The role of cocaine- and amphetamine-regulated transcript in endocrine cells
Type 2 diabetes (T2D) is a metabolic disease characterized by insufficient insulin secretion and insulin resistance. Disturbed beta-cell function is the final culprit that leads to the development of T2D. In addition, impaired incretin effect is likely a part of the pathogenesis of T2D. The aim of this thesis was to study the role of CART in beta-cells and incretin producing L- and K-cells.
Endogenous beta-cell CART was found to regulate insulin secretion and production, ATP levels and beta-cell exocytosis in INS-1 (832/13) cells. CART silencing (KD) decreased expression of genes important for exocytosis, glucose sensing, and insulin processing, likely as a consequence of reduced expression of beta-cell transcription factors Mafa, Pdx-1, Isl1, NeuroD1, Nkx2.2 and Nkx6.1. Moreover, in human islets, CARTPT expression correlated with insulin, exocytosis genes and beta-cell transcription factors. To increase our understanding of the function of CART in beta-cells, we performed RNAseq on CART-silenced INS-1 (832/13) cells. Differential expression analysis revealed that CART KD affected expression of 25.5% of all detected genes, including genes with important roles for insulin secretion and exocytosis. The most strongly upregulated and downregulated genes after CART KD were AABR07068253.1, Jun, Srp14 and Adam11, Pak3, Ppp1r17 respectively. Stxa1, Gnas and Stxbp1 were the top differentially expressed genes related to insulin secretion. In a follow-up study, we established the role of one of the strongest CART-regulated genes, SCRT1 in beta-cell function. SCRT1 expression was found in rodent and human beta-cells. SCRT1 was translocated from the nucleus to the cytosol in diabetic DEX rats and SCRT1 mRNA expression was reduced by increasing glucose concentrations in vitro. SCRT1 KD in INS-1 (832/13) cells resulted in decreased insulin expression and secretion, as well as reduced expression of Tcf7l2, Pdx-1, Isl1, Neurod1 and Mafa. In human islets, SCRT1 expression correlated with insulin, glucagon, beta-cell transcription factors and exocytotic genes. Finally, we established CART expression in K- and L-cells in the human duodenum and jejunum and CART plasma levels were increased after a meal in humans. In incretin-producing GLUTag and STC-1 cells, CART mRNA was increased by fatty acids and GIP. In addition, CART KD in GLUTag cells reduced GLP-1 expression and secretion. CART also increased GLP-1 and GIP secretion during an oral glucose-tolerance test in vivo in mice. Collectively, our data imply that CART is an important regulator of insulin and incretin-producing cell function and glucose homeostasis. Thus, the therapeutic potential of CART-based therapy for treatment of T2D should be evaluated.