AbstractThe advent of antiretroviral therapy (ART) for the management of the human immunodeficiency virus (HIV) was, and to date still is, seminal in reducing the morbidity and mortality associated with HIV infection and subsequent acquired immunodeficiency syndrome (AIDS). Key ART drug classes such as non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside/tide reverse transcriptase inhibitors (NRTIs) are commonly prescribed in people living with HIV (PLWH), but treatment-related complications include an increased risk of developing type 2 diabetes (T2D). Direct damaging effects on pancreatic beta cell function and survival by NNRTIs or NRTIs may predispose PLWH who are also type 2 diabetic to impaired glycaemic control and loss of beta cell mass, hence increasing the risk of diabetic complications and insulin dependency. The aim of this study was to investigate the direct effects of the NNRTIs efavirenz, rilpivirine and doravirine, and the NRTIs tenofovir, emtricitabine and lamivudine, on beta cell function and survival.
The rat insulinoma INS-1E beta cell line and isolated rat islets of Langerhans were exposed to increasing concentrations of NNRTIs or NRTIs for 24 hours. Beta cell function was assessed by measuring glucose-stimulated insulin secretion (GSIS) from INS-1E cells and isolated rat islets of Langerhans with an ELISA. Beta cell survival was assessed by measuring cell viability using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell death by flow cytometric analysis following Annexin V and propidium iodide staining. Then, intracellular ROS generation was measured using the 2′,7′-dichlorodihydrofluorescein diacetate probe followed by confocal microscopy. Mitochondrial function was assessed by measuring the activity of mitochondrial complex I by spectrophotometric analysis and cellular ATP levels by luminometry. Flow cytometry was used to assess changes in mitochondrial membrane potential (Δψm) following staining with tetramethylrhodamine ethyl ester, and mitochondrial superoxide generation following MitoSOX staining. mRNA and protein expression of key endoplasmic reticulum (ER) stress markers (CHOP and GRP78) and uncoupling protein 2 (UCP2) were determined by reverse transcription-quantitative polymerase chain reaction and Western blotting or immunocytochemistry, respectively. In silico docking studies were carried out using the UCSF Chimera and SwissDock.
Our results show contrasting intraclass and interclass effects. While doravirine had no effect on INS-1E cell function and survival, efavirenz and rilpivirine significantly reduced GSIS from INS-1E cells and isolated rat islets of Langerhans. Efavirenz and rilpivirine also significantly reduced cell viability and induced apoptosis in INS-1E cells, likely through ER stress as shown by the upregulated expression of CHOP and GRP78. Furthermore, efavirenz and rilpivirine significantly increased intracellular and mitochondrial ROS generation, disrupted Δψm and decreased cellular ATP levels in INS-1E cells. In silico docking studies predict a possible inhibition of the mitochondrial ATP synthase by rilpivirine while efavirenz directly inhibited mitochondrial complex I activity and upregulated the expression of UCP2. On the other hand, the NRTIs had no effect on INS-1E cell function and survival.
This study has identified, for the first time, that the first- and second-generation NNRTIs efavirenz and rilpivirine can impair beta cell function and survival, likely through increased oxidative stress and contrasting mitotoxic effects. Therefore, although these drugs are effective in the management of HIV/AIDS, they potentially increase the risk of impaired glycaemic control and loss of beta cell mass, hence increasing the risk of diabetic complications and insulin dependency in PLWH and T2D.
|Date of Award||May 2023|
|Supervisor||Jon Mabley (Supervisor) & Wendy Macfarlane (Supervisor)|