Overexpression of the insulin receptor inhibitor PC-1/ENPP1 induces insulin resistance and hyperglycemia

The ectoenzyme PC-1 is an insulin receptor inhibitor that is elevated in cells and tissues of humans with type 2 diabetes (T2D). We have recently shown that acute PC-1 overexpression in liver causes insulin resistance and glucose intolerance in mice (3), but the chronic effects of PC-1 overexpression on these functions are unknown. Herein we produced transgenic mice overexpressing the potent q allele of human PC-1 in muscle and liver. Compared with controls, these mice had 2- to 3-fold elevations of PC-1 content in liver and 5- to 10-fold elevations in muscle. In the fed state, the PC-1 animals had 100 mg/dl higher glucose levels and sixfold higher insulin levels compared with controls. During glucose tolerance tests, these PC-1 animals had peak glucose levels that were >150 mg/dl higher than controls. In vivo uptake of 2-deoxy-d-glucose in muscle during insulin infusion was decreased in the PC-1 animals. These in vivo data support the concept, therefore, that PC-1 plays a role in insulin resistance and hyperglycemia and suggest that animals with overexpression of human PC-1 in insulin-sensitive tissues may be important models to investigate insulin resistance.     

Socs1 deficiency enhances hepatic insulin signaling

Suppressor of cytokine signaling 1 (SOCS1) is an intracellular inhibitor of cytokine, growth factor, and hormone signaling. Socs1-/- mice die before weaning from a multiorgan inflammatory disease. Neonatal Socs1-/- mice display severe hypoglycemia and hypoinsulinemia. Concurrent interferon gamma gene deletion (Ifng-/-) prevented inflammation and corrected the hypoglycemia. In hyperinsulinemic clamp studies, however, Socs1-/- Ifng-/- mice had enhanced hepatic insulin sensitivity demonstrated by greater suppression of endogenous glucose production compared with controls with no difference in glucose disposal. Socs1-/- Ifng-/- mice had elevated liver insulin receptor substrate 2 expression (IRS-2) and IRS-2 tyrosine phosphorylation. This was associated with lower phosphoenolpyruvate carboxykinase mRNA expression. These effects were not associated with elevated hepatic AMP-activated protein kinase activity. Hepatic insulin sensitivity and IRS-2 levels play central roles in the pathogenesis of type 2 diabetes. Socs1 deficiency increases IRS-2 expression and enhances hepatic insulin sensitivity in vivo indicating that inhibition of SOCS1 may be a logical strategy in type 2 diabetes.     

Prkar1a in the regulation of insulin secretion

The incidence of type 2 diabetes mellitus (T2DM) is rapidly increasing worldwide with significant consequences on individual quality of life as well as economic burden on states' healthcare costs. While origins of the pathogenesis of T2DM are poorly understood, an early defect in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is considered a hallmark of T2DM 1.Upon a glucose stimulus, insulin is secreted in a biphasic manner with an early first-phase burst of insulin, which is followed by a second, more sustained phase of insulin output 2. First phase insulin secretion is diminished early in T2DM as well is in subjects who are at risk of developing T2DM 3 4 5 6.An effective treatment of T2DM with incretin hormone glucagon-like peptide-1 (GLP-1) or its long acting peptide analogue exendin-4 (E4), restores first-phase and augments second-phase glucose stimulated insulin secretion. This effect of incretin action occurs within minutes of GLP-1/E4 infusion in T2DM humans. An additional important consideration is that incretin hormones augment GSIS only above a certain glucose threshold, which is slightly above the normal glucose range. This ensures that incretin hormones stimulate GSIS only when glucose levels are high, while they are ineffective when insulin levels are below a certain threshold 7 8.Activation of the GLP-1 receptor, which is highly expressed on pancreatic β-cells, stimulates 2 -distinct intracellular signaling pathways: a) the cAMP-protein kinase A branch and b) the cAMP-EPAC2 (EPAC=exchange protein activated by cAMP) branch. While the EPAC2 branch is considered to mediate GLP-1 effects on first-phase GSIS, the PKA branch is necessary for the former branch to be active 9 10. However, how these 2 branches interplay and converge and how their effects on insulin secretion and insulin vesicle exocytosis are coordinated is poorly understood.Thus, at the outset of our studies we have a poorly understood intracellular interplay of cAMP-dependent signaling pathways, which - when stimulated - restore glucose-dependent first phase and augment second phase insulin secretion in the ailing β-cells of T2DM.     

Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC(50) for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t(1/2) of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.     

Silencing insulin resistance through SIRT1

Insulin resistance is an important risk factor for the development of type 2 diabetes and the metabolic syndrome. A new study in this issue of Cell Metabolism (Sun et al., 2007) shows that SIRT1, a mammalian sirtuin homolog and histone deacetylase, can ameliorate insulin resistance by silencing expression of protein tyrosine phosphatase 1B, a major negative regulator of insulin action.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment.

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1_G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1_G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1_G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF- (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1_G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1_G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1_G subunit site-specific mutations will help clarify the exact role and regulation of PP-1_G site-specific phosphorylations on PP-1 catalytic function.     

Sphingosine 1-phosphate metabolism and insulin signaling

Insulin is the main anabolic hormone secreted by β-cells of the pancreas stimulating the assimilation and storage of glucose in muscle and fat cells. It modulates the postprandial balance of carbohydrates, lipids and proteins via enhancing lipogenesis, glycogen and protein synthesis and suppressing glucose generation and its release from the liver. Resistance to insulin is a severe metabolic disorder related to a diminished response of peripheral tissues to the insulin action and signaling. This leads to a disturbed glucose homeostasis that precedes the onset of type 2 diabetes (T2D), a disease reaching epidemic proportions. A large number of studies reported an association between elevated circulating fatty acids and the development of insulin resistance. The increased fatty acid lipid flux results in the accumulation of lipid droplets in a variety of tissues. However, lipid intermediates such as diacylglycerols and ceramides are also formed in response to elevated fatty acid levels. These bioactive lipids have been associated with the pathogenesis of insulin resistance. More recently, sphingosine 1-phosphate (S1P), another bioactive sphingolipid derivative, has also been shown to increase in T2D and obesity. Although many studies propose a protective role of S1P metabolism on insulin signaling in peripheral tissues, other studies suggest a causal role of S1P on insulin resistance. In this review, we critically summarize the current state of knowledge of S1P metabolism and its modulating role on insulin resistance. A particular emphasis is placed on S1P and insulin signaling in hepatocytes, skeletal muscle cells, adipocytes and pancreatic β-cells. In particular, modulation of receptors and enzymes that regulate S1P metabolism can be considered as a new therapeutic option for the treatment of insulin resistance and T2D.     

Association of insulin receptor substrate 1 (IRS-1) y895 with Grb-2 mediates the insulin signaling involved in IRS-1-deficient brown adipocyte mitogenesis

We have recently generated immortalized fetal brown adipocyte cell lines from insulin receptor substrate 1 (IRS-1) knockout mice and demonstrated an impairment in insulin-induced lipid synthesis as compared to wild-type cell lines. In this study, we investigated the consequences of IRS-1 deficiency on mitogenesis in response to insulin. The lack of IRS-1 resulted in the inability of insulin-stimulated IRS-1-deficient brown adipocytes to increase DNA synthesis and enter into S/G2/M phases of the cell cycle. These cells showed a severe impairment in activating mitogen-activated protein kinase kinase (MEK1/2) and p42-p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. IRS-1-deficient cells also lacked tyrosine phosphorylation of SHC and showed no SHC-Grb-2 association in response to insulin. The mitogenic response to insulin could be partially restored by enhancing IRS-2 tyrosine phosphorylation and its association with Grb-2 by inhibition of phosphatidylinositol 3-kinase activity through a feedback mechanism. Reconstitution of IRS-1-deficient brown adipocytes with wild-type IRS-1 restored insulin-induced IRS-1 and SHC tyrosine phosphorylation and IRS-1-Grb-2, IRS-1-SHC, and SHC-Grb-2 associations, leading to the activation of MAPK and enhancement of DNA synthesis. Reconstitution of IRS-1-deficient brown adipocytes with the IRS-1 mutant Tyr895Phe, which lacks IRS-1-Grb-2 binding, restored SHC-IRS-1 association and SHC-Grb-2 association. However, the lack of IRS-1-Grb-2 association impaired MAPK activation and DNA synthesis in insulin-stimulated mutant cells. These data provide strong evidence for an essential role of IRS-1 and its direct association with Grb-2 in the insulin signaling pathway leading to MAPK activation and mitogenesis in brown adipocytes.     

Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action

Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1^−/− and IRS-2^−/− mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins.     

Insulin signalling: the role of insulin receptor substrate 1

The insulin receptor is a ligand-activated tyrosine kinase that phosphorylates its major substrate protein, insulin receptor substrate 1 (IRS1), at multiple sites. Tyrosine-phosphorylated IRS1 then serves as a docking/effector protein for at least four Src homology 2 (SH2)-domain proteins involved in signal transduction. This initial step in signalling distinguishes the insulin receptor from other receptor tyrosine kinases, which directly bind several SH2-domain proteins, and establishes IRS1 as a founding member of a group of proteins whose function is to link activated tyrosine kinases to SH2-domain proteins.     

Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.     

Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes

Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-a-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70-75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells.     

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.     

Differential activation of insulin receptor substrates 1 and 2 by insulin-like growth factor-activated insulin receptors

The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.     

The CB1 endocannabinoid system modulates adipocyte insulin sensitivity

Mounting evidence suggests that the endocannabinoid system regulates energy metabolism through direct effects on peripheral tissues as well as central effects that regulate appetite. Here we examined the effect of cannabinoid receptor 1 (CB1) signaling on insulin action in fat cells. We examined effects of the natural CB1 agonist, 2-Arachidonoylglycerol (2-AG), and the synthetic CB1 antagonist, SR141716, on insulin action in cultured adipocytes. We used translocation of glucose transporter GLUT4 to plasma membrane (PM) as a measure of insulin action. 2-AG activation of the CB1 receptor promoted insulin sensitivity whereas antagonism by SR141716 reduced insulin sensitivity. Neither drug affected GLUT4 translocation in the absence of insulin or with high doses of insulin. Consistent with these results we found that insulin-stimulated phosphorylation of the protein kinase Akt was increased by 2-AG, attenuated by SR141716, and unaffected in the absence of insulin or by addition of high-dose insulin. These data provide a functional and molecular link between the CB1 receptor and insulin sensitivity, because insulin-stimulated phosphorylation of Akt is required for GLUT4 translocation to the PM. The sensitizing effects of 2-AG were abrogated by SR141716 and Pertussis toxin, indicating that the effects are mediated by CB1 receptor. Importantly, neither 2-AG nor SR141716 alone or in combination with maximal dose of insulin had effects on GLUT4 translocation and Akt phosphorylation. These data are consistent with a model in which the endocannabinoid system sets the sensitivity of the insulin response in adipocytes rather than directly regulating the redistribution of GLUT4 or Akt phosphorylation.     

Suppression of insulin receptor substrate 1 (IRS-1) promotes mammary tumor metastasis

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptors that organize signaling complexes downstream of activated cell surface receptors. Here, we show that IRS-1 and IRS-2, despite significant homology, play critical yet distinct functions in breast cancer, and we identify specific signaling pathways that are influenced by IRS-1 using the polyoma virus middle-T (PyV-MT) transgenic mouse model of mammary carcinoma and Irs-1 null (Irs1(-/-)) mice. The absence of Irs-1 expression enhanced metastatic spread significantly without a significant effect on primary tumor growth. Orthotopic transplant studies revealed that the increased metastatic potential of Irs1-deficient tumor cells is cell autonomous. Mammary tumors that developed in PyV-MT::Irs1(-/-) mice exhibited elevated Irs-2 function and enhanced phosphatidylinositol 3-kinase/Akt/mTor activity, suggesting that one mechanism by which Irs-1 impedes metastasis is to suppress Irs-2-dependent signaling. In support of this mechanism, reduction of Irs-2 expression in Irs1(-/-) tumor cells restored mTor signaling to wild-type levels. PyV-MT::Irs1(-/-) tumors also exhibited a significant increase in vascular endothelial growth factor expression and microvessel density, which could facilitate their dissemination. The significance of our findings for human breast cancer is heightened by our observation that Irs-1 is inactivated in wild-type, metastatic mammary tumors by serine phosphorylation. Collectively, our findings reveal that inactivation of IRS-1 enhances breast cancer metastasis and support the novel hypothesis that IRS-1 has metastasis suppressor functions for breast cancer.