Probes for INSULIN

Thioredoxin-interacting protein (Txnip) has emerged as a key factor in pancreatic beta cell biology and its upregulation by glucose and diabetes contributes to the impairment in functional beta cell mass and glucose homeostasis. In addition, beta cell deletion of Txnip protects against diabetes in different mouse models. However, while Txnip is ubiquitously expressed, its role in pancreatic alpha cells has remained elusive. We therefore now generated an alpha cell Txnip knockout (aTKO) mouse and assessed the effects on glucose homeostasis. While no significant changes were observed on regular chow, after a 30-week high-fat diet, aTKO animals showed improvement in glucose tolerance and lower blood glucose levels compared to their control littermates. Moreover, in the context of streptozotocin (STZ)-induced diabetes, aTKO mice showed significantly lower blood glucose levels compared to controls. While serum insulin levels were reduced in both control and aTKO mice, STZ-diabetes significantly increased glucagon levels in control mice, but this effect was blunted in aTKO mice. Moreover, glucagon secretion from aTKO islets was >2-fold lower than from control islets, while insulin secretion was unchanged in aTKO islets. At the same time, no change in alpha cell or beta cell numbers or mass was observed and glucagon and insulin expression and content were comparable in isolated islets from aTKO and control mice. Thus, together the current studies suggest that downregulation of alpha cell Txnip is associated with reduced glucagon secretion and that this may contribute to the glucose-lowering effects observed in diabetic aTKO mice.

The majority of mammalian genome is composed of non-coding regions, where numerous long non-coding RNAs (lncRNAs) are transcribed. Although lncRNAs have been identified to regulate fundamental biological processes, most of their functions remain unknown, especially in metabolic homeostasis. Analysis of our recent genome-wide screen reveals that Gm15441, a thioredoxin-interacting protein (Txnip) antisense lncRNA, is the most robustly induced lncRNA in the fasting mouse liver. Antisense lncRNAs are known to regulate their sense gene expression. Given that Txnip is a critical metabolic regulator of the liver, we aimed to investigate the role of Gm15441 in the regulation of Txnip and liver metabolism.We examined the response of Gm15441 and Txnip under in vivo metabolic signals such as fasting and refeeding, and in vitro signals such as insulin and key metabolic transcription factors. We investigated the regulation of Txnip expression by Gm15441 and the underlying mechanism in mouse hepatocytes. Using adenovirus-mediated liver-specific overexpression, we determined whether Gm15441 regulates Txnip in the mouse liver and modulates key aspects of liver metabolism.We found that the expression levels of Gm15441 and Txnip showed a similar response pattern to metabolic signals in vivo and in vitro, but that their functions were predicted to be opposite. Furthermore, we found that Gm15441 robustly reduced Txnip protein expression in vitro through sequence-specific regulation and translational inhibition. Lastly, we confirmed the Txnip inhibition by Gm15441 in vivo (mice) and found that Gm15441 liver-specific overexpression lowered plasma triglyceride and blood glucose levels and elevated plasma ketone body levels.Our data demonstrate that Gm15441 is a potent Txnip inhibitor and a critical metabolic regulator in the liver. This study reveals the therapeutic potential of Gm15441 in treating metabolic diseases.

Free fatty acid receptor 1 (FFAR1) is highly expressed in enteroendocrine cells of the small intestine and pancreatic beta cells, where FFAR1 agonists function as GLP-1 and insulin secretagogues, respectively. Most efficacious are so-called second-generation synthetic agonists such as AM5262, which, in contrast to endogenous long-chain fatty acids are able to signal through both IP3/Ca2+ and cAMP pathways. Whereas IP3 signaling is to be expected for the mainly Gq-coupled FFAR1, the mechanism behind FFAR1-induced cAMP accumulation remains unclear, although originally proposed to be Gs mediated.When stimulated with AM5262, we observe that FFAR1 can activate the majority of the Gα proteins, except - surprisingly - members of the Gs family. AM5262-induced FFAR1-mediated transcriptional activation through cAMP response element (CREB) was blocked by the specific Gq inhibitor, YM253890. Furthermore, in Gq-deficient cells no CREB signal was observed unless Gq or G11 was reintroduced by transfection. By qPCR we determined that adenylate cyclase 2 (Adcy2) was highly expressed and enriched relative to the nine other Adcys in pro-glucagon expressing enteroendocrine cells. Co-transfection with ADCY2 increased the FFAR1-induced cAMP response 4-5-fold in WT HEK293 cells, an effect fully inhibited by YM253890. Moreover, co-transfection with ADCY2 had no effect in Gq-deficient cells without reintroduction of either Gq or G11. Importantly, although both AM5262/FFAR1 and isoproterenol/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR response was rescued by Gs transfection, whereas co-transfection with ADCY2 was required to rescue the FFAR1 cAMP response. In situ hybridization demonstrated a high degree of co-expression of ADCY2 and FFAR1 in enteroendocrine cells throughout the intestine. Finally, in the enteroendocrine STC-1 and GLUTag cell lines AM5262-induced cAMP accumulation and GLP-1 secretion were both blocked by YM253890.Our results show that Gq signaling is responsible not only for the IP3/Ca2+ but also the cAMP response, which together are required for the highly efficacious hormone secretion induced by second-generation FFAR1 agonists - and that ADCY2 presumably mediates the Gq-driven cAMP response.