Role Of Insulin Receptor Substrate-2 Research Articles

E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance.

SUMMARYPrevention or amelioration of declining β cell mass is a potential strategy to cure diabetes. Here, we report the pathways utilized by β cells to robustly replicate in response to acute insulin resistance induced by S961, a pharmacological insulin receptor antagonist. Interestingly, pathways that include CENP-A and the transcription factor E2F1 that are independent of insulin signaling and its substrates appeared to mediate S961-induced β cell multiplication. Consistently, pharmacological inhibition of E2F1 blocks β-cell proliferation in S961-injected mice. Serum from S961-treated mice recapitulates replication of β cells in mouse and human islets in an E2F1-dependent manner. Co-culture of islets with adipocytes isolated from S961-treated mice enables β cells to duplicate, while E2F1 inhibition limits their growth even in the presence of adipocytes. These data suggest insulin resistance-induced proliferative signals from adipocytes activate E2F1, a potential therapeutic target, to promote β cell compensation.

Abstract 2854: Adaptation of colorectal cancer cells to the brain microenvironment: the role of IRS2

Abstract Colorectal cancer (CRC) reflects the fourth most frequent etiology of brain metastasis (BM), with rising incidence. Yet, molecular mechanisms supporting the formation of these lesions from CRC are unknown. We aimed to explore drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed the FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence of insulin receptor substrate 2 (IRS2) gene amplification was noted in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. We subsequently constructed an in vitro system mimicking the brain microenvironment using cultured human astrocytes or their conditioned media (CM). Under these conditions using InSphero system, IRS2-overexpressed CRC cells survived better and formed larger 3D spheres. IRS2-silenced CRC cells showed a mirror image. Moreover, in an intracranial CRC surgical implantation BMs mouse model, IRS2-overexpressed CRC cells generated larger brain lesions, while silencing IRS2 in IRS2-amplified CRC cells dramatically decreased tumor outgrowth and survival. Interestingly, transcriptomic analysis revealed significant enrichment of the oxidative phosphorylation pathway by IRS2. Indeed, IRS2-expressing CRC cells showed increased mitochondrial activity using Seahorse extracellular flux analysis and glycolysis-independent viability. Furthermore, IRS2-expressing cells showed enhanced AKT phosphorylation and inhibition of PI3K or AKT using Alpelisib or iAKT, respectively, slightly decreased their proliferation in CM, suggesting that mechanisms additional to the AKT pathway may mediate IRS2 activity. The Wnt/β-catenin pathway was among the most significantly enriched pathways in the transcriptome. Indeed, IRS2-expressing cells had increased transcriptional activity of the β-catenin. In addition, iAKT or Alpelisib, and most significantly the IRS2 inhibitor (NT219), decreased the transcriptional activity of β-catenin in IRS2-expressing CRC cells, suggesting relevance of IRS2 in activating β-catenin. Moreover, NT219 demonstrated significant and dose-dependent inhibition of CRC cells viability. These data reveal, for the first time, the unique genomic profile of CRC BMs and imply the IRS2 role in promoting CRC BMs. These effects may be mediated, at least in part, by modulation of the AKT and β-catenin pathway. Given the molecular signature described, the approach to patients with BMs may be significantly impacted by agents such as NT219. Citation Format: Inbal Greenberg, Anat Klein, Rachel Grossman, Ethan Sokol, Eilam Yeini, Paula Ofek, Ronit Satchi-Fainaro, Bertrand Liang, Hadas Reuveni, Tami Rubinek, Ido Wolf. Adaptation of colorectal cancer cells to the brain microenvironment: the role of IRS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2854.

Abstract PO-003: Adaptation of colorectal cancer cells to the brain microenvironment: The role of IRS2

Abstract Brain metastases (BMs) from colorectal cancer (CRC) are currently the fourths leading cause of BMs, and their incidence is on the rise. Yet, mechanisms mediating the formation of BMs from CRC are unknown. We aimed to explore genomic drivers enabling tropism and adaptation of CRC cells to the brain environment and decipher mechanisms facilitating the process. We analyzed FoundationOne database, which contains genomic alterations data of cancer-related genes in over 16,000 human CRC primary and metastasis samples. Increased prevalence was noted in the insulin receptor substrate 2 (IRS2) gene, which was significantly amplified in BMs relative to primary tumors and other metastatic sites. IRS2 is a cytoplasmic adaptor mediating effects of insulin and IGF-1 receptors and is known to be involved in more aggressive behavior of different cancer types. In agreement with the genomic data, immunohistochemistry of human clinical samples showed increased expression of IRS2 protein in BMs. Next, we created an in-vitro system mimicking the brain microenvironment using human astrocytes or their conditioned media (CM). Under these conditions, IRS2-overexpressed CRC cells survived better and formed larger 3D spheres. IRS2-silenced CRC cells showed a mirror image. Furthermore, IRS2-overexpressed CRC cells generated larger brain lesions in an intracranial CRC BMs mice model. Interestingly, a transcriptomic analysis revealed significant enrichment of the AKT and β-catenin pathways by IRS2. Indeed, IRS2-amplified cells showed enhanced AKT phosphorylation and inhibition of PI3K or AKT using Alpelisib or Akt1/2 kinase inhibitor, respectively, effectively decreased their proliferation in CM. Importantly, IRS2-amplified cells had increased expression and transcriptional activity of the β-catenin, and AKT pathway inhibitors or IRS2 inhibitor (NT-219) had a more pronounced effect on the activity of β-catenin on those cells. These data indicate, for the first time, the unique genomic profile of CRC BMs and imply the IRS2 role in promoting CRC BMs. These effects may be mediated, at least in part, by modulation of the AKT and β-catenin pathway. These findings may pave the way for the development of novel therapeutic strategies to prevent BMs formation. Citation Format: Inbal Greenberg, Anat Klein, Rachel Grossman, Ethan Sokol, Eilam Yeini, Paula Ofek, Ronit Satchi-Fainaro, Hadas Reuveni, Tami Rubinek, Ido Wolf. Adaptation of colorectal cancer cells to the brain microenvironment: The role of IRS2 [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-003.

Insulin receptor substrate-2 (Irs2) in endothelial cells plays a crucial role in insulin secretion.

Endothelial cells are considered to be essential for normal pancreatic β-cell function. The current study attempted to demonstrate the role of insulin receptor substrate-2 (Irs2) in endothelial cells with regard to insulin secretion. Endothelial cell-specific Irs2 knockout (ETIrs2KO) mice exhibited impaired glucose-induced, arginine-induced, and glucagon-induced insulin secretion and showed glucose intolerance. In batch incubation and perifusion experiments using isolated islets, glucose-induced insulin secretion was not significantly different between the control and the ETIrs2KO mice. In contrast, in perfusion experiments, glucose-induced insulin secretion was significantly impaired in the ETIrs2KO mice. The islet blood flow was significantly impaired in the ETIrs2KO mice. After the treatment of these knockout mice with enalapril maleate, which improved the islet blood flow, glucose-stimulated insulin secretion was almost completely restored to levels equal to those in the control mice. These data suggest that Irs2 deletion in endothelial cells leads to a decreased islet blood flow, which may cause impaired glucose-induced insulin secretion. Thus, Irs2 in endothelial cells may serve as a novel therapeutic target for preventing and ameliorating type 2 diabetes and metabolic syndrome.

IRS-2 deficiency in macrophages promotes their accumulation in the vascular wall

The aim of this study was to investigate the role of insulin receptor substrate-2 (IRS-2) mediated signal in macrophages on the accumulation of macrophages in the vascular wall. Mice transplanted with IRS-2 −/− bone marrow, a model of myeloid cell restricted defect of IRS-2, showed accumulation of monocyte chemoattractant protein-1-expressing macrophages in the vascular wall. Experiments using cultured peritoneal macrophages showed that IRS-2-mediated signal pathway stimulated by physiological concentrations of insulin, not by IL-4, contributed to the suppression of monocyte chemoattractant protein-1 expression induced by lipopolysaccharide. Our data indicated that IRS-2 deficiency in macrophages enhanced their accumulation in the vascular wall accompanied by increased expression of proinflammatory mediators in macrophages. These results suggest a role for insulin resistance in macrophages in early atherosclerogenesis.

Expression of insulin receptor substrate-2 and its tyrosine phosphorylation in the hepatic tissue of chronic hepatitis B patients

To observe the expression of insulin receptor substrate-2 (IRS-2) mRNA and protein, and its tyrosine phosphorylation in hepatic tissue of chronic hepatitis B (CHB) patients, and to explore the role of IRS-2 on insulin resistance in CHB patients. Eighteen patients with CHB were included, and 6 individuals with normal liver function were enrolled as control. Based on the insulin resistance index determined by homeostasis model assessment (HOMA), CHB patients were further divided into CHB without insulin resistance group (<2.69, n=10) and CHB with insulin resistance group (>2.69, n=8). Hepatic tissues were harvested from all patients during operation or with liver biopsy. The mRNA expression of IRS-2 in liver tissue was assessed by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression of IRS-2 was detected by Western blotting. Immunoprecipitation and enhanced chemiluminescent technique were used to measure the tyrosine phosphorylation of IRS-2. In CHB without insulin resistance group, the mRNA expression (0.38+/-0.06), the protein expression (0.94+/-0.18) and the tyrosine phosphorylation (0.78+/-0.09) of IRS-2 in hepatic tissue were decreased, but without statistically significant difference (all P>0.05), as compared to those in control group (0.45+/-0.11, 0.99+/-0.20, 1.00+/-0.23, respectively). In CHB with insulin resistance group, the mRNA expression (0.26+/-0.08), the protein expression (0.67+/-0.11) and the tyrosine phosphorylation (0.63+/-0.14) of IRS-2 in hepatic tissue were significantly decreased compared with those of the control group with statistically significant difference (P<0.05 or P<0.01). The decreased expression of mRNA and protein and the reduced tyrosine phosphorylation of IRS-2 in CHB patients with insulin resistance inducing impairment of the insulin signal pathway may be one of the mechanisms underlying insulin resistance.

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.

Essential Role of Insulin Receptor Substrate-2 in Insulin Stimulation of Glut4 Translocation and Glucose Uptake in Brown Adipocytes

Insulin and insulin-like growth factor I signals are mediated via phosphorylation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study the metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals and characterized insulin action in these cells in vitro. Preadipocytes derived from both wild type and IRS-2 KO mice could be fully differentiated into mature brown adipocytes. In differentiated IRS-2 KO adipocytes, insulin-induced glucose uptake was decreased by 50% compared with their wild type counterparts. This was the result of a decrease in insulin-stimulated Glut4 translocation to the plasma membrane. This decrease in insulin-induced glucose uptake could be partially reconstituted in these cells by retrovirus-mediated re-expression of IRS-2, but not overexpression of IRS-1. Insulin signaling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3-kinase by 30% (p < 0.05) in the KO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependent phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not affected by the lack of IRS-2; however, there was a decrease in insulin stimulation of Akt associated with the plasma membrane. These results provide evidence for a critical role of IRS-2 as a mediator of insulin-stimulated Glut4 translocation and glucose uptake in adipocytes. This occurs without effects in differentiation, total activation of Akt and its downstream effectors, but may be caused by alterations in compartmentalization of these downstream signals.

Growth Hormone, Interferon-γ, and Leukemia Inhibitory Factor Utilize Insulin Receptor Substrate-2 in Intracellular Signaling

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.