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A novel lipodystrophy syndrome characterized by truncal adiposity, peripheral fat atrophy, type 2 diabetes mellitus, and dyslipidemia occurs in HIV-infected individuals, and may be aggravated by HIV-1 protease inhibitors. The increase in truncal fat could be due to enhanced preadipocyte differentiation. Using the 3T3-L1 preadipocyte model, we reported that ritonavir enhances adipocyte differentiation in culture. The goal of this study was to characterize the molecular mechanism of ritonavir on preadipocyte differentiation.Time course studies of 3T3-L1 preadipocytes placed in standard differentiation medium (insulin, dexamethasone, and isobutylmethylxanthine) were performed. Glycerol phosphate dehydrogenase (GPDH) was assayed enzymatically, and triacylglycerol (TG) mass was quantified. The adipogenic transcription factors adipocyte determination and differentiation-dependent factor 1 (ADD-1)/sterol regulatory element binding protein 1 (SREBP-1), CCAAT/enhancer-binding protein-α (CEBPα), and peroxisome proliferator activated receptor-γ (PPARγ), were measured by Western analysis.Ritonavir (10 μg/ml) enhanced 3T3-L1 preadipocyte differentiation (30% increase in TG mass; 50% increase in GPDH activity), and transiently raised levels of the 68 kDa active mature form of ADD-1/SREBP-1 during adipogenesis by threefold, compared with standard differentiation. In contrast, ritonavir attenuated the differentiation-induced increase in CEBPα and PPARγ.Our data suggest that ritonavir enhances 3T3-L1 adipogenesis by increasing the level of active mature ADD-1/SREBP-1. This effect may be due to reduced proteolysis of ADD-1/SREBP-1, as ritonavir inhibits an N-acetyl-leucyl-leucyl-norleucinal (ALLN)-sensitive proteosomal degradation pathway in lymphocytes, and ALLN itself inhibits the breakdown of mature ADD-1/SREBP-1. As mature ADD-1/SREBP-1 regulates several lipogenic enzymes, higher levels may explain the effect of ritonavir on TG accumulation and GPDH activity. Studying ADD-1/SREBP-1 may lead to better understanding and prevention of the lipodystrophy syndrome.