Glyceroneogenesis is inhibited through HIV protease inhibitor-induced inflammation in human subcutaneous but not visceral adipose tissue

Abstract

Glyceroneogenesis, a metabolic pathway that participates during lipolysis in the recycling of free fatty acids to triglycerides into adipocytes, contributes to the lipid-buffering function of adipose tissue. We investigated whether glyceroneogenesis could be affected by human immunodeficiency virus (HIV) protease inhibitors (PIs) responsible or not for dyslipidemia in HIV-infected patients. We treated explants obtained from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots from lean individuals. We observed that the dyslipidemic PIs nelfinavir, lopinavir and ritonavir, but not the lipid-neutral PI atazanavir, increased lipolysis and decreased glyceroneogenesis, leading to an increased release of fatty acids from SAT but not from VAT. At the same time, dyslipidemic PIs decreased the amount of perilipin and increased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion in SAT but not in VAT. Parthenolide, an inhibitor of the NFκB pathway, counteracted PI-induced increased inflammation and decreased glyceroneogenesis. IL-6 (100 ng) inhibited the activity of phosphoenolpyruvate carboxykinase, the key enzyme of glyceroneogenesis, in SAT but not in VAT. Our data show that dyslipidemic but not lipid-neutral PIs decreased glyceroneogenesis as a consequence of PI-induced increased inflammation in SAT that could have an affect on adipocytes and/or macrophages. These results add a new link between fat inflammation and increased fatty acids release and suggest a greater sensitivity of SAT than VAT to PI-induced inflammation.

Fig. 1.

Effect of PIs on TG content and glycerol and FFA release in subcutaneous and visceral human fat explants. SAT and VAT fat pads were pretreated from day 1 to 3 with either ethanol (control) or NFV, 10 µM; LPV, 10 µM; RTV, 10 µM; and ATV, 10 µM as described in Material and Methods and according to the scheme presented in A. The number of subjects are shown in brackets, and all experiments were performed in duplicate. Bars represents means ± SD relative to respective controls; *, P < 0.05; and **, P < 0.01. B: Variation of TG content expressed per gram of tissue relative to respective controls values. C: Glycerol concentration was measured in the culture medium after a 1 h incubation in order to evaluate the lipolytic rate in response to antiretroviral agents in fat explants. Results are expressed per gram of TG and are relative to their respective control values. D: FFA release was simultaneously evaluated in the medium after 1 h of basal lipolysis in the presence of 2.5 mM pyruvate. Results are expressed per gram of TG and are relative to their respective control values.

Fig. 2.

Effects of PIs on ATGL and HSL lipases and on perilipin protein levels in subcutaneous and visceral human fat pads. SAT and VAT fat pads were pretreated as shown in Fig. 1A. Upper panels: representative Western blots presented for each protein. Blotting was performed on whole fat explants after removing fat coat and fragments. Lower panels: Corresponding quantifications were performed versus β-actin amount and expressed in fold changes versus control. Data are means ± SD from three individuals; *, P < 0.05; and **, P < 0.01 versus control.

Fig. 3.

GNG-dependent re-esterification of FFA liberated during lipolysis in subcutaneous and visceral human fat pads. SAT and VAT fat pads were pretreated from day 1 to 3 with either ethanol (control) or NFV, 10 µM; LPV, 10 µM; RTV, 10 µM; and ATV, 10 µM, as described in Material and Methods and according to the scheme in Fig. 1A. The number of subjects are in brackets, and all experiments were performed in duplicate. Results are expressed as means ± SD and are relative to the level of their respective control, *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus control A. FFA/glycerol ratio was calculated from the determination of both parameters in the incubation medium of each fat explant. The GNG-dependent fatty acid re-esterification process decreases the FFA/glycerol ratio because it affects only one parameter, the FFA concentration. B: PEPCK-C activity was evaluated in human SAT and VAT cytosolic fractions in response to the different treatments. C: Evaluation of the GNG-dependent re-esterification of FFA liberated during lipolysis in fat pads incubated for 1 h in KRPB-BSA containing 2.5 mM pyruvate and 1 µCi ¹⁴C₁-radiolabeled pyruvate. Lipids were then extracted, and radioactivity was measured as described in Materials and Methods.

Fig. 4.

Effect of PIs on proinflammatory cytokines and chemokine secretion from human subcutaneous and visceral fat pads. SAT and VAT fat pads were pretreated from day 1 to 3 with either ethanol (control) or NFV, 10 µM; LPV, 10 µM; RTV, 10 µM; and ATV, 10 µM, as described in Material and Methods and according to the scheme in Fig. 1A. IL-6, TNF-α, and MCP1 secretion were measured in the last 18 h incubation medium. The number of subjects are in brackets, and results are expressed in nanograms or pg/g of tissue ± SD for each reproduced in duplicate; *, P < 0.05; and **, P < 0.01 versus control.

Fig. 5.

Effect of NFV on IL-6, TNF-α, and PEPCK-C gene expression in human subcutaneous and visceral adipose tissue. Fat pads were treated for 14 h in DMEM, glucose 5 mM, 0.5% low-fat BSA. mRNA concentrations were analyzed by quantitative RT-PCR, normalized to 18S rRNA, and expressed relative to their respective controls. Amounts of IL-6, TNF-α, and PEPCK-C mRNA were about 64, 4, and 2 higher in VAT than in SAT. Data are means ± SD from six individuals. **, P < 0.01; and ***, P < 0.001 versus control.

Fig. 6.

Effect of the inhibitor of NFκB activation, parthenolide, on NFV-induced inflammation and NFV-altered GNG in human subcutaneous adipose tissue. Parthenolide (P), 30 μM, was added 1 h before NFV, 10 µM. mRNA concentrations were analyzed by quantitative RT-PCR, normalized to 18S rRNA, and expressed relative to their respective control values. Cytokine secretion and PEPCK-C enzyme activity levels were measured as in Materials and Methods. Data are means ± SD from six individuals. #, P < 0.05; ##, P < 0.01; ###, P < 0.001 NFV versus control and *, P < 0.05; and **, P < 0.01 NFV+P versus NFV. A: Analysis of the mRNA amounts (duration of the treatment was 6 h for IL-6 and TNF-α and 18 h for PEPCK-C gene expression analysis). B: Measure of secretion of cytokines in culture medium and PEPCK-C enzyme activity in fat pads after 28 h. C: Measure of glycerol and FFA in the culture medium after a 2 h period of isoproterenol-induced lipolysis.

Fig. 7.

Effect of purified human IL-6 on basal and cAMP-induced PEPCK-C enzyme activity in subcutaneous and visceral adipose tissue. Fat pads were treated with 0.1 and/or 1μg/ml IL-6 over 24 h in the presence (or not) of D-butyril cAMP (0.5 mM), which was added 1 h after IL-6 and then homogenized for measurement of PEPCK-C enzyme activity. Each value represents the mean of duplicate determinations from three different explants and were expressed in fold changes versus their respective control values. *, P < 0.05; and **, P < 0.01 versus control.