Research

Signal Transduction by Insulin and Mammalian Lectins

A. Insulin signaling and insulin resistance.

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 insulin sensitivity of target organs (e.g. muscle, adipose tissue and liver). Insulin resistance is defined as a failure of target organs to respond to insulin 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.

I. 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 which 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-2^5A) improves β cell function. Cytokine-treated islets overexpressing IRS-2^5A secreted significantly more insulin in response to glucose, compared to islets overexpressing IRS-2^WT. Moreover, transplantation of a limited number of islets overexpressing IRS-2^5A into Streptozotocin-induced diabetic mice (Fig. 1) restored their ability to respond to glucose loads. Taken together, these studies may open new pharmacological approaches to improve islets engraftment.

Fig. 1. Transplantation of islets overexpressing IRS-25A. Murine slets infected with an ‘empty’ vector control were transplanted under the left renal capsule of STZ-treated hyperglycemic syngeneic mouse. Immunohistochemistry, carried out 20 days post transplantation of the grafted islets reveal an overlap between GFP-expressing cells and insulin positive cells (C-renal capsule, G-grafted islets, K-kidney)

II. Another project in the lab focuses on Selective Serotonin Reuptake Inhibitors (SSRIs) which are common antidepressants. These drugs were found by us to act as inducers of insulin resistance. We could show that treatment of Min6, a β-cell line, with SSRIs activates IRS-kinases (e.g. JNK and GSK3β) that inhibit insulin signaling. Insulin-stimulated Tyr-phosphorylation of IRS-2 was decreased concomitantly with a reduction in Akt activity. Inhibition of IRS-2 action by SSRIs was associated with inhibition of glucose-stimulated insulin secretion in primary cultures of pancreatic islets. These results implicate IRS kinases, activated by SSRIs, as inhibitors of insulin secretion and beta cell function. Given that SSRIs promote insulin resistance while inhibiting insulin secretion, these drugs might accelerate the transition from an insulin resistant state to overt diabetes.

III. Pro-inflammatory cytokines are involved in inducing pancreatic b cell death, leading to the development of Type 1 diabetes. To elucidate the mechanisms underlying this process, a high throughput screen of 3850 mouse siRNAs was performed in cytokine-treated Min6 b cell line. Cells were transfected with the various siRNAs and then treated with a combination of TNFα, IL-1β, and IFNγ. Apoptotic and necrotic activities were monitored by assessing caspase-3/7 activity, cellular ATP content, and cell morphology. Several gene families were identified as promoting cytokine-induced b cell apoptosis (Fig. 2), the most prominent being the cytokine receptor proteins involved in intracellular trafficking, and the ubiquitin ligases. Conversely, deubiquitinating enzymes appeared to reduce cytokine-induced b cell apoptosis. The screen confirmed the involvement of genes such as IRF7, CEBPB and JAK2, previously implicated in cytokine-induced b cell death, and revealed the involvement of several novel genes, among them Fbox 27, Nedd-4 binding partner-1, CAMKK2β, and FOXP3 (forkhead-box P3) in this process. These results indicate that b cell death occurs via a combination of apoptotic and necrotic processes, mediated by novel as well as known genes.

Fig. 2. Distribution of Z-scores in individual siRNA libraries. The sorted Z-scores of the entire 3850 siRNAs screened were divided into 10 bins, each representing a range of Z-scores from lowest to highest. The distributions of Z-scores of the individual libraries were then compared to the overall distribution and were diagramed using MATLAB R2007a. Dark red represents siRNAs that had the lowest Z-score values, representing inhibited pro-apoptotic genes, while yellow represents siRNAs having high Z-scores, representing genes that are anti-apoptotic. Width of each section represents the fraction of siRNAs from the indicated library that harbor Z-scores in an indicated range.

B. Secretion and Action of Galectin-8, a mammalian lectin

Galectins are a family of mammalian lectins, implicated in cell adhesion, cancer and immunity. Our lab studies galectin-8, originally cloned by us. Galectin-8 is a secreted, integrin-binding protein that lacks a signal peptide suitable for ER/ Golgi mediated secretion. Therefore, it is assumed to be secreted by an atypical secretory mechanism. Studies conducted in the lab provide evidence that secretion of galectin-8 is mediated by the endocytic cycle, suggesting that internalization and secretion of galectin-8 are tightly coupled. Some of the components involved in secretion of galectin-8 were revealed. These include PKC, Ca²⁺ and Rab11. We could also demonstrate that secretagogues known to enhance neurotransmitters release, increases secretion of galectin-8. These findings suggest that part of the molecular machinery involved in galectin-8 secretion is shared with that of triggered exocytosis.

II. Another aspect of our work deals with the mode of action of galectin-8 on cancer cells. Galectin-8, known as Prostate Cancer Tumor Antigen-1 (PCTA-1) is high expressed in human prostate cancer. Recent findings in the lab revealed that galectin-8 triggers transcription of a unique set of genes, some of which are associated with bone remodeling and prostate cancer progression.