Role Of Insulin Receptor Substrate Research Articles

Roles of Insulin Receptor Substrates (IRS) in renal function and renal hemodynamics.

We have reported previously that renal hemodynamic abnormalities exist in the prediabetic stage of type II diabetic rats. At this prediabetic stage these rats have hyperinsulinemia, insulin resistance and metabolic syndrome. It is well known that insulin resistance is frequently associated with renal abnormalities, but the mechanism underlying this association has remained speculative. Although insulin is known to modify renal hemodynamics, little is known about the roles of insulin receptor substrates (IRS1, IRS2) in the renal actions of insulin. To address this issue, the effects of insulin on renal function and renal hemodynamics were investigated in C57BL/6 (WT: wild type), insulin receptor substrate 1- knockout (IRS1–/–), and IRS2-knockout (IRS2–/–) mice. IRS2–/–mice had elevated glucose level as expected. 24-h urine collections and serum creatinine revealed that creatinine clearance did not significantly differ between these groups. Albuminuria was found in IRS1–/–and IRS2–/–groups. We examined the effects on the IRS during the administration of Losartan, which is widely used for diabetic nephropathy. After the administration of Losartan the IRS displayed improved renal hemodynamics. Moreover, the subjects were also given Pioglitazone, which improves insulin resistance. Losartan significantly reduced albuminuria in both groups. Pioglitazone also showed similar results. We assessed the autoregulatory responses of the total renal blood flow (RBF), the superficial (SBF) and the deep renal cortical blood flow (DBF) with stepwise reductions of renal perfusion pressure (RPP), which was induced by a manual clamp on the abdominal aorta. During the clamp induced reductions of the RPP by 10 to 20mm HG, RBF, SBF and the DBF fell significantly more in the IRS1 and IRS2 than in the WT mice. Furthermore micropuncture studies showded that compared to the WT tubuloglomerular feedback (TGF) responses of the stop flow pressure (Psf) were reduced in both the IRS1 -/- and IRS2 -/-. The results of the IRS1 and IRS2 mice displayed the pressence of hemodynamic abnormalities. Losartan and Pioglitazone have shown the potential to improve these abnormalities. In conclusion the results indicate that IRS plays a major role in the stimulation of renal functions and renal hemodynamics in type type II diabetes.

Catch-Up Growth in Zebrafish Embryo Requires Neural Crest Cells Sustained by Irs1 Signaling

Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as "catch-up" growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1-mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia.

Role of insulin receptor substrates in the progression of hepatocellular carcinoma

Several cellular signaling pathways, including insulin/IGF signaling, are known to be activated in hepatocellular carcinoma (HCC). Here, we investigated the roles of insulin receptor substrate (Irs) 1 and Irs2, both of which are the major molecules to be responsible for transducing insulin/IGF signaling in the liver, in the development of HCC by inducing chemical carcinogenesis using diethylnitrosamine (DEN) in mice. The Irs1 mRNA and protein expressions were upregulated in the tumors, along with enhanced insulin signaling. Liver-specific Irs1-knockout (LIrs1KO) mice exhibited suppression of DEN-induced HCC development, accompanied by reduced cancer cell proliferative activity and reduced activation of Akt. Gene expression analyses revealed that the tumors in the DEN-treated LIrs1KO mice showed modest metabolic alterations during hepatocarcinogenesis as well as decreased inflammation and invasion potentials. On the other hand, liver-specific Irs2-knockout (LIrs2KO) mice showed a similar pattern of HCC development to the DEN-treated control wild-type mice. Based on the knowledge that Wnt/β-catenin signaling is activated in HCC, we focused on Wnt/β-catenin signaling and demonstrated that Irs1 expression was induced by Wnt3a stimulation in the primary hepatocytes, associated with insulin-stimulated Akt activation. These data suggest that upregulated Irs1 by Wnt/β-catenin signaling plays a crucial role in the progression of HCC.

The role of insulin receptor substrate (IRS) proteins in oncogenic transformation.

Insulin Receptor Substrate (IRS) proteins are the main cytoplasmic adaptor molecules involved in transducing extracellular signals from receptors to downstream proteins. This protein family have pivotal roles on maintenance, distribution and regulation of signaling networks. Since IRS1/2 interact with and transmits signals from the receptors of insulin, Insulin Like Growth Factor 1 (IGF1), prolactin, growth hormone (GH), leptin, Vascular Endothelial Growth Factor (VEGF), TrkB, ALK and integrins this promoted scientist to think that IRS1 may have functions in cell proliferation, tumorigenesis and metastasis. Therefore, over the past decade, studies on IRS proteins and their functions in cancer has been increased and these studies provided valuable results claiming the involvement of IRS1/2 in cancer development. In this review, we discuss the function and contributions of IRS1 and IRS2 in development of breast cancer.

Insulin signaling pathway in the oriental fruit fly: The role of insulin receptor substrate in ovarian development

Insulin signaling pathways have integral roles in regulating organ growth and body size of insects. Here, we identified and characterized six insulin signaling pathway components—InR, IRS, PI3K92E, PI3K21B, Akt, and PDK—from Bactrocera dorsalis. Quantitative real-time polymerase chain reaction was used to establish gene expression profiles for the insulin signaling pathway components for different developmental stages and tissues, and in response to 20-hydroxyecdysone (20E) and starvation. IRS, PI3K92E, and PI3K21B were highly expressed in the head, while InR, Akt, and PDK were most abundant in Malpighian tubules. Both IRS and PI3K92E were highly expressed during the larval–pupal and pupal–adult transition, while the remaining four genes were highly expressed only during the pupal–adult transition. Following initial exposure to 20E, the expression levels of most genes were significantly decreased. However, the expression levels of IRS, PI3K92E, and PI3K21B were significantly increased at 8 and 12h post-treatment compared with the control. Moreover, we found that most insulin signaling pathway genes in B. dorsalis were up-regulated in response to starvation, but decreased when re-fed. On the contrary, transcript levels of PI3K21B decreased significantly during starvation. Furthermore, injection of IRS dsRNA into adult females significantly reduced IRS transcript levels. Suppression of IRS expression inhibited ovarian development, and the average ovary size was reduced by 33% compared with the control. This study provides new insight into the roles of insulin signaling pathway components in B. dorsalis, and demonstrates an important role for IRS in ovarian development.

Role of insulin receptor substrate-1 for diethylnitrosamine plus high-fat diet-induced hepatic tumorigenesis in mice.

We investigated the role of insulin receptor substrate (Irs)-1 for diethylnitrosamine (DEN) plus high-fat (HF) diet-induced hepatic tumorigenesis in mice. We gave DEN by intraperitoneal injection at the dose of 80mg/kg to 18-week-old wild-type (WT) and Irs1-knockout (Irs1 (-/-) ) mice, which were fed a HF diet from 8weeks-of-age until they were killed (52weeks). The Irs1 (-/-) mice showed significantly lower plasma alanine aminotransferase levels, triglyceride contents in the liver and also lower expression levels of the genes encoding inflammatory cytokines than the WT mice. The incidence of DEN plus HF diet-induced hepatic tumors was 71.4% in the WT mice, whereas it was just 14.3% in the Irs1 (-/-) mice. The present study showed that Irs1 played an important role in DEN plus HF diet-induced hepatic tumorigenesis.

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Downregulation of IRS-1 promotes metastasis of head and neck squamous cell carcinoma

Lymph node metastasis is responsible for the high morbidity of head and neck squamous cell carcinoma (HNSCC). To date, the role of insulin receptor substrate1 (IRS-1) in tumorigenesis and metastasis of head and neck cancer has not been elucidated. In this study, we found a negative correlation of IRS-1 expression with tumor metastasis both in human tissue samples and in cell lines. Furthermore, we found that knockdown of IRS-1 expression enhanced cell invasive potency and induced EMT in parallel with upregulation of miR-9 expression. We propose that IRS-1 suppresses metastasis of head and neck cancer possibly through miR-9.

Depletion of insulin receptor substrate 2 reverses oncogenic transformation induced by v-src

To investigate the role of insulin receptor substrate 2 (IRS-2) in oncogenic transformation induced by v-src. IRS-2 gene was silenced using small interfering RNAs (siRNAs). Nuclear translocation and interaction of IRS-2 with v-src was determined using subcellular fractionation, confocal microscopy, and immunoprecipitation. The activity of the cyclin D1 promoter and r-DNA promoter was measured with a luciferase assay. Depletion of IRS-2 inhibited R-/v-src cell growth and reverse the oncogenic transformation. IRS-2 bound to src via its two PI3-K binding sites, which are critical for activities involved in the transformation. Nuclear IRS-2 occupied the cyclin D1 and rDNA promoters. The combination of IRS-2 and v-src increased the activity of the two promoters, especially the rDNA promoter. Depletion of insulin receptor substrate 2 could reverse oncogenic transformation induced by v-src.

Upregulation of IRS-1 Expression in Goto-Kakizaki Rats Following Roux-en-Y Gastric Bypass Surgery: Resolution of Type 2 Diabetes?

Type 2 diabetes mellitus (T2DM) is an endocrine disorder that is rapidly growing in prevalence within China and throughout the world. Roux-en-Y gastric bypass (RYGB) surgery, widely used in the treatment of obesity, has been recognized as an effective and long-term treatment for T2DM in recent years. However, the underlying mechanisms responsible for glycemic control remain unclear. This study was designed to investigate the roles of insulin receptor substrates (IRSs) in glucose tolerance and insulin resistance following RYGB surgery. Goto-Kakizaki (GK) rats, a model of T2DM, were randomly allocated into three groups: RYGB surgery, sham surgery, and control (10 animals/group). Wistar rats were also used as non-diabetic control. Daily food intake, body weight, glucose and insulin were measured pre- and post-operatively. Insulin receptor substrate 1 (IRS-1) and insulin receptor substrate 2 (IRS-2) content, the main subtypes of IRSs, were measured in skeletal muscle, adipose tissue and liver using western immunoblot analyses on postoperative day 28. Following surgery, RYGB-treated rats showed markedly improved oral glucose tolerance, as judged by lower peak and area-under-the-curve glucose values (p < 0.01 vs. GK or GK sham). Improved insulin resistance was also observed in RYGB-treated rats. Western immunoblot analyses showed that IRS-1 and its phosphorylation levels were significantly increased in skeletal muscle and adipose tissues in RYGB group (p < 0.01 vs. GK or GK sham), whereas IRS-2 levels were downregulated in liver. These findings suggest that improvements in glucose tolerance and insulin resistance following RYGB surgery are associated with upregulation of IRS-1.

Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.

Adaptor Proteins as Targets for Cancer Prevention

This perspective on the report by McCampbell et al. in this issue of the journal (beginning on page 290) addresses the role of insulin receptor substrate (IRS) proteins in cancer progression. The IRS proteins link many cell-surface receptors to signal transduction pathways. Activation of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin axis normally results in serine phosphorylation and subsequent downregulation of these adaptor proteins. The authors show that changes in the negative feedback regulation of IRS proteins is associated with the progression of endometrial epithelial cells to hyperplasia and cancer. Therefore, understanding the function of adaptor proteins could provide additional strategies for cancer prevention and treatment.

Roles of Insulin Receptor Substrates (IRS) in renal function and renal hemodynamics.

We have reported previously that renal hemodynamic abnormalities exist in the prediabetic stage of type II diabetic rats. At this stage these rats have insulin resistance. It is well known that insulin resistance is frequently associated with renal abnormalities, but the mechanism underlying this association has remained speculative. Although insulin is known to modify renal hemodynamics, little is known about the roles of insulin receptor substrates (IRS1, IRS2) in the renal actions of insulin. To address this issue, we investigated in wild type (WT), IRS1‐deficient (IRS1‐/‐), and IRS2‐deficient (IRS2‐/‐) mice. IRS2‐/‐ mice had elevated blood pressure and glucose level as expected. 24‐h urine collections revealed that creatinine clearance did not significantly differ between these groups. Fractional excretion of sodium was significantly lower in IRS1‐/‐ and IRS2‐/‐ compared to WT mice. Albuminuria was found in IRS1‐/‐ and IRS2‐/‐ groups. We assessed autoregulatory responses of total renal blood flow (RBF), and of superficial (SBF) and deep renal cortical blood flow (DBF) to stepwise reductions of renal perfusion pressure (RPP) induced by a manual clamp on the abdominal aorta. RBF, SBF and DBF fell significantly more in IRS1‐/‐ and IRS2‐/‐ than in WT mice. These results indicate that IRS plays a major role in the stimulation of renal functions and renal hemodynamics in type II diabetes.

Critical Role for Hypothalamic mTOR Activity in Energy Balance

The mammalian target of rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.

IRS Cancer Connection

Two groups (Dearth et al . and Ma et al .) examined the role of insulin receptor substrate (IRS) in mouse mammary gland tumorigenesis and metastasis. Dearth et al . used transgenic mice forced to overexpress IRS-1 or IRS-2, or an immortalized but nontumorigenic cell line (MCF-10A) also forced to overexpress one of these two proteins. In the MCF-10A cells, overexpression of either protein caused an increase in phosphorylation (and activation) of Akt and an increase in proliferation in response to insulin growth factor-1 (IGF-1). Only the IRS-1-overexpressing cells showed enhanced phosphorylation of extracellular signal-related kinases 1 and 2. Both IRS proteins disrupted acinar formation in three-dimensional Matrigel cultures, and both IRS proteins coimmunoprecipitated with β-catenin. In the transgenic mice, there was no effect on lactational capacity, but both groups of mice formed tumors in the mammary glands. The IRS-2-overexpressing mice had more rapid tumor formation than did the IRS-1-overexpressing mice; however, IRS-2 was overexpressed to a greater extent than was IRS-1. Both groups exhibited lung metastasis. The histology of the tumors was quite diverse, and many contained cells that were highly differentiated, which is similar to phenotypes associated with activation of β-catenin. In the tumors from the IRS-1- or IRS-2-overexpressing mice, β-catenin coimmunoprecipitated with the IRS proteins, and there was increased abundance of cyclin D, which is encoded by a β-catenin target gene. In the overexpressing MCF-10A cells, IGF-1 stimulated the association between IRS-1 or IRS-2 and β-catenin. Ma et al . examined the different functions of IRS-1 and IRS-2 in tumorigenesis and metastasis by generating tumors in IRS-1-deficient mice using polyoma virus middle-T (PyV-MT:: Irs1 −/− ) and comparing these tumors to those formed in wild-type mice (PyV-MT) or in IRS-2-deficient mice (PyV-MT:: Irs2 −/− ). It was previously known that mammary tumors formed in PyV-MT:: Irs2 −/− mice exhibited decreased metastasis. The tumors from the PyV-MT:: Irs1 −/− mice showed increased lung metastasis compared with PyV-MT mice. This effect was intrinsic to the PyV-MT:: Irs1 −/− tumors, because the tumors grew faster and showed increased lung metastasis even when injected into wild-type mice. The PyV-MT:: Irs1 −/− tumors showed elevated microvascular density compared with the PyV-MT tumors and the PyV-MT:: Irs1 −/− tumors, and serum from these mice had increased concentrations of vascular endothelial growth factor A (VEGF-A). The tumors from the PyV-MT:: Irs1 −/− mice also showed increased signaling through the mTOR pathway, which regulates protein production and may explain the increase in VEGF-A production. The tumors from the PyV-MT:: Irs1 −/− mice also showed decreased abundance of apoptotic cells compared with tumors from the PyV-MT mice. IRS-2 abundance and tyrosine phosphorylation were increased in the tumors from the PyV-MT:: Irs1 −/− mice, and there was increased interaction between IRS-2 and the p85 regulatory subunit of phosphinositide 3-kinase, suggesting that in the absence of IRS-1, signaling through IRS-2 is enhanced. Further, these results suggest that IRS-1 may have an inhibitory effect of tumor metastasis, yet promote tumor formation and growth. Metastatic tumors from PyV-MT:: Irs2 −/− or PyV-MT mice had increased serine phosphorylation of IRS-1, which prevents tyrosine phosphorylation and thereby inhibits IRS-1 function. Small interfering RNA (SiRNA) used to decrease IRS-1 in cells from PyV-MT tumors increased IRS-2 abundance in those cells, suggesting that IRS-1 may inhibit IRS-2 signaling, and siRNA of IRS-2 in cells from PyV-MT:: Irs1 −/− decreased the phosphorylation of S6 kinase, a marker for activation of the mTOR pathway. Thus, it appears that IRS-1 and IRS-2 antagonize each other's activity and that inactivation of IRS-1 may contribute to the increased metastatic potential of mammary tumors. R. K. Dearth, X. Cui, H.-J. Kim, I. Kuiatse, N. A. Lawrence, X. Zhang, J. Divisova, O. L. Britton, S. Mohsin, D. C. Allred, D. L. Hadsell, A. V. Lee, Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2. Mol. Cell. Biol. 26 , 9302-9314 (2006). [Abstract] [Full Text] Z. Ma, S. L. Gibson, M. A. Byrne, J. Zhang, M. F. White, L. M. Shaw, Suppression of insulin receptor substrate 1 (IRS-1) promotes mammary tumor metastasis. Mol. Cell. Biol. 26 , 9338-9351 (2006). [Abstract] [Full Text]

Roles of Insulin Receptor Substrates in Insulin-Induced Stimulation of Renal Proximal Bicarbonate Absorption

Insulin resistance is frequently associated with hypertension, but the mechanism underlying this association remains speculative. Although insulin is known to modify renal tubular functions, little is known about roles of insulin receptor substrates (IRS) in the renal insulin actions. For clarifying these issues, the effects of insulin on the rate of bicarbonate absorption (JHCO3-) were compared in isolated renal proximal tubules from wild-type, IRS1-deficient (IRS1-/-), and IRS2-deficient (IRS2-/-) mice. In wild-type mice, physiologic concentrations of insulin significantly increased JHCO3-. This stimulation was completely inhibited by wortmannin and LY-294002, indicating that the phosphatidylinositol 3-kinase pathway mediates the insulin action. The stimulatory effect of insulin on JHCO3- was completely preserved in IRS1-/- mice but was significantly attenuated in IRS2-/- mice. Similarly, insulin-induced Akt phosphorylation was preserved in IRS1-/- mice but was markedly attenuated in IRS2-/- mice. Furthermore, insulin-induced tyrosine phosphorylation of IRS2 was more prominent than that of IRS1. These results indicate that IRS2 plays a major role in the stimulation of renal proximal absorption by insulin. Because defects at the level of IRS1 may underlie at least some forms of insulin resistance, sodium retention, facilitated by hyperinsulinemia through the IRS1-independent pathway, could be an important factor in pathogenesis of hypertension in insulin resistance.

The role of insulin receptor substrate 2 in hypothalamic and β cell function

Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in beta cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and beta cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced beta cell mass. Overt diabetes did not ensue, because beta cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced beta cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in beta cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis.

The role of insulin receptor substrate 2 in hypothalamic and β cell function

Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in β cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and β cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced β cell mass. Overt diabetes did not ensue, because β cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced β cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in β cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis.

Insulin-induced cell cycle progression is impaired in chinese hamster ovary cells overexpressing insulin receptor substrate-3.

To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.

Differential roles of insulin receptor substrates in brown adipocyte differentiation.

Insulin promotes adipocyte differentiation via a complex signaling network involving multiple insulin receptor substrates (IRSs). In cultured brown preadipocytes, expression of IRS-1 and IRS-2 mRNAs and proteins was at relatively high levels before and after differentiation into mature fat cells, while IRS-3 transcript was not detectable in preadipocytes but increased during the course of differentiation, and IRS-4 mRNA was barely detected in both states. To determine more precisely the roles of various IRS proteins in adipogenesis, we established and characterized brown preadipocyte cell lines from wild-type and IRS knockout (KO) animals. While wild-type, IRS-2 KO, and IRS-4 KO cells fully differentiated into mature adipocytes, IRS-3 KO cells showed a moderate defect in differentiation and IRS-1 KO cells exhibited a severe defect in the process. Cells lacking both IRS-1 and IRS-3 completely failed to differentiate. Expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha, fatty acid synthase, glucose transporter 4, and the transcription factor signal transducer and activator of transcription 5, as well as the brown-fat-specific markers PPARgamma coactivator 1 alpha and uncoupling protein 1, mirrored the differentiation pattern. Reconstitution of the IRS-1 KO cells with IRS-1 and IRS-4, but not IRS-2 or IRS-3, compensated for the lack of differentiation in IRS-1 KO cells. A chimeric molecule containing the N terminus of IRS-1 and the C terminus of IRS-2, but not one with the N terminus of IRS-2 and the C terminus of IRS-1, also rescued differentiation. Expression of Wnt 10a, a molecule known to inhibit adipogenesis, was dramatically increased in the IRS-1 KO cells, and this could be reduced by overexpression of IRS-1 or IRS-4, which was correlated with restoration of differentiation. These data indicate that both IRS-1 and -3 play important roles in the differentiation of brown adipocytes and that the N terminus of IRS-1 is more important for this function of the molecule. Although IRS-4 is not essential for the process, overexpression of IRS-4 can compensate for the deficiency in differentiation in IRS-1 KO cells.