Effects of the human immunodeficiency virus-protease inhibitor, ritonavir, on basal and catecholamine-stimulated lipolysis

Abstract

Several of the aspartic acid protease inhibitors used to treat HIV infection increase basal lipolysis in adipocytes, but the cellular mechanisms leading to this augmentation are not well understood. We therefore studied the effects of chronic exposure to the HIV protease inhibitor, ritonavir, on the lipolytic cascade in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with ritonavir for 14 d (during and after differentiation) enhanced basal, isoproterenol (Iso)-stimulated, and cAMP analog-stimulated lipolysis. Enhancement of lipolysis was observed after Iso at concentrations between 0.1 and 10 mum. Despite a significant decrease in cyclic nucleotide phosphodiesterase (PDE)3B activity and protein levels, there were no changes in Iso-stimulated intracellular cAMP, protein kinase A (PKA) expression, or PKA activity. Ritonavir-augmented lipolysis was also observed under conditions that reversed the effect on PDE3B activity via preincubation with 1 mum (-)-N(6)-(2-phenylisopropyl)adenosine. In ritonavir-treated cells, protein expression of the lipid droplet-protective protein, perilipin, was significantly decreased, whereas there was no change in hormone-sensitive lipase. Activation of ERK1/2 by Iso did not play a role in the augmentation. We conclude that ritonavir decreases PDE3B and perilipin protein expression and affects both basal and catecholamine-stimulated lipolysis in 3T3-L1 adipocytes primarily through actions at sites downstream of PKA.

Fig. 1

Total cellular triglyceride (A) and AP-2 protein expression (B and C) in 3T3-L1 adipocytes treated with ritonavir (Rit) or control medium (0.1% ethanol) for 14 d. Cells were grown in 100-mm dishes. AP-2 protein expression by Western blot was measured in whole-cell lysates collected at 6, 10, and 14 d after initiation of differentiation (as described in Materials and Methods). A, n = 5–6 from two independent experiments. B and C, n = 6 from two independent experiments.

Fig. 2

Basal and Iso-stimulated lipolysis in 3T3-L1 adipocytes treated with ritonavir or control medium (0.1% ethanol) for 14 d. Release of glycerol (A) and NEFA (B) in response to stimulation with 10 μm Iso for 2 h. Glycerol and NEFA released into the media were normalized for total cellular DNA; n = 15 from three independent experiments for A and B. Release of glycerol (C) and NEFA (D) in response to 0.001–10 μm Iso; n = 11–17 from four independent experiments for C and D. *, P < 0.05; **, P < 0.01; †, P < 0.001 for ritonavir vs. ethanol.

Fig. 3

PDE3B protein expression and enzymatic activity in 3T3-L1 adipocytes treated with ritonavir for 14 d. A, PDE3B enzyme activity measured after 2 h incubations; n = 13–15 from three independent experiments. B–D, PDE3B protein expression and phosphorylation in adipocytes that were ³²P loaded for 2.5 h and subsequently treated with 1 μm PIA or 10 μm Iso for 15 min. Membrane fractions were isolated and immunoprecipitated with PDE3B antibody as described in Materials and Methods. Immunoprecipitated proteins were analyzed by Western blot followed by autoradiography. A representative Western blot is shown in B, followed by quantitation of total PDE3B protein (C) and phosphorylated PDE3B (D); n = 6 from two independent experiments. *, P < 0.05; **, P < 0.01; †, P < 0.001 for ritonavir vs. ethanol.

Fig. 4

PDE3B activity (A), intracellular cAMP (B), and glycerol release (C) in 3T3-L1 adipocytes treated with ritonavir for 14 d and preincubated with 1 μm PIA for 2 h followed by stimulation for 1 h. Intracellular cAMP was not determined (ND) in cells treated with N⁶cAMP (N⁶), because the analog interfered with the cAMP assay. Data are from three independent experiments. For A, n = 8; for B, n = 10–12; for C, n = 15–19. *, P < 0.05; **, P < 0.01; †, P < 0.001 for ritonavir vs. ethanol.

Fig. 5

Time course of Iso-activated lipolysis in 3T3-L1 adipocytes treated with ritonavir for 14 d. A, Intracellular cAMP from four independent experiments. Data points are least-square means ± sem; n = 20. B, Glycerol release from four independent experiments; n = 13–21. *, P < 0.05; **, P < 0.01 for ritonavir vs. 0.1% ethanol (EtoH).

Fig. 6

PKA protein expression and activity in 3T3-L1 adipocytes treated with ritonavir for 14 d. A, Whole-cell lysates were collected from cells treated with 10 μm Iso or medium alone for 2 h at 37 C, and blotted as specified in Materials and Methods. B, 14-d cells were treated with either 10 μm Iso or medium alone for 10, 20, or 30 min at 37 C, and 12,000 × g cytosolic fractions were used for kemptide assays. A, Representative Western blot for RIIβ (PKA regulatory subunit IIα, RIIβ (PKA regulatory subunit IIβ), and cat-α. B, Quantitation of signal intensity from Western blots. C, Endogenous PKA activity, expressed as percentage of maximal potential PKA activity. All data are from three independent experiments. For A and B, n = 12–15; for C, n = 8–9.

Fig. 7

Peri and HSL protein expression in 3T3-L1 adipocytes treated with ritonavir for 14 d. Whole-cell lysates were collected from cells treated with 10 μm Iso or medium alone for 2 h at 37 C, and blotted as specified in Materials and Methods. A, Representative Western blots; B, quantitation of signal intensity from Western blots for Peri; C, HSL; n = 13–15 from three independent experiments. For Peri, P < 0.01 for ritonavir vs. ethanol groups; for HSL, no significant differences.

Fig. 8

Activation of ERK 1 and 2 in 3T3-L1 adipocytes treated with ritonavir for 14 d. Samples from 2-h incubations were collected after 1-h preincubation with the ERK inhibitor PD 98059 [dissolved in 0.1% dimethylsulfoxide (DMSO)] or 0.1% DMSO. A, Representative Western blot; B, glycerol release; C, NEFA release; n = 14–15 from three independent experiments. †, P < 0.001, ritonavir vs. ethanol.