Inputs and outputs of insulin receptor

The insulin receptor (IR) is an important hub in insulin signaling and its activation is tightly regulated. Upon insulin stimulation, IR is activated through autophosphorylation, and consequently phosphorylates several insulin receptor substrate (IRS) proteins, including IRS1-6, Shc and Gab1. Certain adipokines have also been found to activate IR. On the contrary, PTP, Grb and SOCS proteins, which are responsible for the negative regulation of IR, are characterized as IR inhibitors. Additionally, many other proteins have been identified as IR substrates and participate in the insulin signaling pathway. To provide a more comprehensive understanding of the signals mediated through IR, we reviewed the upstream and downstream signal molecules of IR, summarized the positive and negative modulators of IR, and discussed the IR substrates and interacting adaptor proteins. We propose that the molecular events associated with IR should be integrated to obtain a better understanding of the insulin signaling pathway and diabetes.     

The insulin receptor substrate (IRS) proteins

Increasing evidence supports a connection between cancer and metabolism and emphasizes the need to understand how tumors respond to the metabolic microenvironment and how tumor cell metabolism is regulated. The insulin receptor (IR) and its close family member the insulin-like growth factor-1 receptor (IGF-1R) mediate the cellular response to insulin in normal cells and their function is tightly regulated to maintain metabolic homeostasis. These receptors are also expressed on tumor cells and their expression correlates with tumor progression and poor prognosis. Understanding how the IR/IGF-1R pathway functions in tumors is increasing in importance as the efficacy of drugs that target metabolic pathways, such as metformin, are investigated in prospective clinical trials. This review will focus on key signaling intermediates of the IR and IGF-1R, the Insulin Receptor Substrate (IRS) proteins, with an emphasis on IRS-2, and discuss how these adaptor proteins play a pivotal role at the intersection of metabolism and cancer.     

The insulin receptor substrate (IRS) proteins: At the intersection of metabolism and cancer

Increasing evidence supports a connection between cancer and metabolism and emphasizes the need to understand how tumors respond to the metabolic microenvironment and how tumor cell metabolism is regulated. The insulin receptor (IR) and its close family member the insulin-like growth factor-1 receptor (IGF-1R) mediate the cellular response to insulin in normal cells and their function is tightly regulated to maintain metabolic homeostasis. These receptors are also expressed on tumor cells and their expression correlates with tumor progression and poor prognosis. Understanding how the IR/IGF-1R pathway functions in tumors is increasing in importance as the efficacy of drugs that target metabolic pathways, such as metformin, are investigated in prospective clinical trials. This review will focus on key signaling intermediates of the IR and IGF-1R, the Insulin Receptor Substrate (IRS) proteins, with an emphasis on IRS-2, and discuss how these adaptor proteins play a pivotal role at the intersection of metabolism and cancer.Key words: IRS proteins, insulin receptor, IGF-1 receptor, metabolism, cancer, metformin     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

A high-cholesterol diet increases the association between caveolae and insulin receptors in rat liver

Caveolin-1, a component of caveolae, regulates signaling pathway compartmentalization by interacting with tyrosine (Tyr) kinase receptors and their substrates. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. The role of caveolin-1 in insulin receptor (IR) signaling has been widely investigated in vitro mainly in 3T3-L1 adipocyte cells. However, in vivo experiments investigating this connection in liver tissue have not been carried out. The objective of the present study was to investigate the effects of a high-cholesterol diet on caveolin-1 expression and IR localization and activity in the rat liver. Compared with a standard diet, rats fed with diet rich in cholesterol significantly altered liver caveolae by increasing both caveolin-1 (66%, P < 0.05) and caveolin-2 (55%, P < 0.05) expression while caveolin-1 mRNA levels were reduced. Concomitantly, a 25% increase in localization of the caveolae-resident signaling protein IR was observed. The distribution of caveolar and noncaveolar phosphorylated IR was unaffected but insulin-induced IR activation was significantly enhanced following consumption of the high-cholesterol diet (120%, P < 0.001). However, the downstream molecules IRS-1 and Akt have shown impaired activity in cholesterol-fed rats suggesting insulin resistance condition. Insulin stimulation failed to induce Tyr phosphorylation of caveolin-1 in cholesterol-fed rats. These findings suggest a mechanism by which a high-cholesterol diet altered caveolin-1 expression in vivo accompanied by altered IR localization and activity.     

Insulin resistance: Review of the underlying molecular mechanisms

Most human cells utilize glucose as the primary substrate, cellular uptake requiring insulin. Insulin signaling is therefore critical for these tissues. However, decrease in insulin sensitivity due to the disruption of various molecular pathways causes insulin resistance (IR). IR underpins many metabolic disorders such as type 2 diabetes and metabolic syndrome, impairments in insulin signaling disrupting entry of glucose into the adipocytes, and skeletal muscle cells. Although the exact underlying cause of IR has not been fully elucidated, a number of major mechanisms, including oxidative stress, inflammation, insulin receptor mutations, endoplasmic reticulum stress, and mitochondrial dysfunction have been suggested. In this review, we consider the role these cellular mechanisms play in the development of IR.     

Asymmetric dimethylarginine targets MAPK pathway to regulate insulin resistance in liver by activating inflammation factors

Insulin resistance is associated with impaired glucose uptake and altered protein kinase B (Akt) signaling. Previous studies have suggested asymmetric dimethylarginine (ADMA) and inflammation are two distinguish factors that correlate with insulin resistance (IR). How ADMA and inflammation factors interact and synchronize in the regulation of IR in liver remain to be elucidated. In this study, we systematically investigated whether ADMA is involved in IR using primary hepatocytes, if yes, by via which molecular mechanism. Our results demonstrated that ADMA inhibits insulin sensitivity in a concentration-dependent manner by activating inflammation factors tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 in primary hepatocytes. Further analysis revealed that mitogen-activated protein kinase (MAPK) signaling pathway act downstream of ADMA and inflammation factors, and inhibition of MAPK pathway rescued the IR. Furthermore, metformin effects has been found which could reverse ADMA-induced IR by suppressing MAPK signaling pathway. To our knowledge, we, for the first time, unveiled the complicated regulatory network and interactions among ADMA, inflammation, and MAPK signaling pathway, which advanced current research on the development and regulation of IR in liver. This study also certainly provided novel insights on comprehensive diagonistics roles of ADMA as a potential biomarker.     

Caloric restriction impacts plasma microRNAs in rhesus monkeys

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR‐regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR‐125a‐5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR‐associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR‐induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA‐based homeostatic mechanisms to coordinate a program of delayed aging.     

A non‐canonical rhodopsin‐mediated insulin receptor signaling pathway in retinal photoreceptor neurons

We previously reported a ligand‐independent and rhodopsin‐dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein–protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep? density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep? is a non‐ionic iodixanol‐based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.     

Detailed role of microRNA-mediated regulation of PI3K/AKT axis in human tumors

The regulation of signal transmission and biological processes, such as cell proliferation, apoptosis, metabolism, migration, and angiogenesis are greatly influenced by the PI3K/AKT signaling pathway. Highly conserved endogenous non-protein-coding RNAs known as microRNAs (miRNAs) have the ability to regulate gene expression by inhibiting mRNA translation or mRNA degradation. MiRNAs serve key role in PI3K/AKT pathway as upstream or downstream target, and aberrant activation of this pathway contributes to the development of cancers. A growing body of research shows that miRNAs can control the PI3K/AKT pathway to control the biological processes within cells. The expression of genes linked to cancers can be controlled by the miRNA/PI3K/AKT axis, which in turn controls the development of cancer. There is also a strong correlation between the expression of miRNAs linked to the PI3K/AKT pathway and numerous clinical traits. Moreover, PI3K/AKT pathway-associated miRNAs are potential biomarkers for cancer diagnosis, therapy, and prognostic evaluation. The role and clinical applications of the PI3K/AKT pathway and miRNA/PI3K/AKT axis in the emergence of cancers are reviewed in this article.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Insulin receptor signaling regulates actin cytoskeletal organization in developing photoreceptors

The insulin receptor (IR) and IR signaling proteins are widely distributed throughout the CNS. IR signaling provides a trophic signal for transformed retinal neurons in culture and we recently reported that deletion of IR in rod photoreceptors by Cre/ lox system resulted in stress-induced photoreceptor degeneration. These studies suggest a neuroprotective role of IR in rod photoreceptor cell function. However, there are no studies available on the role of insulin-induced IR signaling in the development of normal photoreceptors. To examine the role of insulin-induced IR signaling, we analyzed cultured neuronal cells isolated from newborn rodent retinas. In insulin-lacking cultures, photoreceptors from wild-type rat retinas exhibited an abnormal morphology with a wide axon cone and disorganization of the actin and tubulin cytoskeleton. Photoreceptors from IR knockout mouse retinas also exhibited a similar abnormal morphology. A novel finding in this study was that addition of docosahexaenoic acid, a photoreceptor trophic factor, restored normal axonal outgrowth in insulin-lacking cultures. These data suggest that IR signaling pathways regulate actin and tubulin cytoskeletal organization in photoreceptors; they also imply that insulin and docosahexaenoic acid activate at least partially overlapping signaling pathways that are essential for the development of normal photoreceptors.     

Growth factor receptor-binding protein 10 (Grb10) as a partner of phosphatidylinositol 3-kinase in metabolic insulin action

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.     

脓毒症分子机制及miRNA在脓毒症中的作用

脓毒症是外科重症监护病房(ICU)的主要死亡原因。近年来其发病呈上升趋势,且住院费用极昂贵,并缺乏有效的救治手段,已成为重症医学研究的重点。目前,关于脓毒症的发病机制并不清楚。研究表明,细胞内及细胞间多种信号通路如核因子κB(NF-κB)通路、丝裂原激活的蛋白激酶(MAPK)通路、JAK激酶/信号转导和转录激活子(JAK/STAT)通路、磷脂酰肌醇3激酶(PI3K/Akt)通路、胆碱能抗炎通路等及其下游的分子都参与脓毒症的发生发展。微小RNA(miRNA)作为小分子非编码RNA,通过转录后水平抑制靶基因的表达而参与细胞的多种过程。miRNA可以调控免疫细胞的分化及免疫反应,其不仅可以直接调控炎症因子的表达,还可作用于炎症信号传导通路的其他关键分子而间接调控脓毒症的发生发展。因此深入研究miRNA在脓毒症中的调节作用,可能为脓毒症的预防和治疗开拓新的思路。本文就参与脓毒症的信号通路及其下游分子以及miRNA进行总结,以利于进一步阐明脓毒症的病理生理机制,为脓毒症的预防和治疗找到合理有效的切入点。     

Insulin and insulin-like growth factor I receptors utilize different G protein signaling components

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.     

胰岛素信号通路、炎症信号通路与泛素-蛋白酶体系统的交互作用

胰岛素抵抗是肥胖和2型糖尿病的主要表征。胰岛素信号通路根据是否需要胰岛素受体底物（insulin receptor substrate, IRS）介导可分为IRS介导和非IRS介导的信号通路,其中以IRS介导的信号通路为主。肥胖可增强炎性细胞因子表达并活化IKKβ/NF κB和JNK等炎症信号通路,抑制IRS酪氨酸磷酸化,从而阻止胰岛素的信号转导,降低胰岛素的敏感性,表现为胰岛素抵抗。泛素 蛋白酶体系统作为机体蛋白降解的主要途径,与胰岛素和炎症信号通路联系密切,一方面胰岛素信号通路的阻断可活化泛素依赖的蛋白降解,另一方面,泛素依赖的蛋白降解系统也可直接降解胰岛素和炎症信号通路的关键蛋白,影响胰岛素的作用。本文拟综述肥胖时,胰岛素信号通路、炎症相关信号通路和泛素 蛋白酶体系统之间的交互作用,在分子水平上探讨胰岛素抵抗的发生机制。     

Postprandial regulation of hepatic microRNAs predicted to target the insulin pathway in rainbow trout

Rainbow trout are carnivorous fish and poor metabolizers of carbohydrates, which established this species as a model organism to study the comparative physiology of insulin. Following the recent characterisation of key roles of several miRNAs in the insulin action on hepatic intermediary metabolism in mammalian models, we investigated the hypothesis that hepatic miRNA expression is postprandially regulated in the rainbow trout and temporally coordinated in the context of insulin-mediated regulation of metabolic gene expression in the liver. To address this hypothesis, we used a time-course experiment in which rainbow trout were fed a commercial diet after short-term fasting. We investigated hepatic miRNA expression, activation of the insulin pathway, and insulin regulated metabolic target genes at several time points. Several miRNAs which negatively regulate hepatic insulin signaling in mammalian model organisms were transiently increased 4 h after the meal, consistent with a potential role in acute postprandial negative feed-back regulation of the insulin pathway and attenuation of gluconeogenic gene expression. We equally observed a transient increase in omy- miRNA-33 and omy-miRNA-122b 4 h after feeding, whose homologues have potent lipogenic roles in the liver of mammalian model systems. A concurrent increase in the activity of the hepatic insulin signaling pathway and the expression of lipogenic genes (srebp1c, fas, acly) was equally observed, while lipolytic gene expression (cpt1a and cpt1b) decreased significantly 4 h after the meal. This suggests lipogenic roles of omy-miRNA-33 and omy-miRNA-122b may be conserved between rainbow trout and mammals and that these miRNAs may furthermore contribute to acute postprandial regulation of de novo hepatic lipid synthesis in rainbow trout. These findings provide a framework for future research of miRNA regulation of hepatic metabolism in trout and will help to further elucidate the metabolic phenotype of rainbow trout.     

Is Acetylation a Metabolic Rheostat that Regulates Skeletal Muscle Insulin Action?

Skeletal muscle insulin resistance, which increases the risk for developing various metabolic diseases, including type 2 diabetes, is a common metabolic disorder in obesity and aging. If potential treatments are to be developed to treat insulin resistance, then it is important to fully understand insulin signaling and glucose metabolism. While recent large-scale “omics” studies have revealed the acetylome to be comparable in size to the phosphorylome, the acetylation of insulin signaling proteins and its functional relevance to insulin-stimulated glucose transport and glucose metabolism is not fully understood. In this Mini Review we discuss the acetylation status of proteins involved in the insulin signaling pathway and review their potential effect on, and relevance to, insulin action in skeletal muscle.