Publications by Year: 2021
The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity.
Dysregulated glucagon secretion deteriorates glycemic control in type 1 and type 2 diabetes. Although insulin is known to regulate glucagon secretion via its cognate receptor (insulin receptor, INSR) in pancreatic alpha cells, the role of downstream proteins and signaling pathways underlying insulin's activities are not fully defined. Using in vivo (knockout) and in vitro (knockdown) studies targeting insulin receptor substrate (IRS) proteins, we compared the relative roles of IRS1 and IRS2 in regulating alpha cell function. Alpha cell-specific IRS1-knockout mice exhibited glucose intolerance and inappropriate glucagon suppression during glucose tolerance tests. In contrast, alpha cell-specific IRS2-knockout animals manifested normal glucose tolerance and suppression of glucagon secretion after glucose administration. Alpha cell lines with stable IRS1 knockdown could not repress glucagon mRNA expression and exhibited a reduction in phosphorylation of AKT Ser/Thr kinase (AKT, at Ser-473 and Thr-308). AlphaIRS1KD cells also displayed suppressed global protein translation, including reduced glucagon expression, impaired cytoplasmic Ca2+ response, and mitochondrial dysfunction. This was supported by the identification of novel IRS1-specific downstream target genes, Trpc3 and Cartpt, that are associated with glucagon regulation in alpha cells. These results provide evidence that IRS1, rather than IRS2, is a dominant regulator of pancreatic alpha cell function.