfeld C. Fat distribution and metabolic changes in patients with HIV infection. AIDS 1999; 13:2493-2505
2. Carr A, Samaras K, Burton S, et al.A syndrome of peripheral lipodystrophy, hyperlipidemia and insulin resistance in patients receiving HIV pretease inhibitors. AIDS 1998; 12:F21-58
3. Viraben R, Aquilina C. Indinavir-associated lipodystrophy. AIDS 1998; 12: F37-39
4. Chene G, Angelini E, Cotte L, et al.Role of long-term nucleoside-analogue therapy in lipodystrophy and metabolic disorders in human immunodeficiency virus-infected patients. Clin Infect Dis 2002; 34:649-657
5. Mallal SA, John M, Moore CB, et al. Contribution of nucleoside analogue reverse transcriptase inhibitors to aubcutaneous fat wasting in patients with HIV infection. AIDS 2000; 14:1309-1316
6. Walker UA, Bickel M, Lutke Volksbeck SI, etal. Evidence of nucleoside analogue reverse transcriptase inhibitor-associated genetic and structural defects of mitochondria in adipose tissue of HIV-infected patients. JAIDS 2002; 29:117-121
7. Shikuma CM, Hu N, Milne C, et al. Mitochondrial DNA decrease in subcutaneous adipose tissue of HIV-infected individuals with peripheral lipoatrophy. AIDS 2001; 15:1801-1809
8.Nolan D, Hammond E,Martin A, et al.Mitochondrial DNA depletion and morphologic changes in adipocytes associated with nucleoside reverse transcriptase inhibitor therapy. AIDS 2003; 17:1329-1338
9. Cherry CL, Ganhan ME, Mcarthur JC, et al. Exposure to dideoxynucleosides is reflected in lowered mitochondrial DNA in subcutaneous fat. Journal of Acquired Immune Deficiency Syndromes. 2002; 30: 271-277
10. Gougeon ML, Penicaud L, Fromenty B, et al.Adipocytes targets and actors in the pathogenesis of HIV-associated lipodystrophy and metabolic alterations.Antiviral Therapy, 2004; 9:161-177
11.Lagathu C, Bastard JP, Auclair M, et al. Antiretroviral drugs wit adverse effects on adipocyte lipid metabolism and survival alter the expression and secretion of proinflammatory cytokines and adiponectin in vitro. Antiviral Therapy 2004; 9:911-920
12. Caron M, Auclair M, Lagathu C, et al.The HIV-nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro.AIDS 2004; 18: 2127-2136
13. Stankov MV, Schmidt RE, Behrens GM, et al. Zidovudine impairs adipogenic differentiation through inhibition of clonal expansion. Antimicrob Agents Chemother 2008 May 12, Epub ahead of print, abstract
14. Stankov M, Schmidt RE, Behrens GMN. Zidovudine inhibits adipogenic differentiation of 3T3-L1 cells though inhibition of clonal expansion. Antiviral Therapy 2005; 10:L20 (abstract no. 26)
15. Jones SP, Janneh 0, Back DJ, et al. Altered adipokine response in murine 3T3-F442A adipocytes treated with protease inhibitors and nucleoside reverse transcriptase inhibitors. Antiviral Therapy 2005; 10:207-213
16. Pilgrim C. DNA synthesis and differentiation in developing wh-ite adipose tissue.Dev Biol 1971; 26:69-76
17. Ailhand G, Anne E, Bardon S, et al. Growth and differentiation of adipose tissue: molecular and hormonal mechenisms. Int J Obes 1991; 15:87-90
18. Goegrioe FM, Smas CM, Sul HS. Underlying adipocyte differentiation. Physiol Rev 1998; 78:783-809
19. Prins JB, 0' Rahilly S: Regulation of adipose cell number in man. Clin Sci( C o 1 c h ) 1997; 92:3-11
20. Ailhaud G, Grimaldi P, Negrel R. Cellular and molecular aspects of adipose tissue development. Annu Rev Nutr 1992; 12:207-233
21. Janneh 0, Hoggard PG, Tjia JF, et al. Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes. Antiviral Therapy 2003; 8:417-426
22. Kosmiski LA, Miller HL, Klemm DJ. In combination, nucleoside reverse transcriptase inhibitors have significant effects on 3T3-L1 adipocyte lipid accumulation and survival. Antiviral Therapy 2006; 11:187-195
23. Lyons AB , Parish CR. Determination of lymphocyte division by follow cytometry. J Immunol Methods 1994; 171 (1) :131-137
24. Nose, A, M. Takeichi.A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos. J Cell Biol 1986; 103: 2649-2658
25. Hodgkin, P. D., J. H. Lee, et al.B cell differentiation and isotype switching is related to division cycle number. J Exp Med 1996; 184: 277-281
26. Fulcher D , Wong S. Carboxyfluorescein succinimidyl ester2based pro21iferative assays for assessment of T cell function in the diagnostic 1ab2oratory. Immunol Cell Biol 1999; 77(6): 559-564
27. Lyons AB , Parish CR. Determination of lymphocyte division by follow cytometry. J Immunol Methods 1994; 171 (1):131-137
28. Karsten Hemmrich K, Meersch M, Heimburg DV et al. Applicability of the Dyes CFSE, CM-DiI and PKH26 for Tracking of Human Preadipocytes to Evaluate Adipose Tissue Engineering. Cells Tissues Organs 2006; 184:117-127
29. Moyer TP, Temesgen Z, Enger R, et al. Drug Monitoring of Antiretroviral Therapy for HIV-1 Infection: Method Validation and Results of a Pilot Study. Clinical Chemistry 1999; 45 (9) : 1465-1476
30. Hansen MB, Niesen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth cell kill. J Immunol Method 1989; 119:203-210
31. Jan Veronique, Cervera P, Maachi M, et al. Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients. Antiviral Therapy 2004; 9:555-564
32. Ramirez ZJL, Castro MF, Kuri HW. Quantization of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red 0. Histochemistry 1992; 97: 493-512
33. Yamauchi T, Kamon J, Ito Y, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 2003; 423(6941): 762-769
34. Yamauchi T, Kamon J, Waki H,et al.The fat derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 2001; 7(8): 941-946
35. Ouchi N, Nihara S, Arita Y, et al. Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 2001; 103(8): 1057-1063
36. Addy CL, Gavrila A, Tsiodras S, et al. Hypoadiponectinemia is associated with insulin resistance, hypertriglyceridemia, and fat redistribution in human immunodeficiency virus-infected patients treated with highly active antiretroviral therapy. J Clin Endocrinol Metab 2003; 88:627-636
37. Estrada V, Martinez-Larrad MT, Gonzalez-Sanchez JL,et al.Lipodystrophy and metabolic syndrome in HIV-infected patients treated with antiretroviral therapy. Metabolism 2006; 55:940-945
38.Stankov MV, Lucke T, Das AM,et al. Relationship of mitochondrial DNA depletion and respiratory chain activity in preadipocytes treated with nucleoside reverse transcriptase inhibitors. Antiviral Therapy 2007; 12:205-216
39. Yamauchi T, Kamon J, Waki H,et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 2001; 7:941-946
40. Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 1995; 16:3-34
1.Lundgren JD, Mocroft A.The impact of qntiretroviral therapy on AIDS and surviral.J HIV Ther 2006,11:36-8
2. Safrin S,Grunfeld C. Fat distribution and metabolic changes in patients with HIV infection. AIDS 1999; 13:2493-2505
3. Bernasconi E, Boubaker K, Junghans C, et al. Abnormalities of body fat distribution in HIV-infected persons treated with antiretroviral drugs: the Swiss HIV Cohort Study. J Acquir Immune Defic Syndr 2002; 31: 50-5
4. Saint-Marc T, Partisani M, Poizot-Martin I, et al. A syndrome of peripheral fat wasting (lipodystrophy) in patients receiving long-term nucleoside analogue therapy. AIDS 1999; 13: 1659-67
5. Mallal SA, John M, Moore CB, et al. Contribution of nucleoside analogue reverse transcriptase inhibitors to subcutaneous fat wasting in patients with HIV infection. AIDS 2000; 14: 1309-16
6. Lichtenstein KA, Ward DJ, Moorman AC,et al. Clinical assessment of HIV-associated lipodystrophy in an ambulatory population. AIDS 2001; 15: 1389-98
7. Miller J, Carr A, Emery S, et al. HIV lipodystrophy: prevalence, severity and correlates of risk in Australia. HIV Med 2003; 4: 293-301
8. Pilgrim C. DNA synthesis and differentiation in developing wh-ite adipose tissue.Dev Biol 1971; 26:69-76
9. Ailhand G,Anne E, Bardon S, et al. Growth and differentiation of adipose tissue: molecular and hormonal mechenisms. Int J Obes 1991; 15:87-90
10.Goegrioe FM, Smas CM, Sul HS. Underlying adipocyte differentiation. Physiol Rev 1998; 78:783-809
11.Prins JB, 0' Rahilly S. Regulation of adipose cell number in man. Clin Sci( C o 1 c h ) 1997; 92:3-11
12. Ailhaud G, Grimaldi P, Negrel R. Cellular and molecular aspects of adipose tissue development.Annu Rev Nutr 1992; 12:207-233
13. Gougeon ML, Penicaud L, Fromenty B, et al. Adipocytes targets and actors in the pathogenesis of HIV-associated lipodystrophy and metabolic alterations. Antiviral Therapy 2004; 9:161-177
14.Carr A, Samaras K, Chisholm DJ , et al. Pathogenesis of HIV protease inhibitor-associated syndrome of peripheral lipodystrophy , hyperlipidemia and insulin resistance. Lancet 1998; 351: 1881-1883
15. Li E, Norris AW. Structure/function of cytoplasmic vitamin A-binding proteins. Annu Rev Nutr 1996; 16:205-34
16. Caron M, Auclair M, Vigouroux C, et al. The HIV protease inhibitor indinavir impairs sterol regulatory element binding protein-1 intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance. Diabetes 2001; 50:1378-1388
17. Stankov MV, Behrens GMN. HIV-therapy associated lipodystrophy:experimental and clinical evidence for the pathogenesis and treatment. Endocrine, Metabolic&Immune Disorders-Drug Targets 2007; 7:237-249
18. Domingo P, Matias-Guiu X, Pujol RM,et al. Subcutaneous adipocyte apoptosis in HIV-1 protease inhibitor-associated lipodystrophy. AIDS 1999; 13(16):2261-7
19. Kosmiski LA, Miller HL, Klemm DJ.In combination, nucleoside reverse transcriptase inhibitors have significant effects on 3T3-L1 adipocyte lipid accumulation and survival. Antiviral Therapy 2006; 11:187-195
20. Mynarcik D, Wei LX, Komaroff E, et al. Loss of subcutaneous adipose tissue in HIV-associated lipodystrophy is not due to accelerated apoptosis. J Acquir Immune Defic Syndr. 2005; 38(1):53-6
21.Brinkman K, Smeitink JA, Romijn JA, Reiss P. Mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a keyfactor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. Lancet. 1999; 354:1112-5
22.Walker UA,Bickel M, Lutke Volksbeck SI, et al. Evidence of nucleoside analogue reverse transcriptase inhibitor-associated genetic and structural defects of mitochondria in adipose tissue of HIV-infected patients. JAIDS 2002; 29:117-121
23. Shikuma CM, Hu N, Milne C, et al. Mitochondrial DNA decrease in subcutaneous adipose tissue of HIV-infected individuals with peripheral lipoatrophy. AIDS 2001; 15:1801-1809
24. Cote HC. Possible ways nucleoside analogues can affect mitochondrial DNA content and gene expression during HIV therapy. Antivir Ther 2005; 10, Suppl 2:M3-11
25. Nolan D, Hammond E, Martin A, et al. Mitochondrial DNA depletion and morphologic changes in adipocytes associated with nucleoside reverse transcriptase inhibitor therapy.AIDS 2003; 17:1329-1338
26. Lagathu C, Bastard JP, Auclair M, et al. Antiretroviral drugs wit adverse effects on adipocyte lipid metabolism and survival alter the expression and secretion of proinflammatory cytokines and adiponectin in vitro. Antiviral Therapy 2004; 9:911-920
27. Caron M, Auclair M, Lagathu C, et al.The HIV-nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro.AIDS 2004; 18: 2127-2136
28. Stankov M, Schmidt RE, Behrens GMN. Zidovudine inhibits adipogenic differentiation of 3T3-L1 cells though inhibition of clonal expansion. Antiviral Therapy 2005 10:L20 (abstract no. 26)
29. Lindegaard B, Keller P, Bruunsgaard H, et al.Low plasma level of adiponectin is associated with stavudine treatment and lipodystrophy in HIV-infected patients. Clin Exp Immunol 2004; 135(2):273-9
30. Vigouroux C, Maachi M, Nguyen TH, et al. Serum adipocytokines are related to lipodystrophy and metabolic disorders in HIV-infected men under antiretroviral therapy. AIDS 2003; 17(10):1503-11
31.Henner T, Richter C, Peterhans E. Tumor necrosis factor-α induses superoxide anion generation in mitochondria of L929 cells. Biochemical 1993; 289:587-592
32. Ledgerwood EC,Prins JB, Bright NA, et al. Tumor necrosis factor is dilivered to mitochondria where a tumor necrosis factor-binding protein is localized. Laboratory Investigation 1998; 78:1583-1589
33. Christeff N, Melchior JC, de Truchis P, et al. Increased serum interferon alpha in HIV—1 associated lipodystrophy syndrome.Eur J Clin Invest 2002; 32(1): 43-50
34. Christeff N, Melchior JC, de Truchis P, et al. Increased serum interferon alpha in HIV—1 associated lipodystrophy syndrome. Eur J Clin Invest 2002; 32(1): 43-50
35. Yamauchi T, Kamon J, Ito Y, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 2003; 423(6941): 762-769
36. Yamauchi T, Kamon J, Waki H,et al.The fat derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 2001; 7(8):941-946
37. Ouchi N, Nihara S, Arita Y, et al. Adipocyte-derived plasma protein,adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 2001; 103(8): 1057-1063
38.Sweeney LL, Brennan AM, Mantzoros CS.The role of adipokines in relation to HIV lipodystrophy. AIDS 2007; 21:895-904
39.Addy CL, Gavrila A, Tsiodras S, et al. Hypoadiponectinemia is associated with insulin resistance, hypertriglyceridemia, and fat redistribution in human immunodeficiency virus-infected patients treated with highly active antiretroviral therapy. J Clin Endocrinol Metab. 2003; 88(2):627-36
40. Estrada V, Martinez-Larrad MT, Gonzdlez-Sanchez JL, et al. Lipodystrophy and metabolic syndrome in HIV-infected patients treated with antiretroviral therapy.Metabolism 2006; 55(7):940-5
41.Nagy GS, Tsiodras S, Martin LD, et al. Human Immunodeficiency Virus Type 1 - Related Lipoatrophy and Lipohypertrophy Are Associated with Serum Concentrations of Leptin. Clin Infect Dis 2003; 36:795-802
42.Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996; 34:292-295