J Physiol Pharmacol. 2017 Apr;68(2):283-293.

The human immunodeficiency virus (HIV1) protease inhibitor sanquinavir activates autophagy and removes lipids deposited in lipid droplets.

Polus A1, Bociaga-Jasik M2, Czech U1, Goralska J1, Cialowicz U1, Chojnacka M1, Polus M3, Jurowski K4, Dembinska-Kiec A1.

1Department of Clinical Biochemistry Jagiellonian University Medical College; Cracow, Poland;

2Department of Gastroenterology, Hepatology and Infectious Diseases, Jagiellonian University Medical College, Cracow, Poland;

3Institute of Water Supply and Environmental Protection, Faculty of Environmental Engineering, Cracow University of Technology, Cracow, Poland;

4Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, Cracow, Poland

 

Abstract

Reduction in mortality and increased average life span of the human immunodeficiency virus (HIV)-infected patients treated with antiretroviral therapy (ART) are associated with the risk of unwanted effects, such as insulin resistance and dyslipidemia with cardiovascular complications. Antiretroviral therapy may also be associated with lipodystrophy characterized by peripheral lipoatrophy with central fat accumulation. Understanding the molecular mechanisms of lipodystrophy caused by ART is important for therapeutic strategy and prediction of side effects. Influence of protease inhibitor saquinavir (SQV) on preadipocyte differentiation was analyzed in in vitro on a human Chub-S7 cell line model. To evaluate the effects of SQV the drug was added to differentiated and non-differentiated cells. The influence of SQV on changes in the gene expression profile was verified by microarray and changes in lipid species content analyzed using GC-MS/MS. Results were confirmed by real-time PCR and autophagy analysis. Addition of SQV to differentiated Chub-S7 cells led to removal of lipids deposited in lipid droplets, down-regulation of expression of transcription factors and markers of adipocyte differentiation. Antiviral activity of SQV based on its non-selective inhibition of proteases resulted in proteasome inhibition, induction of endoplasmic reticulum stress and induction of macroautophagy. This activity was accompanied by an increase in PI, PEPL, PC lipid species especially with MUFA and PUFA. Additionally up-regulation of miR-100-3p, miR-222-5p, miR-483-5p was found, which correlated with obesity, insulin resistance, increasing insulin secretion and activation of lipolysis. Our results indicated that SQV, by inhibition of proteasome protein degradation, activate the unfolded protein response resulting in autophagic breakdown of lipids deposited in adipose tissue causing lipodystrophy.

PMID: 28614778

 

Supplement

Lipodystrophy is one of the known side effect of protease inhibitors (PIs) used in the treatment of patients infected with HIV. Introducing highly active antiretroviral therapy (HAART) resulted in decreased mortality and increased average life expectancy of HIV-infected patients. Unfortunately, this therapy is associated with the change in the adipose tissue functioning and severe lipodystrophy characterized by peripheral lipoatrophy, a symptom that does not disappear after the end of treatment.

The relationship of cellular processes with gene expression and changes in lipid structure allowed to explain the mechanisms of SQV influence on differentiation of adipose tissue cells

In in vitro experiments conducted on the Chub-S7 preadipocyte cell line (immortalized SVF cells) SQV inhibited the formation of LDs (lipid droplets) stimulated by differentiation conditions and suppressed the expression of PAT protein (Perilipin, Adipophilin, TIP47) genes, adipogenesis markers and enzymes involved in fatty acid metabolism. Our results indicated that inhibition of de novo lipid production may be related to the activity of transcription factors ETS1, SREBP, PPARg and CREB1 involved in the regulation of fatty acid synthesis and expression of adipogenesis-related proteins.

Lipodystrophy caused by protease inhibitors may result from the inhibition of proteasome activity. Accumulation of protein aggregates can stimulate autophagy or apoptosis. Protein degradation in the proteasome pathway is largely responsible for regulating the level of SIRT deacetylases in the cells. Increased level of SIRTs protein by SQV inhibited proteasome degradation or up-regulated the observed SIRTs gene expression leading to deacetylation of many metabolic enzymes, and resulting in loss of their activity (1). On the contrary, deacetylation of autophagy related proteins stimulates this process (2). During autophagy initiation, specific lipids from various organelles are recruited to form autophagosomes. Obtained results indicated activation of autophagy by SQV in differentiated Chub-S7 cells by upregulation of the expression of proteins involved in various stages of autophagy and triglycerides depletion in LDs (3). These results were confirmed by a biological assay. Our previous results indicated that SQV affects also the mitochondrial function, connected with the decrease of oxygen consumption, mitochondrial membrane potential and ATP production (4). Reducing the amount of mitochondria helps to diminish the production of reactive oxygen species. Changes found in the gene expression level of mitochondrial proteins (TOMM20, SLC25A4, HSP60, COX4-IV, electron transport proteins, etc.) are often showed as an indicator of damaged mitochondria removal during mitophagy. Mitophagy was probably induced as an adaptive response to SQV-induced stress. Results indicated that SQV stimulated different types of autophagy not only lipophagy as degradation of lipids deposited in LDs but also mitophagy.

Earlier reports have shown that SQV and other antiretroviral drugs can cause lipid disorders, increase insulin resistance and inhibit insulin secretion. Our results did not show any the effect of SQV on the expression of protein genes involved in the insulin receptor pathway in adipocytes. Only a significant reduction in gene expression for GLUT4 glucose transporter was observed. However, the observed activation of the NFκB pathway, the synthesis of ceramides, increased levels of free fatty acids released during lipolysis can induce insulin resistance of adipocytes and other tissues.

SQV increased the expression of miR-100-3p, miR-222-5p, miR-483-5p in differentiated adipocytes which was previously found in the serum of patients with obesity and insulin resistance.

SQV stimulated miRNA could enhance insulin gene transcription and insulin secretion (miR-483-5p), activate lipolysis in adipose tissue (miR-222-5p), and inhibit the mTOR pathway, thereby protecting cells from apoptosis by activating autophagy (miR-100-3p) (5, 6, 7).

The results show that SQV inhibited the expression of genes associated with adipocyte differentiation and the formation of LDs. Inhibiting cellular protease activity stimulates autophagy leading to the removal of lipids deposited in LDs. In addition, SQV stimulated miRNA expression, which activates autophagy and intensifies insulin resistance.

 

 

Figure 1. Changes in the gene expression of proteins related to autophagy in non-differentiated and differentiated Chub-S7 cells in the presence of saquinavir (SQV 30 μM) (microarray data (Agilent), n = 3, one-way ANOVA followed by Benjamini-Hochberg multiple test correction FDR and Tukey post-hoc test, ratio with p < 0.05 in post-hoc test). Autophagy pathway was created using Pathvisio tools, red-upregulation, green- downregulation

 

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