7th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV 13–16 November 2005, Dublin, Ireland

Adipocyte ATP levels contribute to the aetiology of HIV lipoatrophy

M Gerschenson, S Steele, DE LiButti and CM Shikuma
Hawaii AIDS Clinical Research Program and the Department of Medicine, John A. Burns School of Medicine, University of Hawaii-Manoa, Honolulu, HI, USA

Antiviral Therapy 2005; Supplement 3:L8 (abstract no. 9)

OBJECTIVES: HIV lipoatrophy has been linked to the use of NRTIs and depletion of subcutaneous adipose tissue mitochondrial DNA (mtDNA). While this is suggestive of NRTI-mediated mitochondrial toxicity, the specific underlying pathophysiologic mechanisms whereby NRTIs mediate this loss of fat tissue are not well understood. We hypothesize that a decrease in mtDNA leads to a decrease in ATP production and that ATP depletion results in the induction of adipocyte apoptosis and lipoatrophy. We have examined these hypotheses by measuring ATP production in adipocytes and preadipocytes from HIV+ subjects with lipoatrophy and seronegative controls. ATP levels were also correlated with mtDNA copies/cell by real-time PCR.

METHODS: 300 mg of subcutaneous adipose tissue was obtained from gluteal fold in the lateral buttock–thigh area from: 11 lipoatrophic HIV-1-infected patients receiving a regimen with NRTIs as part of HAART for >6 months; three non-lipoatrophic HIV-1-infected patients receiving NRTIs-containing HAART; and seven HIV-1-negative participants. Total DNA was isolated from 20 mg and mtDNA copies/cell quantitated by real-time PCR. The rest of the tissue was minced into small pieces and digested with collagenase and then filtered through a 300 µm nylon mesh and centrifuged at 350×g. The top layer is adipocytes and the pellet is a preadipocyte fraction with preadipocytes, erythrocytes, and macrophages. The erythrocytes were removed using erythrocyte lysis buffer. An aliquot of the adipocytes or preadipocytes were counted using Trypan Blue and a haemacytometer. Another aliquot was stained with Oil-Red-O and visualized using an inverted microscope to confirm adipocyte isolation. ATP amount was measured using the Roche ATP Bioluminescence assay kit HS (Roche Molecular Biochemicals, Mannheim, Germany).

CONCLUSIONS: HIV lipoatrophic adipocyte ATP levels were 0.047 ±0.048 pM/cell and decreased significantly compared to HIV non-lipoatrophic at 0.160 ±0.009 (P=0.011) and HIV seronegative at 0.115 ±0.161 (P=0.013). The preadipocyte fraction ATP amounts were similar among the three cohorts. There was mtDNA depletion in the HIV lipoatrophic SC fat: 165 ±78 mtDNA copies/cell compared with the seronegative at 715 ±615 (P<0.001) but not compared with the non-lipoatrophic. This data demonstrates that HIV lipoatrophy is associated with ATP depletion in adipocytes but not in the preadipocyte fraction.

Acknowledgement: This research was supported by NIH, USA (AI060409, RR16467 and MD000173).

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2005-11-13
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