Is oxygen a key factor in the lipodystrophy phenotype?

Abstract

Background: The lipodystrophic syndrome (LD) is a disorder resulting from selective damage of adipose tissue by antiretroviral drugs included in therapy controlling human-immunodeficiency-virus-1. In the therapy cocktail the nucleoside reverse transcriptase inhibitors (NRTI) contribute to the development of this syndrome. Cellular target of NRTI was identified as the mitochondrial polymerase-gamma and their toxicity described as a mitochondrial DNA (mtDNA) depletion resulting in a mitochondrial cytopathy and involved in fat redistribution. No mechanisms offer explanation whatsoever for the lipo-atrophic and lipo-hypertrophic phenotype of LD. To understand the occurrence we proposed that the pO2 (oxygen partial pressure) could be a key factor in the development of the LD. For the first time, we report here differential effects of NRTIs on human adipose cells depending on pO2 conditions.

Results and discussion: We showed that the hypoxia conditions could alter adipogenesis process by modifying expression of adipocyte makers as leptin and the peroxisome proliferator-activated receptor PPARgamma and inhibiting triglyceride (TG) accumulation in adipocytes. Toxicity of NRTI followed on adipose cells in culture under normoxia versus hypoxia conditions showed, differential effects of drugs on mtDNA of these cells depending on pO2 conditions. Moreover, NRTI-treated adipocytes were refractory to the inhibition of adipogenesis under hypoxia. Finally, our hypothesis that variations of pO2 could exist between adipose tissue from anatomical origins was supported by staining of the hypoxic-induced angiopoietin ANGPTL4 depended on the location of fat.

Conclusion: Toxicity of NRTIs have been shown to be opposite on human adipose cells depending on the oxygen availability. These data suggest that the LD phenotype may be a differential consequence of NRTI effects, depending on the metabolic status of the targeted adipose tissues and provide new insights into the opposite effects of antiretroviral treatment, as observed for the lipo-atrophic and lipo-hypertrophic phenotype characteristic of LD.

Figure 1

Effects of hypoxia on adipogenesis. (A) Hypoxia inhibits TG accumulation. Cells were allowed to differentiate for 15 days with differentiation mix (black bars) under normoxia (20%), gaseous hypoxia (1%) or chemical hypoxia (100 μM DFO). TG accumulation was quantified by colorimetric determination of Red-Oil staining. Data are presented as optical density at 495 nm (OD₄₉₅ nm). n = 4, ** p < 0.001. (B) Hypoxia modulated adipocyte gene expression. Confluent cells were treated for 24 hours with 100 μM DFO (+) grey bars or were not treated (-) white bars. RNA was extracted and expression of the PPARγ, leptin and VEGF genes was followed by quantitative PCR. n = 2, ** p < 0.01. (C) Localization of HIF-1α and leptin protein in hypoxia-stimulated cells. Cells were treated for 24 hours with 100 μM DFO (+DFO) or were not treated (-DFO). Proteins were detected with mAB against HIF1-α tagged with a goat anti-mouse IgG coupled to FITC (a) or with mAB against leptin tagged with a goat anti-rabbit IgG coupled to cy3 (b), or a co-staining (c).

Figure 2

Differential effects of NRTI treatment on human adipose cells cultured under different pO₂. (A) Effects of NRTI treatment on mtDNA contents. Cells were cultured for 10 days as preadipocytes (P) or with Mix medium (A) under normoxia (20%) or in the presence of 100 μM DFO (DFO) plus a cocktail of 10 μM AZT and ddC (NRTI, black bars), or in the presence of 100 μM DFO but without the NRTI cocktail (C, hatched bars). Cell mtDNA was quantified and expressed per million cells (mtDNA/10⁶ cells). n = 3, ** p < 0.001. (B) Effects of NRTI treatment on TG accumulation. Cells were cultured in the same experimental conditions than in panel (A) and TG were quantified and data were normalized with respect to the control value obtained in adipocytes allowed to differentiate under normoxia condition (20% A, hatched bar, OD₄₉₅ nm = 0.25). n = 3, ** p < 0.001. (C) Effects of NRTI treatment on adipocyte marker expression. Cells were cultured in the same experimental conditions than in panel (A), RNA was extracted and expression of the PPARγ, leptin genes was followed by quantitative PCR. n = 2.

Figure 3

ANGPTL4 expression study of adipose tissue sections. (A) Angptl4 is a target of hypoxia in human adipose cells. Quantitative PCR analysis of ANGPTL4 mRNA in adipocytes (white bar), cultured for 24 hours in chemical DFO hypoxic condition (grey bar). n = 2, p < 0.001. (B) Differential ANGPTL4 and leptin expression in adipose tissue sections depending on the anatomical origin of the adipose tissue. We assessed ANGPTL4 (left panel) and leptin (right panel) levels by immunostaining subcutaneous (a), omental (b), and mammary (c) fat deposits and adipose tissues around a pheochromocytoma (d) and from the lipo-hypertrophic zone of lipodystrophic patients treated for HIV (e). Immunostaining with an isotypic control was carried out for each sample tested: a', b', c', d' and e'.