ER Stress and Lipid Metabolism in Adipocytes

The role of endoplasmic reticulum (ER) stress is a rapidly emerging field of interest in the pathogenesis of metabolic diseases. Recent studies have shown that chronic activation of ER stress is closely linked to dysregulation of lipid metabolism in several metabolically important cells including hepatocytes, macrophages, β-cells, and adipocytes. Adipocytes are one of the major cell types involved in the pathogenesis of the metabolic syndrome. Recent advances in dissecting the cellular and molecular mechanisms involved in the regulation of adipogenesis and lipid metabolism indicate that activation of ER stress plays a central role in regulating adipocyte function. In this paper, we discuss the current understanding of the potential role of ER stress in lipid metabolism in adipocytes. In addition, we touch upon the interaction of ER stress and autophagy as well as inflammation. Inhibition of ER stress has the potential of decreasing the pathology in adipose tissue that is seen with energy overbalance

Inhibition of P-Glycoprotein by HIV Protease Inhibitors Increases Intracellular Accumulation of Berberine in Murine and Human Macrophages

Background HIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages. Methodology and Principal Findings Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp. Conclusion and Significance HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic

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

<p>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 <b>“</b>Methods”. <b>A–C)</b> Representative images for each treatment at 2,000 ×, 4,000 × or 10,000 × are shown. <b>C)</b> 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.</p

Effect of HIV PIs on adipocyte differentiation.

<p><b>A–B)</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. <b>C–D)</b> 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. <b>E)</b> 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.</p

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

<p>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 <b>A</b>) LPV and <b>B</b>) LPV/RTV are shown. <b>C–D).</b> 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.</p

Effect of HIV PIs on autophagic punctate formation.

<p>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 <b>A)</b> 40×, <b>B)</b> 63 × oil lens with FITC filter. Representative images for each treatment are shown.</p

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

<p>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.</p

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

<p>Cells were induced to differentiate while concurrently treated with HIV PIs (12.5 µM) for 10 days. <b>A).</b> 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.</p