HIV protease inhibitors disrupt lipid metabolism by activating endoplasmic reticulum stress and inhibiting autophagy activity in adipocytes

Abstract

Background: HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART), the most effective treatment for HIV infection currently available. However, HIV PIs have now been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease and metabolic syndrome. Our previous studies have shown that HIV PIs activate endoplasmic reticulum (ER) stress and disrupt lipid metabolism in hepatocytes and macrophages. Yet, little is known on how HIV PIs disrupt lipid metabolism in adipocytes, a major cell type involved in the pathogenesis of metabolic syndrome.

Methodology and principal findings: Cultured and primary mouse adipocytes and human adipocytes were used to examine the effect of frequently used HIV PIs in the clinic, lopinavir/ritonavir, on adipocyte differentiation and further identify the underlying molecular mechanism of HIV PI-induced dysregulation of lipid metabolism in adipocytes. The results indicated that lopinavir alone or in combination with ritonavir, significantly activated the ER stress response, inhibited cell differentiation, and induced cell apoptosis in adipocytes. In addition, HIV PI-induced ER stress was closely linked to inhibition of autophagy activity. We also identified through the use of primary adipocytes of CHOP(-/-) mice that CHOP, the major transcriptional factor of the ER stress signaling pathway, is involved in lopinavir/ritonavir-induced inhibition of cell differentiation in adipocytes. In addition, lopinavir/ritonavir-induced ER stress appears to be associated with inhibition of autophagy activity in adipocytes.

Conclusion and significance: Activation of ER stress and impairment of autophagy activity are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV patients.

Figure 1. Effect of HIV PIs on the UPR activation in mouse pre-adipocytes.

Representative immunoblots against CHOP, ATF-4, and lamin B from the nuclear extracts of mouse 3T3L1 cells treated with different concentrations of HIV PIs for 6 h are shown. A). lopinavir (LPV). B). lopinavir/ritonavir (LPV/RTV = 4∶1). The density of immunoblot was determined by Image J. Relative protein levels of CHOP and ATF-4 were normalized using Lamin B as a loading control.

Figure 2. Effect of HIV PIs on UPR activation in differentiated mouse adipocytes.

Differentiated 3T3-L1 cells were treated with different concentrations of LPV, or LPV/RTV for 6 h. Total cellular RNA was isolated. The mRNA levels of CHOP and ATF-4 were quantified by real-time RT-PCR and normalized using internal control β-actin. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, *p<0.05, and **p<0.01.

Figure 3. Effect of HIV PIs on UPR activation in differentiated mouse adipocytes.

A) Representative immunoblots against CHOP, ATF-4 and Lamin B from nuclear extracts of mouse differentiated 3T3L1 cells treated with different concentrations of LPV and LPR/RTV for 6 h are shown. B–C) The density of immunoblot was determined by Image J. Relative protein levels of CHOP and ATF-4 were normalized using Lamin B as a loading control. Values are mean ± SE of four independent experiments. Statistical significance relative to vehicle control, *p<0.05.

Figure 4. Effect of HIV PIs on the UPR activation in differentiated human adipocytes.

A–B). Relative mRNA levels of CHOP and ATF-4 from differentiated human SGBS cells treated with different concentrations of LPV or LPV/RTV for 6 h and analyzed by real-time RT-PCR. β-Actin was used as an internal control. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, *p<0.05, **p<0.01. C). Representative immunoblots against CHOP, ATF-4 and Lamin B from nuclear extracts of differentiated human SGBS cells treated with different concentrations of LPV or LPV/RTV for 6 h are shown. The density of immunoblot was determined by Image J. Relative protein levels of CHOP and ATF-4 were normalized using Lamin B as a loading control.

Figure 5. HIV PIs induce cell death in differentiated 3T3-L1s.

Differentiated 3T3-L1 cells were treated with different concentrations of HIV PIs or vehicle control (DMSO) for 24 h, and then stained with Annexin V-FITC/propidium iodide. The percentages of apoptotic and necrotic cells were analyzed by flow cytometry. Relative amount of apoptotic and necrotic cells compared to vehicle control (which was set as 1) was calculated. Values are the mean ± SE of four independent experiments. Statistical significance relative to vehicle control, *p<0.05.

Figure 6. Effect of HIV PIs on adipocyte differentiation.

A–B) 3T3-L1 cells were induced to differentiate while concurrently treated with different HIV PIs (12.5 µM). After 8 days, cells were fixed and stained with Oil Red O and Nile red. Representative images of Oil Red O staining and Nile red staining are shown. C–D) 3T3-L1 cells were induced to differentiate while concurrently treated with different HIV PIs (12.5 µM). After two weeks, cells were fixed and 40 × images were processed using MATLAB. The number and size of lipid droplets were measured. Values are mean ± SE for three independent experiments. Statistical significance relative to vehicle control, ***p<0.001. E) Human SGBS cells were induced to differentiate while concurrently treated with different HIV PIs (12.5 µM) for 10 days. The intracellular lipid was stained with Oil Red O. Representative images from three individual experiments are shown.

Figure 7. Effect of HIV PIs on the expression of essential genes involved in lipid metabolism in differentiated 3T3-L1 cells.

Differentiated 3T3-L1 cells were treated for 6 h with increasing concentrations of LPV or LPV/RTV. Total cellular RNA was isolated and the mRNA levels of LPL, LXRα, FABP, and SREBP-1c were quantified by real-time RT-PCR and normalized using internal control β-Actin. Values are mean ± SE of three independent experiments. Statistical significance relative to vehicle control, *p<0.05, and **p<0.01.

Figure 8. Effect of CHOP on HIV PI-induced inhibition of adipocyte differentiation. Primary murine pre-adipocytes were isolated from wild type (WT) and CHOP^−/− mice.

Cells were induced to differentiate while concurrently treated with HIV PIs (12.5 µM) for 10 days. A). The intracellular lipid was stained with Oil Red O. Images were acquired with a 40× objective lens. Representative images for each treatment are shown. B–C) Bright field images were acquired with a 20×objective lens, and images were processed using MATLAB. The number and size of lipid droplets were measured. Values are mean ± SE for three independent experiments. Statistical significance relative to vehicle control, **p<0.01.

Figure 9. Effect of HIV PIs on autophagic punctate formation.

3T3-L1 cells stably expressing GFP-tagged LC3 were treated with different HIV PIs (12.5 µM), rapamycin (RM, 30 nM), or vehicle control (DMSO) for 24 h. The fluorescence images were recorded using a A) 40×, B) 63 × oil lens with FITC filter. Representative images for each treatment are shown.

Figure 10. Effect of HIV PIs on autophagosome formation in 3T3-L1 cells.

Non-differentiated 3T3-L1s were treated with individual HIV PIs (12.5 μM), rapamycin (RM, 30 nM) or vehicle control (DMSO) for 48 h. Cells were processed for transmission electron microscopy as described in “Methods”. A–C) Representative images for each treatment at 2,000 ×, 4,000 × or 10,000 × are shown. C) The density of autophagosomes for each treatment was point counted at 4,000 × and expressed as percentage of cytoplasmic area. Statistical significance relative to vehicle control: *p<0.05, **p<0.01.

Figure 11. Effect of HIV PIs on autophagy flux in 3T3L1 cells.

Non-differentiated 3T3-L1 cells were treated with different concentrations of HIV PIs for 6 h in the absence or presence of ammonium chloride (20 mM)/leupeptin (10 µM) (NH4Cl/Leu). Total cell lysates were isolated for Immunoblot analysis of LC3-I and LC3-II. β-actin was used as loading control. NH4Cl/Leu was added to the cells 2 h prior to harvesting. Representative immunoblots of A) LPV and B) LPV/RTV are shown. C–D). The density of immunoblot was determined by Image J. Relative a protein level of LC3-II was normalized with β-Actin. Values are mean ±SE of three independent experiments. Statistical significance relative to vehicle control, *p<0.05, **p<0.01.

Figure 12. A) Effect of HIV PIs on p62 degradation.

Differentiated 3T3-L1 cells were treated with different amounts of HIV PIs for 48 h, and total cell lysates were used to analyze p62 protein levels. Representative immunoblots of p62 and β-actin are shown. The density of the immunoblots was analyzed using Image J software. B) Effect of HIV PIs on long-lived protein degradation. Differentiated 3T3-L1 cells were metabolically labeled with ¹⁴C-valine, then treated with HIV PIs (12.5 µM) for 24 h. The degradation of long-lived protein was analyzed as described in “Methods”. Results are the mean ± SE of three independent experiments ***p<0.001.

Figure 13. Effect of HIV PIs on ER calcium stores.

Non-differentiated 3T3-L1 cells were treated with HIV PIs (RTV, LPV, or LPV/RTV, 12.5 μM) or vehicle control (DMSO) for 24 h. ER calcium stores were assessed by Fura-2 fluorescence ratio of 340∶380 nm in individual cells before and after addition of thapsigargin (TG, 100 nM). Representative tracing for a summation of at least 15 cells is shown. A) Cells were treated with RTV or LPV. B) Cells were treated with LPV/RTV. C) Relative calcium content was calculated by total area under the curve for each treatment group and compared to vehicle control (which was set as 1). Statistical significance relative to vehicle control, **p<0.01.