The homeostatic control of blood glucose is determined by two major factors: the concentration of insulin in the circulation, which correlates with β cell function, and the sensitivity of target organs (e.g. muscle, adipose tissue and liver) to insulin. Insulin resistance is defined as a failure of target organs to respond to physiological insulin concentrations, thus leading to the development of diabetes, an ever-increasing epidemic of the 21st century. Ongoing studies in our lab focus on the molecular basis of insulin resistance and its effects on growth and survival of the pancreatic insulin-producing β-cells.
We could show that inducers of insulin resistance (e.g. pro-inflammatory cytokines) exploit phosphorylation-based negative feedback control mechanisms, to uncouple the insulin receptor (IR) from its downstream effectors, the IRS proteins (IRS-1 and IRS-2) and thereby terminate insulin signal transduction. Ser/Thr phosphorylation of IRS proteins was shown to be a pivotal player in the termination of insulin’s action in target organs. Indeed, we found that IRS proteins mutated at inhibitory Ser residues were resistant to the inhibitory effects of prolonged insulin treatment or to the action of inducers of insulin resistance. Accordingly, deletion of a specific Ser-rich-domain (we named DIDI) maintained the ability of IRS-1 to undergo ubiquitination while rendering it insensitive to insulin-induced proteasomal degradation. This deletion mutant of IRS-1 better maintained insulin signaling and insulin action. These results identify DIDI as a novel domain, required for insulin-induced proteasomal degradation of IRS-1 at a post-ubiquitination stage. Studies also focus on IRS-2 that plays a pivotal role in β cell function. We could show that a mutated form of IRS-2, in which five inhibitory Ser residues were replaced by Ala (IRS-25A), improves β cell function. Moreover, transplantation of a limited number of islets overexpressing IRS-25A into diabetic mice restored their ability to respond to glucose loads. These studies may open new pharmacological approaches to improve islets engraftment.
Pro-inflammatory cytokines are involved in the induction of pancreatic β cell death, leading to the development of Type 1 diabetes. To elucidate the mechanisms underlying this process, a high throughput screen of 3850 mouse and 750 human siRNAs was performed in cytokine-treated Min6 β cell line and primary human pancreatic islets. Several gene families were identified as promoting cytokine-induced beta cell apoptosis, the most prominent being the ubiquitin ligases and serine/threonine kinases. Conversely, deubiquitinating enzymes appeared to reduce apoptosis while protein phosphatases were mainly associated with lowering cellular reducing power. The screen suggested with a high confidence the involvement of several novel genes in cytokine-induced beta cell death. These included CAMKK2β, EPN3, FOXP3 and TM7SF3, an orphan seven transmembrane receptor. siRNAs to TM7SF3 promoted cytokine-induced death of MIN6 cells and human pancreatic islets, and abrogated insulin secretion in these cells. These findings implicate TM7SF3 as a potential new player involved in the inhibition of cytokine-induced death and in the promotion of insulin secretion from pancreatic beta cells. Another novel ‘hit’ identified in the screens was the deubiquitinating enzyme Otubain 2 (Otub2). Silencing of Otub2 expression increased caspase-3/7 activity both in Min6 cells and primary human islets treated with cytokines; inhibited insulin secretion and increased NF-kB activity. A third novel ‘hit’ was Nedd4 family interacting protein 1 (Ndfip1), an adaptor and activator of Nedd4-family ubiquitin ligases. Silencing of Ndfip1 inhibited cytokine-induced apoptosis of mouse and human pancreatic islets and promoted glucose-stimulated insulin secretion. These effects were associated with an increase in the cellular content of JunB, a potent inhibitor of ER stress and apoptosis. Silencing of Ndfip1 also increased the expression of ATF4, IRE-1α, and the spliced form of XBP that govern the unfolded protein response (UPR) and relieve cytokine-induced ER stress. Overexpression of Ndfip1 exerted opposite effects. These findings implicate Ndfip1 in the degradation of JunB; inhibition of the UPR and insulin secretion; and promotion of cytokine-induced death of pancreatic β-cells. Collectively, the above studies indicate that TM7SF3, Otub2, and Ndfip1.may be considered as novel targets for therapeutic intervention in cases of type 1 diabetes and islet transplantation.
