NYMC Student Theses and Dissertations

Date of Award

5-28-2025

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Dr.Jerry L Nadler

Second Advisor

Dr.Sachin Gupte

Abstract

Inflammation and immune cell activation in islets lead to pancreatic β-cell dysfunction and loss of mass, leading to type 2 diabetes development. Lipoxygenases, group of enzymes, that catalyze the oxygenation of cellular poly-unsaturated fatty acids to form lipid inflammatory mediators. 12-Lipoxygenase protein (12-LOX) converts arachidonic acid to 12-hydroperoxyeicasatetranoic acid (12-HPETE), which is then reduced to a more stable 12-hydroxyeicosatetraenoic acid (12-HETE) by glutathione peroxidase. In the islets, increased expression or activity of 12-LOX catalyzes the production of 12-HETE to accelerate inflammation and promote oxidative stress. 12-LOX has been identified in both rodent and human endocrine pancreas and is upregulated in both species in pre-diabetes and T2D. Also, evidence suggests that SARS-CoV-2 infection aggravates diabetes, and diabetic patients have severe outcomes. In addition, SARS-CoV-2 infection increases 12-LOX products in platelets, lungs, and vascular system. Elevated levels of 12-HETE are reported in serum and lung-derived fluids of patients undergoing severe SARS-CoV-2 infection. With this information, we hypothesized that Increased 12-LOX expression contributes to insulin resistance and β-cell dysfunction, and it plays an important role in exacerbating tissue inflammation and β-cell dysfunction in COVID-19 infection. Specifically, Insulin resistance is associated with increased 12-LOX expression, and 12-LOX participates in the pathology of SARS-CoV2 infection and may lead to β-cell dysfunction associated with diabetic pathology. In this study, I evaluated the molecular mechanisms leading to activation and downstream functions of human 12-LOX in the human beta cell in insulin resistance using an insulin receptor antagonist, S961, in non-diabetic and T2D pancreatic islet donors. To test this hypothesis, we analyzed human islet 12-LOX protein expression, mRNA levels, and eicosanoid levels to link human ALOX12 expression and 12-LOX production to impaired insulin receptor signaling. There was a significant increase of ALOX12 mRNA and 12-LOX in the presence of S961-induced insulin resistance in non-diabetic islets and an increase in 12-HETE in T2D islet donors xiv compared to the non-diabetic donors. Next, to determine the link between insulin resistance and 12-LOX in vivo, I used samples from a β-cell specific insulin receptor knockout mouse that manifests a local β-cell signaling defect and spontaneously develops T2D. Using this novel mouse model, we studied ALOX12 expression by PCR and 12-LOX protein by immunostaining in isolated islets at various time points. Freshly isolated islets from βIRKO and littermate control mice were examined at ages 4, 9-11, and 18 weeks. There was a peak increase of 12-LOX in the islets at 9-11 weeks of age in the βIRKOs compared to control mice. The data also suggests ALOX12 but not ALOX 15 expression in the βIRKO mouse islets compared to control mice. Immune cell activation or inflammation is also observed in βIRKO mice compared to the wild type, specifically in the pancreatic islet. These new data suggest that 12-LOX plays a key role in inducing inflammation and β-cell dysfunction in this mouse model of localized insulin resistance and T2D. In a follow-up aim, we investigated the potential role of 12-LOX in inflammation associated with SARS-CoV-2 infection in decedents with diabetes. We obtained lung and pancreatic sections of COVID-19 decedents without diabetes and with diabetes and determined 12-LOX protein and mRNA levels. The studies demonstrated an elevated expression of 12-LOX protein and mRNA levels in lungs and pancreas samples from COVID-19 + decedents with diabetes. These interesting findings led us to move into in-vivo studies in the K-18hACE2 transgenic mice, a mouse model of COVID-19. Both male and female mice were inoculated with a high dose of SARS-CoV-2 and were treated with a specific 12-LOX small molecule inhibitor, VLX-1005 (30mg/kg; i.p) for 24-48 hours post-infection for 7-12 days. The results revealed significantly higher survival with minimal body weight loss in the VLX-1005-treated male mice compared to the vehicle-treated mice group, especially when the treatment was initiated 48 hours after the infection and continued for 12 days. The survival rates were higher in VLX-1005-treated male than female mice. In VLX-1005-treated male mice, there was a significant reduction in the expression of key chemokines and cytokines known to be associated with severe COVID-19 infection. Histopathological evaluation of the lung showed resolution of inflammation in xv VLX-1005 treated mice 14 days post-infection. 12-LOX expression was also increased in the pancreas of infected mice. These results suggest that inhibition of 12-LOX could provide therapeutic efficacy against COVID-19 infection by reducing hyper-inflammatory response. In conclusion, this study provides evidence that 12-LOX plays a key role in inflammation and may lead to β-cell dysfunction in a diabetic pathology, including COVID-19 infection-associate diabetic complications.

Additional Files
HSL.pdf (790 kB)

Keywords

pharamcology, diabetes, COVID-19, insulin, 12-LOX, ALOX12, inflammation, lungs, pancreas

Disciplines

Animal Experimentation and Research | Circulatory and Respiratory Physiology | Endocrine System Diseases | Endocrinology, Diabetes, and Metabolism | Lipids | Medical Pharmacology | Medicine and Health Sciences | Other Chemicals and Drugs | Translational Medical Research | Virus Diseases

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