Cellular and biochemical effects induced by antiretroviral drugs.

Ph. D. University of KwaZulu-Natal, Durban 2013.In the treatment of HIV/AIDS, protease inhibitors (PIs) and nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs) are the major components of highly active antiretroviral therapy (HAART). The side effects of these drugs include various metabolic disorders including insulin resistance, dyslipidaemia and lipodystrophy. The precise mechanistic basis of these remains largely unknown. In this study we aimed to understand the molecular basis of these metabolic effects by analysing the effects on lipoprotein lipase (LPL) activity, insulin signaling and the cellular metabolic profile. It was previously shown by this group that indinavir inhibits insulin signaling at a proximal level. The study was extended to a wider range of ARVs and in particular, the effects of sodium salicylate (NaSal) and berberine chloride (BBR) were analysed to determine if they could reverse the effects of the drugs on insulin signalling. In addition, Chinese hamster ovary cells transfected with the human insulin receptor (CHO-IR) were used for the first time to study the effects of NRTIs on the insulin signaling pathways. The high level of expression of insulin receptor facilitated sensitive detection of any alteration in the phosphorylation of signaling proteins as compared to 3T3-L1 adipocytes. Three PIs, indinavir, nelfinavir and ritonavir were used in this study. Indinavir and nelfinavir treatment significantly reduced the insulin-stimulated phosphorylation of the IRβ, IRS-1, Akt and MAPK in CHO-IR cells. However phosphorylation of GSK-3α\β was not affected by the PIs. Ritonavir also decreased (not statistically significant) the phosphorylation of IR-β and IRS-1 but its inhibitory effect on MAPK was the same as by the other PIs. NRTI’s did not inhibit insulin-stimulated tyrosine phosphorylation of IRβ and IRS-1 but reduced phosphorylation at MAPK and Akt. In order to understand the role of NFκ-B pathway in blocking insulin-stimulated tyrosine phosphorylation, IKK-16, a selective inhibitor of IkB kinase (IKK) was used but no significant involvement of this pathway was found in blocking tyrosine phosphorylation at IRS-1. Similarly, NaSal and BBR were also used to reverse the effects induced by PIs and NRTIs in CHO-IR cells but no significant change was observed on Akt and MAPK. NaSal and BBR reduced (but not significantly) the effects of PIs (indinavir and nelfinavir) on IR-β and IRS-1. These findings suggest that PIs induce insulin resistance by affecting multiple steps in the signaling pathway. At the proximal end of the insulin signalling pathway, protease inhibitors affect IR-β and IRS-1 while at the distal end they affect phosphorylation of Akt and MAPK. CHO-IR cells were also used to measure LPL activity using a colorimetric method employing pNPB as substrate. The most commonly used ARVs were tested. These included four PIs and six NRTIs .The results showed that NRTIs stavudine and emtricitabine significantly inhibited the LPL activity from the CHO-IR cells. PIs indinavir and nelfinavir were also found to decrease LPL activity extracellularly when added to the assay reaction in vitro. Similarly nelfinavir and atazanavir sulfate inhibited the activity of the LPL from the CHO-IR cells after 16 hour treatment. This suggested that these drugs may interfere with the enzyme activity intracellularly either at the level of its synthesis or its transportation from cytoplasm to the cell surface. These finding suggests that protease inhibitors may play a role in inhibiting lipoprotein lipase activity in vivo, and may thereby induce metabolic disorders in HIV-positive patients being treated with protease inhibitors. Metabolomic analysis was performed on the supernatant of cells treated with PIs and NRTIs, with and without insulin stimulation. Many significant alterations and trends in amino acids and organic acids levels in CHO-IR cells supernatants (treated with PIs) were recorded using 1H-NMR. For example, PIs decreased the synthesis of threonine, phenylalanine, lysine, arginine, isoleucine butyrate, glutamate, histidine and 2-oxo-isovalerate. Furthermore overproduction of lactate and ketones were observed in the nelfinavir treated cells. This may be a consequence of the secondary effects of insulin resistance induced by the PIs. Similarly NRTIs (stavudine and tenofovir) treatment also induced changes in the levels of many amino acids and organic acids .NRTIs decreased the synthesis of acetate, acetoacetate, histidine, methionine, phenylalanine and tryptophan while significant increase in the acetone was observed in stavudine-treated cells. Taken together, the results of this study suggest that PIs and NRTIs, inhibits LPL activity and synthesis, affects insulin signalling pathways at different levels and alters the synthesis of different cellular metabolites which may affect the signalling pathways of insulin