Direct protection and rescue of beta cell function in diabetes

Background: Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia, with more than 500 million people affected worldwide. With rare exceptions, diabetes mellitus is due to the inability of pancreatic islet β cells to produce enough insulin to meet the body’s needs, caused by the interplay of both genetic and environmental factors. Type 1 diabetes (T1D), which accounts for 5-10% of cases, begins more often in childhood and is characterized by autoimmune-based β cells destruction. The most common form of diabetes (80-90% of all cases) is type 2 (T2D), which is typical of adults and is generally associated with overweight/obesity. The key role of β cell dysfunction, usually due to metabolic and/or pro-inflammatory stress, in the onset and progression of any form of diabetes is well recognized. More recently, β cell functional recovery in diabetes has been observed in a few studies, which can contribute to disease remission. Thus, preservation and restoration of β cell function represent crucial goals in diabetes therapy. However, strategies on how to pursue these tasks are still largely undefined. This doctoral thesis aims to assess human β cell capability to recover from functional damage induced by T1D-like or T2D conditions, explore the molecular mechanisms involved, and test new strategies for protection and/or rescue of β cell function. Aims: 1. To assess if human β cells exposed ex vivo to pro-inflammatory cytokines and an increased glucose concentration, mimicking islet microenvironment at T1D onset, can spontaneously recover their function after stressors removal and the possible mechanisms involved; 2. To test a new strategy for human β cell functional protection from prolonged pro-inflammatory cytokines exposure, mimicking a less early stage of T1D, by drug repurposing of metformin, a drug commonly used in T2D treatment; 3. To assess if human β cells from subjects affected by T2D can spontaneously recover their function ex vivo, the possible mechanisms involved and to test strategies for induction of β cell functional recovery by drug repurposing. Materials and methods: 1. Human islets from five non-diabetic (ND) organ donors were exposed to pro-inflammatory cytokines (Cyt: 50 U/ml interleukin-1β, IL-1β, plus 1000 U/ml interferon-γ, INF-γ) at 5.5 (as in control) or 11.1 mM glucose (Cyt+G) for 24 h, followed by 72 h culture in normal medium (5.5 mM glucose, washout). Insulin secretion (IS) was assessed at 3.3 and 16.7 mM G, and the ratio between IS at 16.7 and 3.3 mM glucose (insulin stimulation index, ISI) was used to evaluate β cell function. Islet transcriptomes were evaluated by RNA-seq. 2. Human islets from fourteen ND organ donors exposed to Cyt for 48 h, with or without 2.4 µg/ml metformin. Insulin secretion and caspase 3/7 activity were assessed, and shotgun label free proteomics was performed. Western blot was also carried out for technical validation. 3. Human islets from fifteen ND and twenty-one T2D organ donors were studied shortly after isolation (“basal”) and following an additional period of culture in normal medium (5.5 mM glucose, “cultured”). Insulin secretion was studied, and transcriptome was assessed in subgroups of T2D islets based on glucose responsiveness and culture conditions; insulin secretion changes (“cultured” vs “basal”) were correlated to transcriptome traits; and potential therapeutic compounds that mimic gene signatures of recovered β cell function in T2D islets were sought. Finally, selected validation experiments were performed on a rat β cell line. Results: 1. Cytokines increased insulin secretion by two-fold at 3.3 and 16.7 mM glucose, with an ISI similar to control islets, and this persisted after washout. Cyt+G decreased ISI by >50%, which recovered after washout, due to better insulin secretion at 16.7 mM glucose. Compared to control islets, Cyt and Cyt+G induced, respectively, 3924 and 4203 differentially expressed genes (DEG). Of these, 3,281 were in common between the two comparisons, all regulated in the same direction. In comparison with Cyt alone, the presence of glucose up-regulated specific pathways linked to mammalian target of rapamycin complex 1 (mTORC1) signaling and unfolded protein response. After washout, islets previously treated with Cyt and Cyt+G showed 2506 and 3137 DEG, respectively. These genes identified negatively enriched pathways involved in inflammation, immune response, and cell death in washed out versus Cyt exposed islets. In islets treated with Cyt+G, after washout, we observed positively (e.g. glycolysis/gluconeogenesis, metabolic pathways) and negatively enriched pathways, the latter linked to inflammation, immune response, cell death, and endoplasmic reticulum stress. 2. Met prevented the reduction of ISI and the increase of caspase 3/7 activity induced by cytokine exposure. Proteomics analysis identified more than 3000 proteins in human islets. Cytokines significantly altered 244 proteins (145 up- and 99 downregulated), while, in the presence of Met, cytokines modified 231 proteins (128 up- and 103 downregulated). There were 212 differentially expressed proteins in common. Among the proteins inversely regulated in the two conditions, there were proteins involved in vesicle motility, defense from oxidative stress (e.g. peroxiredoxins, PRDX2 and PRDX5), metabolism, protein synthesis (e.g. 40S ribosomal proteins and eukaryotic translation initiation factor 4E), glycolysis and its regulation, cytoskeletal proteins and proteins interacting with the cytoskeleton. Met inhibited pathways linked to inflammation, immune reactions, mammalian target of rapamycin (mTOR) signaling and cell senescence. Some of the changes were confirmed by Western blot. 3. Insulin secretion was lower with T2D islets compared to ND, with both “basal” and “cultured” cells. However, ISI improved thirteen out of twenty-one T2D islet preparations after culture. Ameliorated β cell glucose responsiveness was accompanied by changes in the expression of 438 genes, many of which were involved in metabolic or inflammatory processes. Of them, 123 were significantly correlated with changes in ISI. Drug repurposing analysis for β cell functional recovery predicted several chemicals, including mTOR inhibitors and anti-inflammatory drugs, such as JAK inhibitors. Baricitinib, a JAK1/2 inhibitor, which preserves β cell function in patients with new-onset T1D, protected a rat β cell line from functional impairment and, in a case, cell death, induced by metabolic and pro-inflammatory stress, potentially relevant in T2D milieu. By super-resolution imaging microscopy, baricitinib showed also to counteract lipotoxic induced alteration of mitochondria morphology, and, possibly, function. Conclusion: These results show that, in certain conditions, β cell functional damage induced by both T1D-like and T2D islet microenvironment is reversible and β cell functional recovery is associated to specific transcriptomics traits, linked to restoration of metabolic processes and reduced inflammation, immune response, and endoplasmic reticulum stress. New effective strategies for β cell protection, based on drug repurposing, are also presented, such as the exploitation of metformin in T1D-like pro-inflammatory condition and baricitinib in T2D. This data demonstrates that β cell functional restoration and protection are indeed possible, encouraging a β cell oriented pharmacological treatment for both T1D and T2D.