Evidence of toxicity in Adipose tissue of HIV-infected patients correlates with NRTI-associated mtDNA depletion

Abstract

Background: The proposed basis for NRTI-associated fat wasting involves cellular mitochondrial DNA (mtDNA) depletion and mitochondrial dysfunction leading to clinical toxicities. We have investigated the relationship between NRTI therapy and mtDNA depletion in adipocytes using cross sectional data from the West Australian HIV cohort. Longitudinal samples were then used to investigate associations between mtDNA depletion and adipose tissue toxicity. Methods: Cross sectional data: Excisional biopsy fat samples (n = 78) were collected from 60 patients (pts). mtDNA copies/cell was determined after collagenase treatment using real-time PCR. Statistical analysis was performed using linear mixed effects models. Longitudinal data: Adipose biopsies (n = 19) were obtained from suprailiac and buffalo hump sites from 9 HIV-infected pts before and after commencing/switching NRTI therapy, and in one healthy control. mtDNA copies/cell was determined as above. Immunohistochemistry, using fluorescent probes and confocal microscopy to assess mitochondrial (I) and nuclear encoded (IV) subunits of cytochrome c oxidase, was performed on frozen biopsies. Results: Cross sectional Data: Current NRTI therapy was the dominant determinant of mtDNA content with reduced average mtDNA associated with d4T (n = 22, 234 copies/cell, p < 0.0001) and AZT therapy (n = 15, 537 copies/cell, p = 0.002) compared with no NRTI (n = 23, 1,622 copies/cell). Longitudinal Data: All tissue with NRTI-associated mtDNA depletion (< 500 copies/cell) demonstrated evidence of tissue toxicity characterized by irregularly shaped adipocytes with reduced lipid droplet volume, mitochondrial proliferation, and increasing numbers of nuclei and stroma. Severity of adipose toxicity correlated with decreasing absolute mtDNA copy number, rather than percentage decrease from baseline. In 3/6 biopsies with mtDNA copies < 300 copies/cell, depletion of mitochondrial encoded subunit COX (I) relative to nuclear encoded COX (IV) was detected. Pts with mtDNA depletion who had switched therapies showed increased mtDNA levels, but this was not accompanied by decreased evidence of tissue toxicity. Conclusions: Increasing adipocyte mtDNA depletion correlates with a gradation of toxicity in adipose tissue. Our findings are consistent with a model in which inhibition of mtDNA synthesis by NRTIs has mild to severe toxic outcomes in adipocytes (depending on choice of NRTI) which ultimately contributes to the severity of fat wasting