Phenotyping of single pancreatic islets reveals a crosstalk between proinsulin intracellular alteration, ER stress and loss of β cell identity in impaired glucose tolerant and type 2 diabetic patients

Type 2 diabetes mellitus is a heterogeneous group of metabolic diseases characterized by increased levels of blood glucose due to insulin resistance and alteration of insulin secretion by pancreatic β cells. Recent studies suggest that β cell loss in T2D results from endoplasmic reticulum stress which can cause an alteration in the processing of PI to INS. In particular, it has been reported that the increased circulating levels of PI and elevated PI/INS ratio are well-known abnormalities in type 2 diabetes. Several studies have hypothesized that an elevated PI/INS ratio was caused by increased secretory demand on β cells due to insulin resistance and hyperglycemia. However, an in-depth analysis of PI/INS expression pattern inside the pancreatic islets during metabolic alteration is not entirely clear. For this reason, the first aim of this work was: (i) to analyze PI and INS expression pattern in pancreatic islets of tissue biopsies of patients undergoing partial pancreatectomy (PP) with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and type 2 diabetes, in order to explore the in-situ alterations that occur in islets during metabolic stress. Given the enormous heterogeneity between pancreatic islets of different donors but also between islets belonging to the same donors, the second aim of project thesis was: (ii) to perform single islet phenotyping by characterizing histological and molecular aspects, in order to investigate the underlying molecular mechanisms driving β cell failure at single islet level with the aim to specifically determine the cues leading to intracellular alteration of PI and insulin during metabolic stress. In order to explore the alterations that occur in PI and INS staining pattern in pancreatic islets, OGTT (Oral Glucose Tolerant Test) was performed in n=19 patients scheduled for PP, classified into n=5 NGT, n=9 IGT and n=5 T2D. Frozen sections of pancreatic tissue biopsy were stained for INS and PI through double immunofluorescence staining. Image analysis was performed through Volocity software. In-situ staining measurements were correlated with patients’ clinical parameters. Subsequently, we evaluated the expression of ER stress genes and β cell mature phenotype-related genes in microdissected pancreatic islets collected from n=4 NGT, n=7 IGT and n=4 T2D patients. Given the high heterogeneity among pancreatic islets we also performed a single islets phenotyping analysis in n=88 islets from n=3 NGT, n=3 IGT and n=3 T2D patients. Results showed that in pancreatic islets of IGT and T2D patients, PI intracellular localization was altered. The colocalization coefficient INS-PI as well as PI levels and PI/INS ratio gradually increased from NGT to IGT and T2D pancreatic islets and were related to the loss of glucose tolerance and impaired β-cell function. PDIA1, GRP78 and XBP1, genes involved in unfolded protein response (UPR) were significantly upregulated in pancreatic islets of IGT and T2D patients compared to NGT and were positively correlated with in-situ PI/INS ratio and colocalization, with in-vivo measurements of glucose intolerance and β cell functional reduction. Single islets phenotyping approach revealed a progressively increased heterogeneity from NGT to IGT and T2D patients. Of note, in-situ PI/INS ratio and colocalization were positively correlated with the expression of UPR genes and negatively with those associated to β cell identity. In conclusion, we demonstrated that: (i) the in-situ expression patterns of proinsulin and insulin are altered in prediabetic and diabetic patients reflecting their metabolic profiles; (ii) the molecular mechanism behind this in-situ alteration involves the ER stress and the establishment of UPR within the β cell; (iii) single islets phenotyping analysis in T2D pancreas reveals a high heterogeneity among pancreatic islets in terms of ER stress and β cell differentiation profile.