Insulin Receptor Tyrosine Kinase Research Articles

Insulin-Like Growth Factor-1 Receptor-Mediated Inhibition of A-type K+ Current Induces Sensory Neuronal Hyperexcitability Through the Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/2 Pathways, Independently of Akt

Although IGF-1 has been implicated in mediating hypersensitivity to pain, the underlying mechanisms remain unclear. We identified a novel functional of the IGF-1 receptor (IGF-1R) in regulating A-type K(+) currents (IA) as well as membrane excitability in small trigeminal ganglion neurons. Our results showed that IGF-1 reversibly decreased IA, whereas the sustained delayed rectifier K(+) current was unaffected. This IGF-1-induced IA decrease was associated with a hyperpolarizing shift in the voltage dependence of inactivation and was blocked by the IGF-1R antagonist PQ-401; an insulin receptor tyrosine kinase inhibitor had no such effect. An small interfering RNA targeting the IGF-1R, or pretreatment of neurons with specific phosphatidylinositol 3-kinase (PI3K) inhibitors abolished the IGF-1-induced IA decrease. Surprisingly, IGF-1-induced effects on IA were not regulated by Akt, a common downstream target of PI3K. The MAPK/ERK kinase inhibitor U0126, but not its inactive analog U0124, as well as the c-Raf-specific inhibitor GW5074, blocked the IGF-1-induced IA response. Analysis of phospho-ERK (p-ERK) showed that IGF-1 significantly activated ERK1/2 whereas p-JNK and p-p38 were unaffected. Moreover, the IGF-1-induced p-ERK1/2 increase was attenuated by PI3K and c-Raf inhibition, but not by Akt blockade. Functionally, we observed a significantly increased action potential firing rate induced by IGF-1; pretreatment with 4-aminopyridine abolished this effect. Taken together, our results indicate that IGF-1 attenuates IA through sequential activation of the PI3K- and c-Raf-dependent ERK1/2 signaling cascade. This occurred via the activation of IGF-1R and might contribute to neuronal hyperexcitability in small trigeminal ganglion neurons.

Overcoming IGF1R/IR Resistance through Inhibition of MEK Signaling in Colorectal Cancer Models

Results from clinical trials involving resistance to molecularly targeted therapies have revealed the importance of rational single-agent and combination treatment strategies. In this study, we tested the efficacy of a type 1 insulin-like growth factor receptor (IGF1R)/insulin receptor (IR) tyrosine kinase inhibitor, OSI-906, in combination with a mitogen-activated protein (MAP)-ERK kinase (MEK) 1/2 inhibitor based on evidence that the MAP kinase pathway was upregulated in colorectal cancer cell lines that were resistant to OSI-906. The antiproliferative effects of OSI-906 and the MEK 1/2 inhibitor U0126 were analyzed both as single agents and in combination in 13 colorectal cancer cell lines in vitro. Apoptosis, downstream effector proteins, and cell cycle were also assessed. In addition, the efficacy of OSI-906 combined with the MEK 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) was evaluated in vivo using human colorectal cancer xenograft models. The combination of OSI-906 and U0126 resulted in synergistic effects in 11 of 13 colorectal cancer cell lines tested. This synergy was variably associated with apoptosis or cell-cycle arrest in addition to molecular effects on prosurvival pathways. The synergy was also reflected in the in vivo xenograft studies following treatment with the combination of OSI-906 and selumetinib. Results from this study demonstrate synergistic antiproliferative effects in response to the combination of OSI-906 with an MEK 1/2 inhibitor in colorectal cancer cell line models both in vitro and in vivo, which supports the rational combination of OSI-906 with an MEK inhibitor in patients with colorectal cancer. Clin Cancer Res; 19(22); 6219-29. ©2013 AACR.

Stimulation of glucose uptake by theasinensins through the AMP-activated protein kinase pathway in rat skeletal muscle cells

Theasinensins, dimeric catechins, have been reported to possess anti-hyperglycemic activity, but the underlying mechanism for this activity remains unknown. In this study, the effect of theasinensins A and B on glucose uptake into rat skeletal muscle cells (L6 myotubes) was investigated. A glucose uptake study using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) indicated that both theasinensins A and B stimulated glucose uptake in a concentration-dependent manner and translocation of glucose transporter 4 (GLUT4) to the plasma membrane. In addition, inhibition studies measuring 2-NBDG uptake in L6 cells revealed that compound C (AMP-activated protein kinase inhibitor) suppressed theasinensin-stimulated glucose uptake, whereas genistein (insulin receptor tyrosine kinase inhibitor) and wortmannin (phosphatidylinositol 3-kinase inhibitor) were inactive. Subsequent experiments on GLUT4-related signaling pathways in L6 cells demonstrated that theasinensins promoted the phosphorylation of AMPK, but not that of Akt, and that the theasinensin-promoted glucose uptake was blocked in the presence of a CaMKK inhibitor. The promotion of AMPK phosphorylation by theasinensins was not blocked in LKB1-knockdown cells. Consequently, it was concluded that theasinensins A and B did in fact promote GLUT4 translocation to the plasma membrane in L6 myotubes through the CaMKK/AMPK signaling pathway, but not through the PI3K/Akt pathway.

Fetuin-A in patients with metabolic syndrome

Metabolic syndrome is a common disorder the prevalence of which is estimated to be about 20% in Polish adult population. Abdominal obesity and insulin resistance are important pathogenetic factors. Metabolic syndrome plays a role as a risk factor for type 2 diabetes and cardiovascular disease. Fetuin-A is a multifunctional plasma glycoprotein. It is a physiological inhibitor of insulin receptor tyrosine kinase and thus associated with insulin resistance, metabolic syndrome and an increased risk for type 2 diabetes. The study was conducted in 62 patients with metabolic syndrome (34F and 28M) aged 35-83. In 47 persons type 2 diabetes was a component of metabolic syndrome, 62% of diabetics had coronary artery disease as a macrovascular complication. Determinations of biochemical parameters and anthropometric measurements were performed in the studied group. We analysed a relationship between serum fetuin-A concentration and components of metabolic syndrome and total cholesterol, LDL-cholesterol, HbA1C, BMI as well. Diabetics had lower fetuin-A concentrations than patients without diabetes (0.550 g/l vs 0.600 g/l). Fetuin-A levels in patients with diabetes and coronary artery disease were significantly lower (0.535 g/l) than in those without macrovascular complications (0.590 g/l) (Z=1.969; p=0.048). Furthermore the correlation between fetuin-A serum concentration and fasting plasma glucose, LDL-cholesterol and triglycerides levels were observed. Patients with higher fasting glucose had lower fetuin-A levels. However, fetuin-A concentration was positive correlated with LDL-cholesterol and triglycerides levels. No association between fetuin-A and waist circumference, blood pressure, HDL-cholesterol, HbA1C and BMI were found. In summary, serum fetuin-A level has a correlation with some components of metabolic syndrome. We concluded that fetuin-A could be used not only as a marker, but also plays some role in pathogenesis of metabolic syndrome, type 2 diabetes and higher risk of cardiovascular disease.

Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor.

Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R) antibody figitumumab in non-small cell lung cancer (NSCLC) patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR) in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R) and IR47-9 (IR), and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

English

An important pathogenic process in enterohemorrhagic Escherichia coli (EHEC)associated diseases is the formation of attaching and effacing (A/E) lesions, which are a typical pathological change in host cells. The classical pathway for A/E lesion formation requires the participation of proteins from both bacteria and host cells, namely intimin, translocated intimin receptor (Tir), insulin receptor tyrosine kinase substrate (IRTKS), Tir coupled cytoskeleton protein (TCCP), ARP3/2, and N-WASP. The interaction between IRTKS and TCCP is mediated by the binding of the SH3 domain of IRTKS (SH3IRTKS) to the proline rich repeat (PRR) domain of TCCP, which is important for the induction of A/E lesions. The inability to efficiently produce the purified target complex has hindered the structural determination of these protein complexes. Here, we report an effective method for the generation of a complex consisting of SH3IRTKS with three PRRTCCP domains. Two recombinant fragments, TCCP3R and TCCP5R, as well as SH3IRTKS, were successfully expressed in soluble form and purified. In addition, methods were established to prepare two different protein complexes, SH3IRTKS-TCCP3R and SH3IRTKS-TCCP5R. These methods are a good foundation for future studies on the crystal structure of TCCP-IRTKS. Meanwhile, recombinant TCCP was shown to directly bind to IRTKS in vitro, which provides additional evidence for the interaction between these two molecules. Key words: Enterohemorrhagic Escherichia coli, Tir coupled cytoskeleton protein, insulin receptor tyrosine kinase substrate, protein complex.

Insulin receptor tyrosine kinase substrate activates EGFR/ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma

Insulin receptor tyrosine kinase substrate (IRTKS) is closely associated with actin remodelling and membrane protrusion, but its role in the pathogenesis of malignant tumours, including hepatocellular carcinoma (HCC), is still unknown. In this study, we showed that IRTKS was frequently upregulated in HCC samples, and its expression level was significantly associated with tumour size. Enforced expression of IRTKS in human HCC cell lines significantly promoted their proliferation and colony formation in vitro, and their capacity to develop tumour xenografts in vivo, whereas knockdown of IRTKS resulted in the opposite effects. Furthermore, the bromodeoxyuridine (BrdU) incorporation analyses and propidium iodide staining indicated that IRTKS can promote the entry into S phase of cell cycle progression. Significantly, IRTKS can interact with epidermal growth factor receptor (EGFR), results in the phosphorylation of extracellular signal-regulated kinase (ERK). By contrast, inhibition of ERK activation can attenuate the effects of IRTKS overexpression on cellular proliferation. Taken together, these data demonstrate that IRTKS promotes the proliferation of HCC cells by enhancing EGFR–ERK signalling pathway.

Involvement of GLUT-4 in the stimulatory effect of rutin on glucose uptake in rat soleus muscle

The aim of this study was to investigate the in-vitro effect of rutin on glucose uptake in an insulin target (soleus muscle) and the mechanism of action involved. Isolated soleus muscles from rats were treated with rutin (500 μm) with or without the following inhibitors; hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester (HNMPA(AM)3 ), an insulin receptor tyrosine kinase activity inhibitor, wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C, colchicine, a microtubule-depolymerizing agent, PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK), and cycloheximide, an inhibitor of protein synthesis on fresh Krebs Ringer-bicarbonate plus [U-(14) C]-2-deoxy-d-glucose (0.1 μCi/ml). Samples of tissue medium were used for the radioactivity measurements. Rutin increased the glucose uptake in rat soleus muscle. In addition, the effect of rutin on glucose uptake was completely inhibited by pretreatment with HNMPA(AM)3 , wortmannin, RO318220, colchicine, PD98059, and cycloheximide. These results suggested that rutin stimulated glucose uptake in the rat soleus muscle via the PI3K, atypical protein kinase C and mitogen-activated protein kinase (MAPK) pathways. Also, rutin may have influenced glucose transporter translocation and may have directly activated the synthesis of the transporter GLUT-4. The similarities of rutin action on glucose uptake compared with the signalling pathways of insulin constitute strong evidence for the insulin-mimetic role of rutin in glucose homeostasis.

Abstract 2321: Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS signaling.

Abstract Hyperactivation of the PI3K/AKT/mTOR pathway in tumors causes feedback inhibition throughout the signaling network, including inhibition of receptor tyrosine kinase (RTK) expression and function. Exposure of these tumors to mTOR or AKT inhibitors relieves this feedback and reactivates RTK signaling. RTK reactivation promotes PI3K activity and attenuates the antitumor effects of the drugs. We have now characterized the details of PI3K-induced feedback using a selective class 1 PI3K inhibitor and compared its effects with those elicited by a selective allosteric inhibitor of AKT. In breast cancer cell lines with dysregulation of PI3K signaling, both drugs cause marked inhibition of AKT phosphorylation and relief of feedback inhibition of HER3, IGF1, and insulin RTKs. Whereas both inhibitors cause tumor cells to undergo growth arrest, only the PI3K inhibitor causes a rapid induction of apoptosis within 2 hours of drug addition. As opposed to the PI3K inhibitor, the AKT inhibitor induces PI3K activity and a set of substrates that are inhibited by the PI3K inhibitor. We therefore sought to identify processes that are selectively inhibited by the PI3K inhibitor to explain its selective induction of apoptosis. AKT inhibition causes the induction of ERK phosphorylation in these cells. In contrast, we found that PI3K inhibition causes rapid inhibition of RAS-GTP levels, RAF activation and ERK phosphorylation in tumor cells with wild type RAS, but not those with mutant RAS. Inhibition of RAS is transient, recovering 2-3 hours after drug addition. We determined that adding MEK inhibitors to AKT inhibitors is similarly associated with a rapid induction of apoptosis within 2 hours. Moreover, addition of EGF to tumor cells co-treated with PI3K inhibitors prevents ERK inhibition and apoptosis, whereas addition of MEK inhibitors to the PI3K inhibitor, EGF treated cells restores the apoptotic phenotype. We conclude that PI3K is upstream of both RAS and AKT in RAS wild type tumors. Inhibitors of PI3K cause sustained inhibition of AKT and transient inhibition of RAS with both effects required for rapid induction of apoptosis. These data suggest that pulsatile inhibition of PI3K may maximize the therapeutic efficacy of this approach. Citation Format: Marie Will, Weiyi Toy, Zhan Yao, Poulikos Poulikakos, Vanessa Rodrik-Outmezguine, Claudia Schneider, Juliane Paul, Jose Baselga, Ningshu Liu, Neal Rosen, Sarat Chandarlapaty. Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2321. doi:10.1158/1538-7445.AM2013-2321

Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle

The aim of the study was to investigate the in vitro effect and the mechanism of action of kaempferitrin on glucose uptake in an insulin target (soleus muscle). A stimulatory effect of kaempferitrin on glucose uptake was observed when rat soleus muscle was incubated with 10, 100 and 1000ηM of this flavonoid glycoside. The presence of specific insulin signaling inhibitors, such as wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C (PKC), PD98059, an inhibitor of mitogen-activated protein kinase (MEK), HNMPA(AM)3, an insulin receptor tyrosine kinase activity inhibitor, colchicine, a microtubule-depolymerizing agent, SB239063, an inhibitor of P38 MAPK and cycloheximide, an inhibitor of protein synthesis showed that kaempferitrin triggers different metabolic and nuclear pathways in skeletal muscle. Besides the influence on glycogen storage, the metabolic action involves the insulin receptor, PI3K, atypical PKC activity and the translocation of GLUT4. Additionally, the nuclear pathways (via MAPK and MEK) provide evidence of the stimulation of the expression of glucose transporters or other signaling proteins, reinforcing proposals that skeletal muscle represents a primary site at which kaempferitrin exerts its effect promoting glucose homeostasis. Also, these similarities with the signaling pathways of insulin constitute strong evidence for the insulin-mimetic role of kaempferitrin in glucose homeostasis.

Novel Link Between Inflammation, Endothelial Dysfunction, and Muscle Insulin Resistance

In response to development of the euglycemic-hyperinsulinemic clamp technique in the 1980s, much emphasis was placed on skeletal muscle as the major site of insulin resistance in patients with type 2 diabetes (1). Following the discovery of the insulin receptor (IR) tyrosine kinase activity by Ron Kahn and colleagues (2) and the importance of IR substrate molecules in the 1990s (3,4), attention was directed on intrinsic alterations in the insulin signaling cascade in skeletal muscle and liver as fundamental causes of insulin resistance. In 1996, I joined Ron Kahn's laboratory as a participant in the gold rush to discover novel mechanisms responsible for insulin resistance. It was a time of intellectual and scientific awakening. With the advent of the Cre/loxP system, came the first surprise: muscle-specific IR knockout (MIRKO) mice showed no alterations in glucose homeostasis. Rather, they developed visceral adiposity (5). MIRKO mice exhibited impaired insulin activation of muscle glycogen synthase resulting in decreased muscle glycogen content (6,7). MIRKO mice also displayed reduced insulin-stimulated muscle glucose uptake during the euglycemic-hyperinsulinemic clamp (6). However, under physiological conditions of a glucose tolerance test, MIRKO mice had near normal skeletal muscle glucose uptake and did not display insulin resistance (7). Conversely, disruption of insulin action in the hepatocyte-specific IR knockout mouse produced severe resistance to the blood glucose–lowering effect of insulin (8). Together with the MIRKO, the hepatocyte-specific IR knockout mouse demonstrated …

English

Diabetic retinopathy is one of the most devastating complications of diabetes. The prevalence of Diabetic retinopathy in India is relatively on the lower side as compared to the western countries. Indian studies have recorded prevalence rates of 34.1% and 37% in two studies from south India . Our aim of the study is to measure and compare the serum levels of zinc and magnesium in normal individuals and in diabetic retinopathy patients. We found that serum magnesium levels in diabetic retinopathy mean is 11.98 ± 2.20 μ gm/ml levels were significantly lower (P< 0.001) than the controls, the value being 18.89 ± 4.26 μ gm/ml. Serum zinc levels in cases were being 0.422 ± 0.182 significantly lower than the control group 1.10 ± 0.20 μ gm/ml. (p< 0.001). Hypomagnesemia may be implicated in the etiology of diabetic retinopathy by causing reduction in the rate of inositol transport, causing subsequent intracellular inositol depletion and inhibition of Na+ K+ ATPase activity thus leading to the development of this complication. Zinc has been shown to stimulate insulin action and insulin receptor tyrosine kinase activity. Its deficiency interferes with its normal physiological enzyme and hormone action causing impairment of insulin action and development of insulin resistance leading to diabetes mellitus and its complications. The decrease in these micro elements could be due to poor glycemic control, osmotic diuresis and altered metabolism.

Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor

We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC50 within the range of single-chain Ahsg concentrations in human serum. Binding of 125I-insulin to living cells overexpressing the InsR shows a dissociation constant (KD) of 250pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.

The effect of demethylasterriquinone B-1 on insulin secretion in rat pancreas

A small nonpeptidyl compoud extracted from Pseudomassaria sp. was found to induce the activity of human insulin receptor tyrosine kinase in vitro. The compound was identified as demethylasterriquinone B-1 (DMAQ-B1). DMAQ- B1 also induced an increase in [Ca2+]i and insulin secretion in mice pancreatic beta-cells at low glucose (3 mM) concentration via insulin receptor substrate-1/phosphatidylinositol-3-kinase (PI3 kinase) pathway. By using rat pancreatic perfusion technique, we found that 10 μM DMAQ-B1 directly stimulated insulin secretion up to 240% in normal rat pancreas. In the dosage from 1 to 20 μM, DMAQ-B1 stimulated insulin secretion in a dose dependent manner. Furthermore, DMAQ-B1 enhanced glucose-induced insulin secretion by 17.6% (first stage) and 19.0% (second stage), respectively. The PI3 kinase inhibitors, LY 294002 (3.9 μM) or wortmannin (100 nM), inhibited DMAQ-B1-induced insulin secretion by 46.3% and 57.4%, respectively. LY 294002 or wortmannin also inhibited DMAQ-B1 with10 mMglucose-induced insulin secretion by 70.3% and 79.0%, respectively. All the results suggested that DMAQ-B1 directly stimulated insulin secretion and enhanced glucose-induced insulin secretion. The effect of DMAQ-B1 may mediate through the activation of PI3 kinase pathway to stimulate insulin secretion in normal rat pancreas.

Tub Has a Key Role in Insulin and Leptin Signaling and Action In Vivo in Hypothalamic Nuclei

Mutation of tub gene in mice induces obesity, suggesting that tub could be an important regulator of energy balance. In the current study, we investigated whether insulin, leptin, and obesity can modulate Tub in vivo in hypothalamic nuclei, and we investigated possible consequences on energy balance, neuropeptide expression, and hepatic glucose metabolism. Food intake, metabolic characteristics, signaling proteins, and neuropeptide expression were measured in response to fasting and refeeding, intracerebroventricular insulin and leptin, and Tub antisense oligonucleotide (ASO). Tub tyrosine phosphorylation (Tub-p-tyr) is modulated by nutritional status. Tub is a substrate of insulin receptor tyrosine kinase (IRTK) and leptin receptor (LEPR)–Janus kinase 2 (JAK2) in hypothalamic nuclei. After leptin or insulin stimulation, Tub translocates to the nucleus. Inhibition of Tub expression in hypothalamus by ASO increased food intake, fasting blood glucose, and hepatic glucose output, decreased O2 consumption, and blunted the effect of insulin or leptin on proopiomelanocortin, thyroid-releasing hormone, melanin-concentrating hormone, and orexin expression. In hypothalamus of mice administered a high-fat diet, there is a reduction in leptin and insulin-induced Tub-p-tyr and nuclear translocation, which is reversed by reducing protein tyrosine phosphatase 1B expression. These results indicate that Tub has a key role in the control of insulin and leptin effects on food intake, and the modulation of Tub may contribute to insulin and leptin resistance in DIO mice.

Severe progressive obstructive cardiomyopathy and renal tubular dysfunction in Donohue syndrome with decreased insulin receptor autophosphorylation due to a novel INSR mutation

Donohue syndrome (leprechaunism; OMIM *246200) is a rare, recessively inherited disorder of extreme insulin resistance due to mutations in the insulin receptor gene (INSR) causing either defects in insulin binding or receptor autophosphorylation and tyrosine kinase activity. We report a patient with pronounced clinical picture of leprechaunism who developed severe progressive hypertrophic obstructive cardiomyopathy (HOCM) and renal tubular dysfunction which improved on continuous subcutaneous infusion of recombinant human insulin-like growth factor-1 (rhIGF-I). INSR gene molecular analysis and insulin receptor (IR) autophosphorylation on cultured fibroblasts were performed. A novel homozygous missense mutation p.Leu795Pro was found, located in the extracellular portion of the β subunit of the insulin receptor. The post-binding defect of the insulin receptor signaling in cultured fibroblasts demonstrated decreased insulin receptor autophosphorylation. Treatment with rhIGF-I partially reversed severe progressive HOCM and renal tubular dysfunction in a patient with Donohue syndrome associated with a novel p.Leu795Pro INSR gene mutation causing a severe decrease in IR autophosphorylation.

Insulin receptor regulates photoreceptor CNG channel activity.

Photoreceptor cyclic nucleotide gated (CNG) channels are critical elements in phototransduction and light adaptation. Here we report that insulin receptor (IR), an integral membrane protein, directly phosphorylates the CNGA1 subunit of CNG channels that in turn affects the function of these channels negatively. The IR phosphorylates Tyr(498) and Tyr(503) residues on CNGA1 that are situated at the membrane-cytoplasmic interface. The IR tyrosine kinase activity is essential for the inhibition of CNG channel. To maintain the channels in an off state, it is necessary not only to have a precise balance of the cGMP levels but also to have a control on the cGMP sensitivity of the CNG channels itself. In this study, we observed that the channel opens at a lower concentration of cGMP in IR(-/-) mice. These studies suggest that IR regulates the modulation of CNG channel activity in vivo.

Effect of Metformin on Tyrosine Kinase Autophosphorylation of Erythrocyte Insulin Receptor from Women with Polycystic Ovary Syndrome

Objective To document the possible effect of tyrosine kinase autophosphorylation on erythrocyte insulin-receptor after 12 weeks of metformin administration from the women with polycystic ovary syndrome (PCOS). Methods Thirty non-obese (BMI 2 ) healthy women with normal reproductive cycles were evaluated by conventional criteria as the control, 30 non-obese women with PCOS were categorized as nob-PCOS group, 40 obese women with PCOS (BMI ⩾ 25 kg/m 2 ) were categorized as ob-PCOS group. Subjects with PCOS were given metformin 850 mg/d for 12 weeks. The autophosphorylation of insulin receptor pY1158, pY1162/1163 and insulin receptor-β subunit (IR-β) from the solubilised erythrocyte were detected by ELISA. Results 1) Only the autophosphorylation level of pY1158 in nob-PCOS and ob-PCOS groups was lower (P 0.05), even insulin-stimulated insulin receptor (P>0.05) from both of PCOS groups. 2) After metformin administration, the pY1158, pY1162/pY1163 autophosphorylation levels were increased (P 0.05). In summary, oral administration of metformin led to an significant increase in tyrosine kinase activity in both groups of PCOS. Conclusion The impairment of tyrosine autophosphorylation at pY1158 may contribute to insulin resistance, the tyrosine kinase activity per receptor of solubilised erythrocytes were significantly increased by metformin administration and the effect of metformin on insulin-receptor tyrosine kinase activity appeared to be independent of either of these variables.

Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2

The insulin receptor substrate proteins IRS1 and IRS2 are key targets of the insulin receptor tyrosine kinase and are required for hormonal control of metabolism. Tissues from insulin-resistant and diabetic humans exhibit defects in IRS-dependent signalling, implicating their dysregulation in the initiation and progression of metabolic disease. However, IRS1 and IRS2 are regulated through a complex mechanism involving phosphorylation of >50 serine/threonine residues (S/T) within their long, unstructured tail regions. In cultured cells, insulin-stimulated kinases (including atypical PKC, AKT, SIK2, mTOR, S6K1, ERK1/2 and ROCK1) mediate feedback (autologous) S/T phosphorylation of IRS, with both positive and negative effects on insulin sensitivity. Additionally, insulin-independent (heterologous) kinases can phosphorylate IRS1/2 under basal conditions (AMPK, GSK3) or in response to sympathetic activation and lipid/inflammatory mediators, which are present at elevated levels in metabolic disease (GRK2, novel and conventional PKCs, JNK, IKKβ, mPLK). An emerging view is that the positive/negative regulation of IRS by autologous pathways is subverted/co-opted in disease by increased basal and other temporally inappropriate S/T phosphorylation. Compensatory hyperinsulinaemia may contribute strongly to this dysregulation. Here, we examine the links between altered patterns of IRS S/T phosphorylation and the emergence of insulin resistance and diabetes.

Anti-hyperglycemic action of apigenin-6-C-β-fucopyranoside from Averrhoa carambola

A stimulatory effect of apigenin-6-C-β-fucopyranoside (1) on glucose uptake was observed when rat soleus muscle was incubated with 1, 10 and 100μM of this flavonoid glycoside. The presence of specific insulin signaling inhibitors, such as wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C (PKC), PD98059, an inhibitor of mitogen-activated protein kinase (MEK), and HNMPA(AM)3, an insulin receptor tyrosine kinase activity inhibitor showed that apigenin-6-C-β-fucopyranoside triggers different metabolic pathways in skeletal muscle. The oral administration of crude extract, fractions and isolated flavonoids (apigenin-6-C-β-fucopyranoside (1) and apigenin-6-C-(2″-O-α-rhamnopyranosyl)-β-fucopyranoside (2)) from Averrhoa carambola leaves exhibited a potential hypoglycemic activity in hyperglycemic normal rats. Additionally, both flavonoids significantly increased the muscle and liver glycogen content after an acute treatment. The results indicate that A. carambola can be regarded as a potent antihyperglycemic agent with insulin secretagogue and insulin mimetic properties.