Investigating molecular interactions between SARS-CoV-2 spike protein variants and glucose metabolic dysregulation in beta-cells

Abstract

The severity of COVID-19 disease onset is strongly linked to co-morbidities such as diabetes, with hyperglycemia an independent risk factor for mortality. Additionally, hyperglycemia alters intracellular mechanisms to induce oxidative stress and subsequently dysregulate glucose metabolic pathways such as the hexosamine biosynthetic pathway (HBP) and the formation of advanced glycation end-products (AGEs). The emergence of new-onset hyperglycemia following SARS-CoV-2 infection is a cause for concern. One possible explanation is the direct infection of the pancreas leading to damage of β-cells. Binding of SARS-CoV-2 spike (S)-protein to its primary receptor, ACE2, can be enhanced via glycosylation and/or glycation of the receptor, rendering it more susceptible to S-protein interactions. The interplay between hyperglycemia and SARS-CoV-2 infection, however, remains to be fully elucidated. The primary objective of this study is therefore to investigate the effects of hyperglycemia and SARS-CoV-2 binding on pancreatic β-cells. More specifically, we aim to determine the role(s) of AGE production and HBP-mediated O-GlcNAcylation in modulation of SARS-CoV-2 entry and accessory proteins, S-protein binding, and downstream molecular effects in cultured INS-1 β-cells under hyperglycemic conditions. We hypothesize that hyperglycemia primes host cells for SARS-CoV-2 S-protein variants via glycation (AGE) and O-GlcNAcylation (HBP) pathways to promote binding to INS-1 β-cells. INS-1 cells were exposed to 1) high glucose, glucosamine (increases HBP), and methylglyoxal (AGE precursor) and 2) recombinant S-protein (Wild type, β and δ-variants) in separate experiments. Metabolic activity (WST-1 assays) and Western blot analyses were performed to assess markers of mitochondrial oxidative phosphorylation, the AGE (RAGE) and HBP (OGT and O-GlcNAc), SARS-CoV-2 entry (ACE2, DPP4, Basigin, EEA1, VCP) and accessory proteins (TMPRSS2, ADAM10, ADAM17, Cathepsin B, CD13). Glucose-stimulated insulin response was determined using an insulin ELISA. Metabolic activity was increased by high glucose (p<0.0001), glucosamine (p<0.0001) and methylglyoxal (p<0.05) versus controls. Recombinant S-protein variants increased metabolic activity in a time, concentration and variant-dependent manner. Here, the lower (10 ng/ml) concentration showed a >4-fold increase for the β- and δ-variants (p<0.0001 versus control, respectively), while the wild type had a similar response with higher concentrations (100 and 1000 ng/ml). The endosomal protein EEA1 was significantly increased (p<0.05 versus control) in response to AGE modulation. We conclude that hyperglycemia and modulation of the HBP and AGE pathways exert differential β-cell metabolic effects and AGE induction may prime endosomal entry mechanisms for SARS-CoV-2 spike protein. The spike variants significantly stimulate metabolic activity, modulating β-cell function in an acute manner. This project is funded by the National Research Foundation (NRF) of South Africa and the South African Medical Research Council (SAMRC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.