Multivalent insulin receptor activation using insulin-DNA origami nanostructures.

Insulin binds the insulin receptor (IR) and regulates anabolic processes in target tissues. Impaired IR signalling is associated with multiple diseases, including diabetes, cancer and neurodegenerative disorders. IRs have been reported to form nanoclusters at the cell membrane in several cell types, even in the absence of insulin binding. Here we exploit the nanoscale spatial organization of the IR to achieve controlled multivalent receptor activation. To control insulin nanoscale spatial organization and valency, we developed rod-like insulin-DNA origami nanostructures carrying different numbers of insulin molecules with defined spacings. Increasing the insulin valency per nanostructure markedly extended the residence time of insulin-DNA origami nanostructures at the receptors. Both insulin valency and spacing affected the levels of IR activation in adipocytes. Moreover, the multivalent insulin design associated with the highest levels of IR activation also induced insulin-mediated transcriptional responses more effectively than the corresponding monovalent insulin nanostructures. In an in vivo zebrafish model of diabetes, treatment with multivalent-but not monovalent-insulin nanostructures elicited a reduction in glucose levels. Our results show that the control of insulin multivalency and spatial organization with nanoscale precision modulates the IR responses, independent of the insulin concentration. Therefore, we propose insulin nanoscale organization as a design parameter in developing new insulin therapies.

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer.

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.

Targeting the IGF-Axis for Cancer Therapy: Development and Validation of an IGF-Trap as a Potential Drug.

The insulin-like growth factor (IGF)-axis was implicated in cancer progression and identified as a clinically important therapeutic target. Several IGF-I receptor (IGF-IR) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signaling and compensatory signaling by the insulin receptor (IR) isoform A that can bind IGF-II and initiate mitogenic signaling. Here we briefly review the current state of IGF-targeting biologicals, discuss some factors that may be responsible for their poor performance in the clinic and outline the stepwise bioengineering and validation of an IGF-Trap-a novel anti-cancer therapeutic that could bypass these limitations. The IGF-Trap is a heterotetramer, consisting of the entire extracellular domain of the IGF-IR fused to the Fc portion of human IgG1. It binds human IGF-I and IGF-II with a three-log higher affinity than insulin and could inhibit IGF-IR driven cellular functions such as survival, proliferation and invasion in multiple carcinoma cell models in vitro. In vivo, the IGF-Trap has favorable pharmacokinetic properties and could markedly reduce metastatic outgrowth of colon and lung carcinoma cells in the liver, outperforming IGF-IR and ligand-binding monoclonal antibodies. Moreover, IGF-Trap dose-response profiles correlate with their bio-availability profiles, as measured by the IGF kinase receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. Our studies identify the IGF-Trap as a potent, safe, anti-cancer therapeutic that could overcome some of the obstacles encountered by IGF-targeting biologicals that have already been evaluated in clinical settings.

Effects of a fructose-rich diet and chronic stress on insulin signaling and regulation of glycogen synthase kinase-3 beta and the sodium-potassium pump in the hearts of male rats.

Both a diet rich in fructose and chronic stress exposure induce metabolic and cardiovascular disturbances. The aim of this study was to examine the effects of the fructose-rich diet and chronic stress, separately and in combination, on insulin signaling and molecules regulating glycogen synthesis and ion transport in the heart, and to reveal whether these effects coincide with changes in glucocorticoid receptor (GR) activation. Male Wistar rats were subjected to 10% fructose in drinking water and/or to chronic unpredictable stress for 9 weeks. Protein expression and/or phosphorylation of the insulin receptor (IR), protein tyrosine phosphatase 1B, insulin receptor substrate 1 (IRS1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (ERK1/2), glycogen synthase kinase-3ß (GSK-3ß) and Na+/K+-ATPase α-subunits in cardiac tissue were analyzed by western blot. GR distribution between cytosolic and nuclear fractions was also analyzed. The fructose-rich diet decreased the level of pERK1/2 (Thr202/Tyr204) and pGSK-3ß (Ser9) independently of stress, while chronic stress increased the IRS1 content and prevented the fructose diet-induced decrease of the pAkt (Ser473) level. The fructose-rich diet in combination with chronic stress reduced the protein content of cardiac IR and attenuated IRS1 upregulation. Separate treatments increased the protein content of Na+/K+-ATPase α1- and α2-subunits, while after combined treatment the α2 content was at the control level and the α1 content was lower than the control level. The effect of combined treatment on cardiac IR and α2-subunit expression could be mediated by increased GR nuclear accumulation. Our study provides new insights into the effects of chronic stress and a combination of the fructose diet and chronic stress on the studied molecules in the heart.

[Effects of AdipoRon orally on the functions of spleen and pancreas in type 2 diabetic mice].

OBJECTIVE: To observe the effects of AdipoRon orally on the functions of spleen and pancreas in type 2 diabetic mice, in order to present data for clinical application. METHODS: Forty C57/BL6 male mice were randomly divided into 2 groups: normal control group (n=10) and model group (n=30), the former group was fed normally, while the later group was fed with high fat and sugar for 4 weeks.After that, type 2 diabetes model was established in DM group induced by intraperitoneal injection of streptozotocin (STZ, 40 mg/kg).As type 2 diabetes model established successfully, the model mice were randomly divided into three groups (n=10): diabetes mellitus (DM) group, high dose of AdipoRon group (DM + H) and low dose of adiponRon group (DM + L).All the four groups were treated with saline, saline, AdipoRon at the doses of 20 mg/kg and 50 mg/kg by gavages respectively, once a day for 10 days.And then put them to death for collecting blood, pancreas and spleen.Pathological changes of pancreas were observed with a light microscope after HE staining.Protein contents of insulin receptor (INSR), insulin receptor substrate 1( IRS-1) and tumor necrosis factor-α(TNF-α) in pancreatic and spleen tissues were detected by ELISA.The protein level of phosphorylation insulin receptor substrate 1(p-IRS-1) in pancreas was determined by Western blot, and the expression of insulin mRNA in pancreas was tested by RT-PCR. RESULTS: Under the light microscope, it was visible that the pancreatic tissue in NC group was full and closely packed, and the islet was big.Pancreatic tissue of DM mice was incompact and the islet of DM mice was smaller than that of normal mice.As for the mice treated with AdipoRon orally, the pancreatic tissue was full and closely arranged, and the islet was slightly smaller.Compared with NC group, the levels of TNF-α in pancreas and spleen of DM group were increased markedly, the levels of INSR and IRS-1 were decreased, the spleen coefficient, p-IR-1 protein level and insulin mRNA expression in pancreas were decreased, all were significant statistically (P＜0.05).Compared with DM group, the levels of TNF-α in pancreas and spleen of AdipoRon groups were decreased, the levels of INSR and IRS-1 in pancreas and spleen of AdipoRon groups were increased, while the spleen coefficient was increased (P＜0.05).The p-IRS-1 protein level and insulin mRNA expression in pancreas in DM+H group were increased (P＜0.05).Compared with DM + L group, the level of TNF-α was decreased, and the levels of INSR and IRS-1 were significantly increased (P＜0.05) in DM + H group (P＜0.05). CONCLUSION: Oral administration of AdipoRon can protect the spleen and pancreas of diabetic mice by decreasing the inflammatory response, up-regulating the expression of INSR, and increasing p-IRS-1 level in diabetic mice.

Antitumor effects of ß-elemene via targeting the phosphorylation of insulin receptor.

Ewing sarcoma family tumors (ESFTs) are a group of aggressive and highly metastatic tumors lacking efficient therapies. Insulin-like growth factor 1 receptor (IGF1R) blockade is one of the most efficient targeting therapy for ESFTs. However, the appliance is obstructed by drug resistance and disease recurrence due to the activation of insulin receptor (IR) signaling induced by IGF1R blockade. Herein ß-elemene, a compound derived from natural plants, exhibited a remarkable proliferation repression on ESFT cells, which was weakened by a caspase inhibitor Z-VAD. ß-elemene in combination with IGF1R inhibitors enhanced markedly the repression on cellular proliferation and mTOR activation by IGF1R inhibitors and suppressed the PI3K phosphorylation induced by IGF1R inhibitors. To investigate the mechanisms, we focused on the effects of ß-elemene on IR signaling pathway. ß-elemene significantly suppressed the insulin-driven cell growth and the activation of mTOR and PI3K in tumor cells, while the toxicity to normal hepatocytes was much lower. Further, the phosphorylation of IR was found to be suppressed notably by ß-elemene specifically in tumor cells other than normal hepatocytes. In addition, ß-elemene inhibited the growth of ESFT xenografts in vivo, and the phosphorylation of IR and S6 ribosomal protein was significantly repressed in the ß-elemene-treated xenografts. These data suggest that ß-elemene targets IR phosphorylation to inhibit the proliferation of tumor cells specifically and enhance the effects of IGF1R inhibitors. Thus, this study provides evidence for novel approaches by ß-elemene alone or in combination with IGF1R blockades in ESFTs and IR signaling hyperactivated tumors.

Effect of metformin and flutamide on insulin, lipogenic and androgen-estrogen signaling, and cardiometabolic risk in a PCOS-prone metabolic syndrome rodent model.

Polycystic ovary syndrome (PCOS) is highly associated with cardiometabolic risk and the metabolic syndrome (MetS), predisposing women to increased risk of developing type 2 diabetes and cardiovascular disease. Metformin is commonly used to treat insulin resistance-glucose intolerance, and flutamide, an androgen receptor (AR) antagonist, is used to target hyperandrogenemia and dyslipidemia. Currently, the physiological mechanism of action of these treatments on androgen, lipidogenic, and insulin signaling pathways remains unclear in PCOS. The aim of this study was to investigate the effects and mechanisms of action of metformin and flutamide on plasma lipid-apolipoprotein (Apo)B-lipoprotein and insulin-glucose metabolism, and endocrine-reproductive indices in a PCOS-prone MetS rodent model. PCOS-prone rodents were treated with metformin (300 mg/kg body wt), flutamide (30 mg/kg body wt), or metformin + flutamide combination treatment for 6 wk. Metformin was shown to improve fasting insulin and HOMA-IR, whereas flutamide and combination treatment were shown to reduce plasma triglycerides, ApoB48, and ApoB100, and this was associated with decreased intestinal secretion of ApoB48/triglyceride. Flutamide and metformin were shown to reduce plasma androgen indices and to improve ovarian primary and preovulatory follicle frequency. Metformin treatment increased hepatic estrogen receptor (ER)α, and metformin-flutamide decreased intestinal AR and increased ERα mRNA expression. Metformin-flutamide treatment upregulated hepatic and intestinal insulin signaling, including insulin receptor, MAPK1, and AKT2. In conclusion, cardiometabolic risk factors, in particular ApoB-hypertriglyceridemia, are independently modulated via the AR, and understanding the contribution of AR and insulin-signaling pathways further may facilitate the development of targeted interventions in high-risk women with PCOS and MetS.

A Novel Strategy to Prevent Advanced Atherosclerosis and Lower Blood Glucose in a Mouse Model of Metabolic Syndrome.

Cardiovascular disease caused by atherosclerosis is the leading cause of mortality associated with type 2 diabetes and metabolic syndrome. Insulin therapy is often needed to improve glycemic control, but it does not clearly prevent atherosclerosis. Upon binding to the insulin receptor (IR), insulin activates distinct arms of downstream signaling. The IR-Akt arm is associated with blood glucose lowering and beneficial effects, whereas the IR-Erk arm might exert less desirable effects. We investigated whether selective activation of the IR-Akt arm, leaving the IR-Erk arm largely inactive, would result in protection from atherosclerosis in a mouse model of metabolic syndrome. The insulin mimetic peptide S597 lowered blood glucose and activated Akt in insulin target tissues, mimicking insulin's effects, but only weakly activated Erk and even prevented insulin-induced Erk activation. Strikingly, S597 retarded atherosclerotic lesion progression through a process associated with protection from leukocytosis, thereby reducing lesional accumulation of inflammatory Ly6Chi monocytes. S597-mediated protection from leukocytosis was accompanied by reduced numbers of the earliest bone marrow hematopoietic stem cells and reduced IR-Erk activity in hematopoietic stem cells. This study provides a conceptually novel treatment strategy for advanced atherosclerosis associated with metabolic syndrome and type 2 diabetes.

A glucose-responsive insulin therapy protects animals against hypoglycemia.

Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.

Prenatal testosterone exposure decreases colocalization of insulin receptors in kisspeptin/neurokinin B/dynorphin and agouti-related peptide neurons of the adult ewe.

Insulin serves as a link between the metabolic and reproductive systems, communicating energy availability to the hypothalamus and enabling reproductive mechanisms. Adult Suffolk ewes prenatally exposed to testosterone (T) display an array of reproductive and metabolic dysfunctions similar to those seen in women with polycystic ovarian syndrome (PCOS), including insulin resistance. Moreover, prenatal T treatment alters neuropeptide expression in KNDy (co-expressing kisspeptin, neurokinin B/dynorphin) and agouti-related peptide (AgRP) neurons in the arcuate nucleus, two populations that play key roles in the control of reproduction and metabolism, respectively. In this study, we determined whether prenatal T treatment also altered insulin receptors in KNDy and AgRP neurons, as well as in preoptic area (POA) kisspeptin, pro-opiomelanocortin (POMC), and gonadotropin-releasing hormone (GnRH) neurons of the adult sheep brain. Immunofluorescent detection of the beta subunit of insulin receptor (IRß) revealed that KNDy, AgRP and POMC neurons, but not GnRH or POA kisspeptin neurons, colocalize IRß in control females. Moreover, prenatal T treatment decreased the percentage of KNDy and AgRP neurons that colocalized IRß, consistent with reduced insulin sensitivity. Administration of the anti-androgen drug, Flutamide, during prenatal T treatment, prevented the reduction in IRß colocalization in AgRP, but not in KNDy neurons, suggesting that these effects are programmed by androgenic and oestrogenic actions, respectively. These findings provide novel insight into the effects of prenatal T treatment on hypothalamic insulin sensitivity and raise the possibility that decreased insulin receptors, specifically within KNDy and AgRP neurons, may contribute to the PCOS-like phenotype of this animal model.

Compounds from the marine sponge Cribrochalina vasculum offer a way to target IGF-1R mediated signaling in tumor cells.

In this work two acetylene alcohols, compound 1 and compound 2, which were isolated and identified from the sponge Cribrochalina vasculum, and which showed anti-tumor effects were further studied with respect to targets and action mechanisms. Gene expression analyses suggested insulin like growth factor receptor (IGF-1R) signaling to be instrumental in controlling anti-tumor efficacy of these compounds in non-small cell lung cancer (NSCLC). Indeed compounds 1 and 2 inhibited phosphorylation of IGF-1Rß as well as reduced its target signaling molecules IRS-1 and PDK1 allowing inhibition of pro-survival signaling. In silico docking indicated that compound 1 binds to the kinase domain of IGF-1R at the same binding site as the well known tyrosine kinase inhibitor AG1024. Indeed, cellular thermal shift assay (CETSA) confirmed that C. vasculum compound 1 binds to IGF-1R but not to the membrane localized tyrosine kinase receptor EGFR. Importantly, we demonstrate that compound 1 causes IGF-1Rß but not Insulin Receptor degradation specifically in tumor cells with no effects seen in normal diploid fibroblasts. Thus, these compounds hold potential as novel therapeutic agents targeting IGF-1R signaling for anti-tumor treatment.

Nanoparticle Delivered Human Biliverdin Reductase-Based Peptide Increases Glucose Uptake by Activating IRK/Akt/GSK3 Axis: The Peptide Is Effective in the Cell and Wild-Type and Diabetic Ob/Ob Mice.

Insulin's stimulation of glucose uptake by binding to the IRK extracellular domain is compromised in diabetes. We have recently described an unprecedented approach to stimulating glucose uptake. KYCCSRK (P2) peptide, corresponding to the C-terminal segment of hBVR, was effective in binding to and inducing conformational change in the IRK intracellular kinase domain. Although myristoylated P2, made of L-amino acids, was effective in cell culture, its use for animal studies was unsuitable. We developed a peptidase-resistant formulation of the peptide that was efficient in both mice and cell culture systems. The peptide was constructed of D-amino acids, in reverse order, and blocked at both termini. Delivery of the encapsulated peptide to HepG2 and HSKM cells was confirmed by its prolonged effect on stimulation of glucose uptake (>6 h). The peptide improved glucose clearance in both wild-type and Ob/Ob mice; it lowered blood glucose levels and suppressed glucose-stimulated insulin secretion. IRK activity was stimulated in the liver of treated mice and in cultured cells. The peptide potentiated function of IRK's downstream effector, Akt-GSK3-(α, ß) axis. Thus, P2-based approach can be used for improving glucose uptake by cells. Also, it allows for screening peptides in vitro and in animal models for treatment of diabetes.

Growth Modulation of Diabetic Factors and Antidiabetic Drugs on Prostate Cancer Cell Lines.

Risk factors for prostate cancer (PCa) include age, hormones, race, family history and diet. Recently, epidemiologic evidence has indicated that history of diabetes mellitus (DM) is inversely associated with risk of PCa. However, epidemiological investigations have yielded inconsistent results. Hence, the exact mechanism of DM-induced reduction in the incidence of PCa has yet to be fully elucidated. The aim of this study was to investigate the effects of DM factors, including glucose, insulin and insulin-like growth factor-1 (IGF-1), on the proliferation of PCa cell lines in vitro. Cell proliferation and expression of hormone receptors was examined in MTT assay and Western blot analysis, respectively. The results showed that DM factors did not affect the viability of androgen receptor (AR)-expressing PCa cell lines. However, cell proliferation increased after treatment with DM factors in androgen-independent PCa cell lines. On PCa tissue arrays, intensities of total AR and nuclear IGF-1R were higher in malignant tissues than in normal prostate glands. In terms of hormonal receptors, androgen-dependent LNCaP cells treated with insulin and IGF-1 in a low-serum medium showed decreased expression of insulin receptor beta (IRß) and elevated expression of IGF-1 receptor beta (IGF-1Rß). Moreover, expression of AR was upregulated after insulin and IGF-1 treatment in LNCaP cells, but not in the other PCa cell lines. Most of the studied antidiabetic drugs promoted the viability of PCa cells. However, metformin decreased the viability of AR-expressing PCa cells. These results suggest that diabetic factors modify the expression of AR, IR and IGF-1R to increase cancer cell proliferation. Moreover, the growth suppressing effects of metformin on PCa may be via the regulation of the AR signaling pathway.

Impaired stimulation of glucose transport in cardiac myocytes exposed to very low-density lipoproteins.

We recently observed that free fatty acids impair the stimulation of glucose transport into cardiomyocytes in response to either insulin or metabolic stress. In vivo, fatty acids for the myocardium are mostly obtained from triglyceride-rich lipoproteins (chylomicrons and Very Low-Density Lipoproteins). We therefore determined whether exposure of cardiac myocytes to VLDL resulted in impaired basal and stimulated glucose transport. Primary adult rat cardiac myocytes were chronically exposed to VLDL before glucose uptake was measured in response to insulin or metabolic stress, provoked by the mitochondrial ATP synthase inhibitor oligomycin. Exposure of cardiac myocytes to VLDL reduced both insulin-and oligomycin-stimulated glucose uptake. The reduction of glucose uptake was associated with a moderately reduced tyrosine phosphorylation of the insulin receptor. No reduction of the phosphorylation of the downstream effectors of insulin signaling Akt and AS160 was however observed. Similarly only a modest reduction of the activating phosphorylation of the AMP-activated kinase (AMPK) was observed in response to oligomycin. Similar to our previous observations with free fatty acids, inhibition of fatty acid oxidation restored oligomycin-stimulated glucose uptake. In conclusions, VLDL-derived fatty acids impair stimulated glucose transport in cardiac myocytes by a mechanism that seems to be mediated by a fatty acid oxidation intermediate. Thus, in the clinical context of the metabolic syndrome high VLDL may contribute to enhancement of ischemic injury by reduction of metabolic stress-stimulated glucose uptake.

Neuregulin improves response to glucose tolerance test in control and diabetic rats.

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

Molecular targets of the multifunctional iron-chelating drug, M30, in the brains of mouse models of type 2 diabetes mellitus.

BACKGROUND AND PURPOSE: Neurodegenerative diseases are now recognized to be multifunctional, whereby a heterogeneous set of reactions acts independently or cooperatively, leading eventually to the demise of neurons. This has led our group to design and synthesize the multifunctional, nontoxic, brain-permeable, iron chelator compound M30 with a range of pharmacological properties. Here, we have characterized the molecular targets of M30 in the brains of animal models of type 2 diabetes mellitus (T2DM). EXPERIMENTAL APPROACH: Effects of M30 on molecular mechanisms associated with neuroprotection in the CNS were investigated-in the high-fat diet (HFD) and ob/ob transgenic mouse models of T2DM, using real-time PCR and Western blotting analyses. Brain monoamine oxidase (MAO) activity and catecholamine levels, and peripheral glucose tolerance were assayed after treatment in vivo. KEY RESULTS: M30 increased cerebral levels of insulin and insulin receptor and phosphorylated-GSK-3ß in HFD mice, compared with vehicle-treated HFD mice. In both T2DM mice models, M30 treatment significantly up-regulated cerebral hypoxia-inducible factor (HIF)-1α protein levels and induced the expression of several HIF-1 target genes involved in neuroprotection, glycolysis, neurogenesis, oxidative stress and anti-inflammation. Additionally, M30 inhibited MAO-A and -B activities in the cerebellum. Accordingly, M30 administration significantly reduced brain levels of dopamine metabolites and increased levels of 5-HT and noradrenaline. Glucose tolerance was also improved after M30 treatment in both models of T2DM. CONCLUSIONS AND IMPLICATIONS: In the brain of HFD and ob/ob transgenic mice, M30 exerted a variety of beneficial neuroprotective regulatory effects that may act synergistically to delay or prevent neurodegenerative processes associated with T2DM.

Zinc ions modulate protein tyrosine phosphatase 1B activity.

Protein tyrosine phosphatases (PTPs) are key enzymes in cellular regulation. The 107 human PTPs are regulated by redox signalling, phosphorylation, dimerisation, and proteolysis. Recent findings of very strong inhibition of some PTPs by zinc ions at concentrations relevant in a cellular environment suggest yet another mechanism of regulation. One of the most extensively investigated PTPs is PTP1B (PTPN1). It regulates the insulin and leptin signalling pathway and is implicated in cancer and obesity/diabetes. The development of novel assay conditions to investigate zinc inhibition of PTP1B provides estimates of about 5.6 nM affinity for inhibitory zinc(II) ions. Analysis of three PTP1B 3D structures (PDB id: 2CM2, 3I80 and 1A5Y) identified putative zinc binding sites and supports the kinetic studies in suggesting an inhibitory zinc only in the closed and cysteinyl-phosphate intermediate forms of the enzyme. These observations gain significance with regard to recent findings of regulatory roles of zinc ions released from the endoplasmic reticulum.

Cocoa polyphenols suppress adipogenesis in vitro and obesity in vivo by targeting insulin receptor.

OBJECTIVE: To investigate the inhibitory effect of cocoa polyphenol extract (CPE) on adipogenesis and obesity along with its mechanism of action. METHODS AND RESULTS: 3T3-L1 preadipocytes were cultured with isobutylmethylxanthine, dexamethasone and insulin (MDI), and male C57BL/6N mice (N=44) were fed a high-fat diet (HFD) for 5 weeks with or without CPE. CPE at 100 or 200 µg ml(-1) inhibited MDI-induced lipid accumulation without diminishing cell viability. In particular, CPE reduced the protein expression levels of PPARγ and CEBPα, and blocked mitotic clonal expansion (MCE) of preadipocytes by reducing proliferating signaling pathways. This in turn attenuates lipid accumulation during the differentiation of 3T3-L1 preadipocytes. CPE effectively suppressed MDI-induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and their downstream signals. We then examined whether CPE regulates insulin receptor (IR), a common upstream regulator of ERK and Akt. We found that although CPE does not affect the protein expression level of IR, it significantly inhibits the activity of IR kinase via direct binding. Collectively, the results suggested that CPE, a direct inhibitor of IR kinase activity, inhibits cellular differentiation and lipid accumulation in 3T3-L1 preadipocytes. Consistently, CPE attenuated HFD-induced body weight gain and fat accumulation in obese mice fed with a HFD. We also found that HFD-induced increased fasting glucose levels remained unaffected by CPE. CONCLUSION: This study demonstrates that CPE inhibits IR kinase activity and its proliferative downstream signaling markers, such as ERK and Akt, in 3T3-L1 preadipocytes, and also prevents the development of obesity in mice fed with a HFD.

Berberine activates Nrf2 nuclear translocation and protects against oxidative damage via a phosphatidylinositol 3-kinase/Akt-dependent mechanism in NSC34 motor neuron-like cells.

Berberine (BBR) is a well-known anti-diabetic herbal medicine in Asia due to its beneficial effects on insulin sensitivity, glucose metabolism and glycolysis. Here, we identified the critical role of phosphatidylinositol 3-kinase (PI3K)/Akt involved BBR cellular defense mechanisms and first revealed the novel effect of BBR on nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2)/heme oxygenase (HO)-1 induction in NSC34 motor neuron-like cells. BBR (0.1-10 nM) led to increasing insulin receptor expression, Akt phosphorylation and enhanced oxidant-sensitive Nrf2/HO-1 induction, which were blocked by a PI3K inhibitor, LY294002. In H(2)O(2)-treated cells, BBR significantly attenuated ROS production and increased cell viability, antioxidant defense (GSH and SOD) and oxidant-sensitive proteins (HO-1 and Nrf2), which also were blocked by LY294002. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential and decreasing the oxygen consumption rate. BBR-induced anti-apoptotic function was demonstrated by increasing anti-apoptotic protein Bcl-2 and survival of motor neuron protein (SMN) and by decreasing apoptotic proteins (cytochrome c, Bax and caspase). These results suggest that BBR, which is active at nanomolar concentration, is a potential neuroprotective agent via PI3K/Akt-dependent cytoprotective and antioxidant pathways.

Periodontal disease decreases insulin sensitivity and insulin signaling.

BACKGROUND: The purpose of this study is to investigate whether local inflammatory events, such as periodontal disease, are able to increase tumor necrosis factor-alpha (TNF-α) plasmatic concentration and decrease insulin sensitivity and insulin signaling in non-diabetic rats. METHODS: Forty-eight male Wistar rats (2 months old) were divided into two groups, with either ligature-induced periodontal disease (LPD) or control conditions (CN). Experiments were performed in both groups 28 days after ligature placement. Plasmatic concentration of glycemia and TNF-α (n = 10) were analyzed by the glucose oxidase and enzyme-linked immunosorbent assay method, respectively. Insulin sensitivity (n = 7) was measured using the insulin tolerance test. Insulin signal transduction (n = 7) was measured by pp185 tyrosine phosphorylation status in insulin-sensitive tissues using the Western blotting method. RESULTS: The LPD group showed decreased insulin sensitivity (P <0.05), although no glycemic alterations were noted (P >0.05). TNF-α plasmatic concentration was higher in LPD rats compared to CN rats. In addition, a decrease in the pp185 tyrosine phosphorylation status was observed after insulin stimulus in both white adipose and skeletal muscle tissues of the LPD group compared with the CN group. CONCLUSIONS: LPD is able to cause alterations to both insulin signaling and insulin sensitivity, probably because of the elevation of TNF-α plasmatic concentration. Thus, the present results emphasize the importance of the prevention of local inflammatory diseases, such as periodontitis, to prevent diabetes mellitus.