10th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV 6-8 November 2008, London, UK

SKELETAL MUSCLE MITOCHONDRIAL PROTEINS DISCORDANTLY REGULATED BY INSULIN IN HIV+ WITH INSULIN RESISTANCE: A MASS SPECTROMETRY-BASED MUSCLE PROTEOMICS STUDY

Antiviral Therapy 2008; 13(Suppl. 4):A8 (abstract no. O-10)

KE Yarasheski, DN Reeds, S Smith, LY Munsell, J Malone, H Rohrs and RR Townsend
Washington University School of Medicine, St Louis, MO, USA

BACKGROUND: Insulin resistance (IR) and diabetes are common metabolic complications in HIV-infected people treated with antiretroviral therapy. Skeletal muscle is mitochondria (mt)-rich and the primary site for insulin-stimulated glucose storage. Impairments in human muscle mt protein expression patterns and activities might be involved in the pathogenesis of HIV+ IR. We aimed to identify and characterize human muscle mt protein expression patterns associated with HIV+ IR.

METHODS: During a hyperinsulinaemic euglycaemic clamp, thigh muscle samples were obtained from six healthy normal men (35 ±5 years, body mass index [BMI]=27 ±2 kg/m² and fat =21 ±3%) and six HIV+ IR men (36 ±2 years, median CD4=544 ±120 copies/µl, VL=Und, HIV+ duration =7 ±3 years, BMI=27 ±1 kg/m² and fat=24 ±4%; all on nucleoside reverse transcriptase inhibitors, 5/6 on non-nucleoside reverse transcriptase inhibitors and 3/6 on protease inhibitors). Glucose disposal rate (M µmol/kg FFM/min/µU/ ml insulin) was lower in HIV+ IR at baseline (-36%) and during hyperinsulinaemia (-14%). One muscle sample was obtained at baseline (insulin 6–10 µU/ml) and the second muscle sample was obtained at the end of the hyperinsulinaemic (77–89 µU/ml) and euglycaemic (100 mg/dl) clamp. In each sample, muscle proteins were separated, visualized and compared using 2D-differential fluorescence gel electrophoresis. Each gel included a protein pool from all samples that was used as an internal standard to normalize protein expression levels between patients and within the same patient under fasting and hyperinsulinaemic conditions. Differentially expressed protein spots that were upregulated under hyperinsulinaemic conditions in controls, but not upregulated in HIV+ IR were excised, digested with trypsin and their peptide accurate mass and amino acid sequence information obtained using matrix-assisted laser desorption ionization tandem mass spectrometry (MS) and liquid chromatography-electrospray ionization-Fourier transform-tandem MS. Mass spectra were searched against the NCBI protein database (Mascot) and gene ontology terms to identify human mt proteins.

RESULTS: Sixty-five protein spots were discordantly regulated by insulin; five were identified as mt proteins: aconitase 2, mitofilin, malate dehydrogenase, calmitine and 3-hydroxyacyl-CoA dehydrogenase. Mitofilin (IMMT; MW=83626, Mascot score=441, 29 peptides matched and 36% protein coverage) is a unique inner mt membrane protein, not previously recognized to be insulin-regulated. In controls, insulin increased mitofilin expression 17-fold more than in HIV+ IR. Mitofilin is crucial for maintaining normal tubular mt cristae and cristae junction morphology. Mitofilin deficiency results in tightly packed cristae, which could impair ion and metabolite exchange between mt membranes.

CONCLUSIONS: These mt proteins represent potential biomarkers and sites of pathogenesis in HIV+ IR. The structure and function of the muscle intramitochondrial membrane and mt cristae might be altered in HIV+ IR. This could impair mt metabolite and ion flux, oxidative phosphorylation and mt ATP production during insulin stimulation.

Supported by NIH.

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2008-11-06
O-10

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