The Role of PTP1B O-GlcNAcylation in Hepatic Insulin Resistance

Protein tyrosine phosphatase 1B (PTP1B), which can directly dephosphorylate both the insulin receptor and insulin receptor substrate 1 (IRS-1), thereby terminating insulin signaling, reportedly plays an important role in insulin resistance. Accumulating evidence has demonstrated that O-GlcNAc modification regulates functions of several important components of insulin signal pathway. In this study, we identified that PTP1B is modified by O-GlcNAcylation at three O-GlcNAc sites (Ser104, Ser201, and Ser386). Palmitate acid (PA) impaired the insulin signaling, indicated by decreased phosphorylation of both serine/threonine-protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) following insulin administration, and upregulated PTP1B O-GlcNAcylation in HepG2 cells. Compared with the wild-type, intervention PTP1B O-GlcNAcylation by site-directed gene mutation inhibited PTP1B phosphatase activity, resulted in a higher level of phosphorylated Akt and GSK3β, recovered insulin sensitivity, and improved lipid deposition in HepG2 cells. Taken together, our research showed that O-GlcNAcylation of PTP1B can influence insulin signal transduction by modulating its own phosphatase activity, which participates in the process of hepatic insulin resistance.     

Fraction from Wax Apple [Syzygium samarangense (Blume) Merrill and Perry] Fruit Extract Ameliorates Insulin Resistance via Modulating Insulin Signaling and Inflammation Pathway in Tumor Necrosis Factor α-Treated FL83B Mouse Hepatocytes

Inflammation is associated with the development of insulin resistance in Type 2 diabetes mellitus. In the present study, mouse FL83B cells were treated with tumor necrosis factor-alpha (TNF-α) to induce insulin resistance, and then co-incubated with a fraction from wax apple fruit extract (FWFE). This fraction significantly increased the uptake of the nonradioactive fluorescent indicator 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) in insulin resistant cells. Western blot analysis revealed that, compared with the TNF-α-treated control group, FWFE increased the expression of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), protein kinase B (Akt/PKB), phosphatidylinositol-3 kinase (PI3K), and glucose transporter 2 (GLUT-2), and increased IR tyrosyl phosporylation, in insulin resistant FL83B cells. However, FWFE decreased phosphorylation of c-Jun N-terminal kinases (JNK), but not the expression of the intercellular signal-regulated kinases (ERK), in the same cells. These results suggest that FWFE might alleviate insulin resistance in TNF-α-treated FL83B cells by activating PI3K-Akt/PKB signaling and inhibiting inflammatory response via suppression of JNK, rather than ERK, activation.     

Comparing the effects of palmitate,insulin, and palmitate-insulin co-treatment on myotube metabolism and insulin resistance

Previous studies have shown various metabolic stressors such as saturated fatty acids (SFA) and excess insulin promote insulin resistance in metabolically meaningful cell types (such as skeletal muscle). Additionally, these stressors have been linked with suppressed mitochondrial metabolism, which is also a common characteristic of skeletal muscle of diabetics. This study characterized the individual and combined effects of excess lipid and excess insulin on myotube metabolism and related metabolic gene and protein expression. C2C12 myotubes were treated with either 500 μM palmitate (PAM), 100 nM insulin (IR), or both (PAM-IR). qRT-PCR and western blot were used to measure metabolic gene and protein expression, respectively. Oxygen consumption was used to measure mitochondrial metabolism. Glycolytic metabolism and insulin-mediated glucose uptake were measured via extracellular acidification rate. Cellular lipid and mitochondrial content were measured using Nile Red and NAO staining, respectively. IR and PAM-IR treatments led to reductions in p-Akt expression. IR treatment reduced insulin mediated glucose metabolism while PAM and PAM-IR treatment showed increases with concurrent reductions in mitochondrial metabolism. All three treatments showed suppression in mitochondrial metabolism. PAM and PAM-IR also showed increases in glycolytic metabolism. While PAM and PAM-IR significantly increased lipid content, expression of inflammatory and lipogenic proteins were unaltered. Lastly, PAM-IR reduced BCAT2 protein expression, a regulator of BCAA metabolism. Both stressors independently reduced insulin signaling, mitochondrial function, and cell metabolism, however, only PAM-IR co-treatment significantly reduced the expression of regulators of metabolism not seen with individual stressors, suggesting an additive effect of stressors on metabolic programming.     

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.     

Cellular inhibition of protein tyrosine phosphatase 1B by uncharged thioxothiazolidinone derivatives

As important regulators of cellular signal transduction, members of the protein tyrosine phosphatase (PTP) family are considered to be promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, thus limiting cellular permeability. Here, we have identified an uncharged thioxothiazolidinone derivative (compound 1), as a competitive inhibitor of a subset of PTPs. Compound 1 effectively inhibited protein tyrosine phosphatase 1B (PTP1B) in two cell-based systems: it sensitized wild-type, but not PTP1B-null fibroblasts to insulin stimulation and prevented PTP1B-dependent dephosphorylation of the FLT3-ITD receptor tyrosine kinase. We have also tested a series of derivatives in vitro against PTP1B and proposed a model of the PTP1B-inhibitor interaction. These compounds should be useful in the elucidation of cellular PTP function and could represent a starting point for development of therapeutic PTP inhibitors.     

C‐reactive protein upregulates lectin‐like oxidized LDL receptor‐1 expression in THP‐1‐derived macrophages through NF‐κB

Lectin‐like oxidized LDL receptor‐1 (LOX‐1), a newly identified scavenger receptor, has been increasingly linked to atherosclerosis. C‐reactive protein (CRP), a prototypic inflammatory marker, has been proven to promote atherogenesis. In this study, we evaluated the in vitro effects of CRP on LOX‐1 expression and the associated signal transduction pathway in THP‐1‐derived macrophages. Our study showed that incubation of macrophages with CRP significantly enhanced expression of LOX‐1 protein and mRNA levels in macrophages in a dose‐dependent manner; this expression could be suppressed by the nuclear factor kappa B (NF‐κB) pathway inhibitor BAY11‐7085. However, LOX‐1 was not inhibited by the inhibitor of mitogen‐activated protein kinase (MAPK) proteins (SP600125‐JNK/SAPK, SB203580‐p38, and U0126‐ERK1/2) in macrophages. In conclusion, human native CRP up‐regulated LOX‐1 expression in THP‐1‐derived macrophages primarily through the NF‐κB signaling pathway. Practical applications: Identification of LOX‐1 and definition of its biological role in pathophysiological states provided a new clue for understanding the nature of oxLDL uptake into macrophages. Internalization of modified lipoprotein by macrophages and foam cell formation are critical events in hypertension, diabetes mellitus, and dyslipidemia, which are the most important risk factors for atherosclerosis. As a characteristic inflammatory marker, CRP has been proven to play a pivotal role in promoting atherogenesis. However, crosstalk between CRP and LOX‐1 on macrophages has not been elucidated. Therefore, determining the regulatory process for LOX‐1 and the underlying signal transduction pathways may provide a new insight into the mechanism of atherosclerosis.     

激活素受体相互作用蛋白在海马神经细胞中的表达及其作用

目的　在发现和克隆新的激活素受体相互作用蛋白 3(ActRIP3)基因的基础上 ,探讨ActRIP3在海马神经细胞介导激活素信号传导的作用。方法　利用GST- ActRIP3融合蛋白免疫家兔制备抗ActRIP3抗体 ,采用RT -PCR及免疫组化染色分析ActRIP3在脑组织中的表达与分布 ,采用pcDNA-ActRIP3与CAGA -lux质粒共转染海马神经细胞 ,分析信号传导作用。结果RT- PCR及免疫组化染色显示ActRIP3在海马神经细胞中高表达 ,构建的pcDNA- ActRIP3表达载体在体外培养海马神经细胞中可有效表达ActRIP3蛋白 ,过表达ActRIP3可促进激活素诱导的海马神经细胞特异信号传导。结论　ActRIP3具有促进激活素信号传导作用 ,是介导激活素作用于海马神经细胞的关键性信号传导蛋白。     

Identification of Molecular Markers of Clozapine Action in Ketamine-Induced Cognitive Impairment: A GPCR Signaling PathwayFinder Study

Background: Cognitive disorders associated with schizophrenia are closely linked to prefrontal cortex (PFC) dysfunction. Administration of the non-competitive NMDA receptor antagonist ketamine (KET) induces cognitive impairment in animals, producing effects similar to those observed in schizophrenic patients. In a previous study, we showed that KET (20 mg/kg) induces cognitive deficits in mice and that administration of clozapine (CLZ) reverses this effect. To identify biochemical mechanisms related to CLZ actions in the context of KET-induced impairment, we performed a biochemical analysis using the same experimental paradigm—acute and sub-chronic administration of these drugs (0.3 and 1 mg/kg). Methods: Since the effect of CLZ mainly depends on G-protein-related receptors, we used the Signaling PathwayFinder Kit to identify 84 genes involved in GPCR-related signal transduction and then verified the genes that were statistically significantly different on a larger group of mice using RT-PCR and Western blot analyses after the administration of acute and sub-chronic drugs. Results: Of the 84 genes involved in GPCR-related signal transduction, the expression of six, βarrestin1, βarrestin2, galanin receptor 2 (GalR2), dopamine receptor 2 (DRD2), metabotropic glutamate receptor 1 (mGluR1), and metabotropic glutamate receptor 5 (mGluR5), was significantly altered. Since these genes affect the levels of other signaling proteins, e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), G protein-coupled receptor kinase 2 (Grk2), and G protein-gated inwardly rectifying potassium 3 (Girk3), we determined their levels in PFC using Western blot. Most of the observed changes occurred after acute treatment with 0.3 mg/kg CLZ. We showed that acute treatment with CLZ at a lower dose significantly increased βarrestin1 and ERK1/2. KET treatment induced the upregulation of βarrestin1. Joint administration of these drugs had no effect on the βarrestin1 level. Conclusion: The screening kit we used to study the expression of GPCR-related signal transduction allowed us to select several important genes affected by CLZ. However, the obtained data do not explain the mechanism of action of CLZ that is responsible for reversing KET-induced cognitive impairment.     

Hyperphosphorylation of Tau Due to the Interference of Protein Phosphatase Methylesterase-1 Overexpression by MiR-125b-5p in Melatonin Receptor Knockout Mice

Melatonin has been indicated to ameliorate tau hyperphosphorylation in the pathogenesis of tau diseases, but the role of melatonin-receptor signal transduction has not been clearly discovered. In this study, we found intensive tau hyperphosphorylation in melatonin receptor knockout mice. Bielschowsky silver staining showed ghostlike neurofibrillary tangles in melatonin receptor-2 knockout (MT2KO) as well as melatonin receptors-1 and -2 knockout (DKO) mice, and an argyrophilic substance was deposited in melatonin receptor-1 knockout (MT1KO) mice. Furthermore, we found significantly decreased activity of protein phosphatase 2A (PP2A) by Western blot and enzyme-linked immunosorbent assay (ELISA), which was partly due to the overexpression of protein phosphatase methylesterase-1 (PME-1), but not glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (CDK5) or protein kinase B (Akt). Finally, we observed a significant increase in cyclic adenosine monophosphate (cAMP) and a decrease in miR-125b-5p levels in MT1KO, MT2KO and DKO mice. Using a luciferase reporter assay, we discovered that miR-125b-5p largely decreased the expression of firefly luciferase by interfering with the 3′UTR of PME-1. Furthermore, miR-125b-5p mimics significantly decreased the expression of PME-1, while miR-125b-5p inhibitor induced tau hyperphosphorylation. These results show that melatonin-receptor signal transduction plays an important role in tau hyperphosphorylation and tangle formation.     

Virtual Screening of Specific Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors from the National Cancer Institute (NCI) Molecular Database

Insulin-like growth factor 1 receptor (IGF1R) is an attractive drug target for cancer therapy and research on IGF1R inhibitors has had success in clinical trials. A particular challenge in the development of specific IGF1R inhibitors is interference from insulin receptor (IR), which has a nearly identical sequence. A few potent inhibitors that are selective for IGF1R have been discovered experimentally with the aid of computational methods. However, studies on the rapid identification of IGF1R-selective inhibitors using virtual screening and confidence-level inspections of ligands that show different interactions with IGF1R and IR in docking analysis are rare. In this study, we established virtual screening and binding-mode prediction workflows based on benchmark results of IGF1R and several kinase receptors with IGF1R-like structures. We used comprehensive analysis of the known complexes of IGF1R and IR with their binding ligands to screen specific IGF1R inhibitors. Using these workflows, 17 of 139,735 compounds in the NCI (National Cancer Institute) database were identified as potential specific inhibitors of IGF1R. Calculations of the potential of mean force (PMF) with GROMACS were further conducted for three of the identified compounds to assess their binding affinity differences towards IGF1R and IR.     

Mechanisms of Insulin Resistance at the Crossroad of Obesity with Associated Metabolic Abnormalities and Cognitive Dysfunction

Obesity mediates most of its direct medical sequelae through the development of insulin resistance (IR). The cellular effects of insulin occur through two main postreceptor pathways that are the phosphatidylinositol 3-kinase (PI3-K) and the mitogen-activated protein kinase (MAP-K) pathways. Obesity-related IR implicates the PI3-K pathway that confers the metabolic effects of insulin. Numerous and complex pathogenic pathways link obesity with the development of IR, including chronic inflammation, mitochondrial dysfunction (with the associated production of reactive oxygen species and endoplasmic reticulum stress), gut microbiota dysbiosis and adipose extracellular matrix remodelling. IR itself plays a key role in the development of metabolic dysfunction, including hypertension, dyslipidaemia and dysglycaemia. Furthermore, IR promotes weight gain related to secondary hyperinsulinaemia, with a resulting vicious cycle of worsening IR and its metabolic sequelae. Ultimately, IR underlies obesity-related conditions such as type 2 diabetes mellitus (T2D) and polycystic ovary syndrome (PCOS). IR also underlies many obesity-related malignancies, through the effects of compensatory hyperinsulinaemia on the relatively intact MAP-K insulin pathway, which controls cellular growth processes and mitoses. Furthermore, the emergent data over recent decades support an important role of obesity- and T2D-related central IR in the development of cognitive dysfunction, including effects on hippocampal synaptic plasticity. Importantly, IR is largely reversible through the optimisation of lifestyle factors that include regular engagement in physical activity with the avoidance of sedentariness, improved diet including increased fibre intake and sleep sufficiency. IR lies at the key crossroad between obesity and both metabolic and cognitive dysfunction. Given the importance of IR in the pathogenesis of many 21st century chronic diseases and its eminent reversibility, it is important that we all embrace and facilitate optimised lifestyles to improve the future health and wellbeing of the populace.     

Small-molecule inhibitors of PDK1

Signal transduction of many growth factors and oncogenes is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1), a master regulator of a number of downstream signal protein kinase cascades. Hence, PDK1 represents a convergence point for receptor tyrosine kinase and cytokine-mediated pathways for the regulation of vital cell processes such as cell survival and proliferation. Pathological upregulation of PDK1 signalling due to constitutive growth factor receptor activation and/or PTEN (phosphatase and tensin homologue) mutations significantly triggers downstream signalling, e.g. PKB/Akt, which subsequently promote proliferative events such as tumour invasiveness, angiogenesis, and progression. Consistent with this, a mouse model expressing low levels of PDK1 is protected from tumourigenesis resulting from loss of PTEN. Because more than 50 % of all human cancers possess significant overstimulation of the PDK1 signalling pathway, inhibition of this protein kinase by small molecules is predicted to result in effective inhibition of cancer cell proliferation and thus be therapeutically beneficial. Various classes of small-molecule PDK1 inhibitors have been published in patents and papers. Herein we present for the first time a comprehensive collection of small molecules reported to interact with PDK1, and we refer to their biological characterisation in terms of activity and selectivity for PDK1.     

促凋亡蛋白BID多克隆抗体的制备与鉴定

目的制备抗BID的多克隆抗体,用于检测凋亡信号转导过程中BID蛋白的表达。方法采用PCR技术合成编码BID特异性肽段的基因,构建GST融合基因表达载体,在E.coli BL21中诱导表达GST-BID多肽的融合蛋白,经Glutathione Sepharose 4B纯化后免疫家兔,免疫血清经纯化后,得到抗BID的多克隆抗体,经Western blot鉴定其特异性。结果通过PCR扩增获得了BID肽段的编码基因;pGEX-6P-1-BID多肽重组质粒经酶切鉴定及测序证明构建正确;在E.coli BL21中表达了GST-BID多肽融合蛋白;纯化后蛋白纯度达95%;制备的抗BID多克隆抗体能够特异地识别BID蛋白。结论所制备的抗BID多克隆抗体可特异性检测BID蛋白。     

Effect of Black Soybean Koji Extract on Glucose Utilization and Adipocyte Differentiation in 3T3-L1 Cells

Adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and progression of diseases such as insulin resistance and obesity. Black soybean koji (BSK) is produced by the fermentation of black soybean with Aspergilllus awamori. Previous study indicated that BSK extract has antioxidative and multifunctional bioactivities, however, the role of BSK in the regulation of energy metabolism is still unclear. We aimed to investigate the effect of glucose utilization on insulin-resistant 3T3-L1 preadipocytes and adipogenesis-related protein expression in differentiated adipocytes with BSK treatment. Cytoxicity assay revealed that BSK did not adversely affect cell viability at levels up to 200 μg/mL. The potential for glucose utilization was increased by increased glucose transporter 1 (GLUT1), GLUT4 and protein kinase B (AKT) protein expression in insulin-resistant 3T3-L1 cells in response to BSK treatment. Simultaneously, BSK inhibited lipid droplet accumulation in differentiated 3T3-L1 cells. The inhibitory effect of adipogenesis was associated with downregulated peroxisome proliferator-activated receptor γ (PPARγ) level and upregulated Acrp30 protein expression. Our results suggest that BSK extract could improve glucose uptake by modulating GLUT1 and GLUT4 expression in a 3T3-L1 insulin-resistance cell model. In addition, BSK suppressed differentiation and lipid accumulation in mature 3T3-L1 adipocytes, which may suggest its potential for food supplementation to prevent obesity and related metabolic abnormalities.