右美托咪定对异丙酚麻醉所致新生大鼠脑损伤的保护作用及机制

高浓度的异丙酚可导致动物和人类发生脑损伤,而右美托咪定对多种脑损伤动物模型具有一定的神经保护作用。为了考察右美托咪定对异丙酚麻醉所致新生大鼠脑损伤的保护作用及机制,本研究对7日龄清洁级SD大鼠分别腹腔注射异丙酚(60 mg/kg)、右美托咪定(80μg/kg)和异丙酚(60 mg/kg)+右美托咪定(80μg/kg)。Morris水迷宫实验发现高剂量的异丙酚可显著增加大鼠的逃避潜伏期并减少穿越平台次数,然而右美托咪定预处理则可显著降低大鼠的逃避潜伏期并提高穿越平台次数(p<0.05)。异丙酚单独处理导致大鼠的海马神经元细胞凋亡程度显著增加,而右美托咪定预处理则可显著抑制神经元细胞的凋亡(p<0.05)。异丙酚单独处理可显著下调PSD95蛋白的表达,但右美托咪定预处理则可有效抑制PSD95蛋白的下调(p<0.05)。高剂量的异丙酚可明显下调大鼠海马组织P13K、Akt和GSK-3βmRNA的表达,而右美托咪定预处理则可抑制P13K、Akt和GSK-3βmRNA的下调。此外,右美托咪定预处理可显著提高p-Akt/Akt和p-GSK-3β/GSK-3β蛋白比值。本研究表明,右美托咪定可有效抑制异丙酚诱导的神经元细胞凋亡,改善大鼠的学习和记忆能力。右美托咪定的神经保护作用与其对PI3K/AKT/GSK-3β信号通路的激活有关。     

Gastrin-releasing peptide and its receptor increase arthritis fibroblast-like synoviocytes invasiveness through activating the PI3K/AKT pathway

Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. The fibroblast-like synoviocytes (FLS) has a central role on the disease pathophysiology. The present study aimed to examine the role of gastrin-releasing peptide (GRP) and its receptor (GRPR) on invasive behavior of mice fibroblast-like synoviocytes (FLS), as well as to evaluate GRP-induced signaling on PI3K/AKT pathway. The expression of GRPR in FLS was investigated by immunocytochemistry, western blot (WB) and qRT-PCR. The proliferation and invasion were assessed by SRB and matrigel-transwell assay after treatment with GRP and/or RC-3095 (GRPR antagonist), and/or Ly294002 (inhibitor of PI3K/AKT pathway). Finally, AKT phosphorylation was assessed by WB. GRPR protein was detected in FLS and the exposure to GRP increased FLS invasion by nearly two-fold, compared with untreated cells (p < 0.05), while RC-3095 reversed that effect (p < 0.001). GRP also increased phosphorylated AKT expression in FLS. When Ly294002 was added with GRP, it prevented the GRP-induced increased cell invasiveness (p < 0.001). These data suggest that GRPR expression in FLS and that exogenous GRP are able to activate FLS invasion. This effect occurs at least in part through the AKT activation. Therefore, understanding of the GRP/GRPR pathway could be relevant in the development of FLS-targeted therapy for RA.     

Prenatal stress causes dendritic atrophy of pyramidal neurons in hippocampal CA3 region by glutamate in offspring rats

A substantial number of human epidemiological data, as well as experimental studies, suggest that adverse maternal stress during gestation is involved in abnormal behavior, mental, and cognition disorder in offspring. To explore the effect of prenatal stress (PS) on hippocampal neurons, in this study, we observed the dendritic field of pyramidal neurons in hippocampal CA3, examined the concentration of glutamate (Glu), and detected the expression of synaptotagmin‐1 (Syt‐1) and N‐methyl‐D ‐aspartate receptor 1 (NR1) in hippocampus of juvenile female offspring rats. Pregnant rats were divided into two groups: control group (CON) and PS group. Female offspring rats used were 30‐day old. The total length of the apical dendrites of pyramidal neurons in hippocampal CA3 of offspring was significantly shorter in PS than that in CON (p < 0.01). The number of branch points of the apical dendrites of pyramidal neurons in hippocampal CA3 of offspring was significantly less in PS (p < 0.01). PS offspring had a higher concentration of hippocampal Glu compared with CON (p < 0.05). PS offspring displayed increased expression of Syt‐1 and decreased NR1 in hippocampus compared with CON (p < 0.001 and p < 0.01, respectively). The expression of NR1 in different hippocampus subfields of offspring was significantly decreased in PS than that in CON (p < 0.05‐0.01). This study shows that PS increases the Glu in hippocampus and causes apical dendritic atrophy of pyramidal neurons of hippocampal CA3 in offspring rats. The decline of NR1 in hippocampus may be an adaptive response to the increased Glu. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

BI‐69A11‐mediated inhibition of AKT leads to effective regression of xenograft melanoma

The AKT/PKB pathway plays a central role in tumor development and progression and is often up‐regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI‐69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI‐69A11 was performed in melanoma cells, a tumor type that commonly exhibits up‐regulation of AKT. Treatment of the UACC903 human melanoma cells, harboring the PTEN mutation, with BI‐69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI‐69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP‐90. BI‐69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI‐69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra‐peritoneal injection of BI‐69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI‐69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.     

Isoform-specific regulation of adipocyte differentiation by Akt/protein kinase Balpha

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1/PKBα in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKBα and Akt2/PKBβ by ectopic expression of Akt1/PKBα but not Akt2/PKBβ. Akt1/PKBα was found to be the major regulator of phosphorylation and nuclear export of FoxO1, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKBα-deficient cells, but was restored after forced expression of Akt1/PKBα. Moreover, expression of p27^Kip1, an inhibitor of the cell cycle, was down regulated in an Akt1/PKBα-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKBα isoform plays a major role in adipocyte differentiation by regulating FoxO1 and p27^Kip1.     

Primordial follicle activation is affected by the absence of Sohlh1 in mice

Previous studies have reported that only primordial follicles and empty follicles can be found in 7.5 days postparturition (dpp) Sohlh1^?/? mouse ovaries and females are infertility. There appears to be a defect in follicle development during the primordial‐to‐primary follicle transition in Sohlh1^?/? mouse ovaries. However, detailed analyses of these phenomena have not been performed. In this study, we used Sohlh1^?/? transgenic mice to explore the role of Sohlh1 in folliculogenesis. The results showed that only primordial follicles and empty follicles can be observed in Sohlh1^?/? ovaries from 0.5 to 23.5 dpp. The expression of Foxo3 and FOXO3 was downregulated; nucleocytoplasmic shuttling of FOXO3 was normal in 7.5‐dpp Sohlh1^+/+ but not Sohlh1^?/? ovaries; and primordial follicle activation (PFA) was not observed in 7.5‐dpp Sohlh1^?/? mice. The expression levels of KIT, AKT, and P308‐AKT were downregulated (p < 0.05), whereas that of P473‐AKT was not significantly changed (p > 0.05). The KIT/PI3K/AKT pathway was inhibited. Furthermore, we conducted a dual luciferase assay and chromatin immunoprecipitation. The results showed that SOHLH1 can upregulate the Kit gene by binding to the ?3698 bp E‐box motif. The absence of Sohlh1 may affect PFA in mouse ovaries via downregulation of Kit and inhibition of the KIT/PI3K/AKT pathway.     

JTC-801 exerts anti-proliferative effects in human osteosarcoma cells by inducing apoptosis

Background: The research of G protein-coupled receptors (GPCRs) is a promising strategy for drug discovery. In cancer therapy, there is a need to discover novel agents that can inhibit proliferation and induce apoptosis in cancer cells. JTC-801 is a novel GPCR antagonist with the function of reversing pain and anxiety symptoms. This study aims to investigate the antitumor effects of JTC-801 on human osteosarcoma cells (U2OS) and elucidate the underlying mechanism.

Materials and methods: The Cell Counting Kit-8 assay was used to detect the viability of U2OS cells treated with JTC-801 in vitro. The cell apoptosis was evaluated using a flow cytometry assay with Annexin V-FITC/PI double staining. The inhibitory effect of JTC-801 on invasion and migration of U2OS cells were determined by the Transwell assays. Western blot assay was performed to measure the levels of proteins related to cell apoptosis and its mechanism.

Results: The JTC-801 significantly decreased the viability of U2OS cells (p?p?p?Conclusions: JTC-801 may exert osteosarcoma cell growth inhibition by promoting cell apoptosis, through PI3K/AKT signaling pathway participation.     

The expression of protein kinase B in gastric cancer cell apoptosis induced by 12-O-tetradecanoylphorbol-1, 3-acetate

Protein kinase B (PKB/Akt) is a serine-threonine kinase functioning downstream of phosphatidylinositol 3-kinase (PI-3 kinase) in response to mitogen or growth factor stimulation. In several cell types, it plays an important anti-apoptotic role. TPA is a potent regulator of the growth of many different cell types. Here, we detected that TPA could induce cell apoptosis in the gastric cancer cell line, BGC-823. We also found that TPA inhibited the expression of PKB/Akt in a TPA concentration- and time-dependent manner. Furthermore, TPA inhibited the phosphorylation of PKB at Ser473, but did not affect the phosphorylation of Thr308. It only attenuated the expression of PKB/Akt and the phosphorylation of Ser473 in the cell nucleus, whereas it did not change the PKB/Akt distribution in BGC-823 cells. These results suggest that PKB/Akt inhibition by TPA may be the important factor in the mechanism of effect of TPA on gastric cell lines.     

Complestatin prevents apoptotic cell death: inhibition of a mitochondrial caspase pathway through AKT/PKB activation

Complestatin, a bicyclo hexapeptide from Streptomyces, was isolated as a possible regulator of neuronal cell death. In this study, we report an anti-apoptotic activity of complestatin and its underlying molecular mechanism. Complestatin blocked TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis and activation of caspase-3 and -8 at micromolar concentration levels without inhibiting the catalytic activities of these caspases. Complestatin potently induced a rapid and sustained AKT/PKB activation and Bad phosphorylation, resulting in inhibition of mitochondrial cytochrome c release. These anti-apoptotic activities of complestatin were significantly abrogated in cells expressing dominant negative AKT/PKB. Taken together, our results suggest that complestatin prevents apoptotic cell death via AKT/PKB-dependent inhibition of the mitochondrial apoptosis signal pathway. The novel property of complestatin may be valuable for developing new pharmaceutical means that will control unwanted cell death.     

Maternal diabetes up‐regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up‐regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose‐tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF‐Akt (protein kinase B)‐mammalian target of rapamycin (mTOR)‐NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose‐induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF‐Akt‐mTOR‐NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.     

Rab11a通过PI3K/AKT和Ras/MEK/ERK信号通路调节胰腺癌细胞凋亡和侵袭

目的:探究Rab11a在胰腺癌中的表达模式及其对肿瘤生长和转移的影响.方法:通过免疫组织化学法、RT-PCR和Western blot检测60例胰腺癌患者的癌组织和癌旁组织中Rab11a的表达.通过对人胰腺癌细胞系PANC1转染靶向Rab11a的小干扰RNA或过表达Rab11a的pcDNA3.1质粒考察Rab11a对细...     

p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.     

Attenuation of ischemia–reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts

Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.     

Molecular mechanism of antidiabetic zinc–allixin complexes: regulations of glucose utilization and lipid metabolism

We previously reported new zinc complexes of allixin [bis(allixinato)zinc] and its derivative bis(thioallixin-N-methyl)zinc that demonstrated excellent antidiabetic activity in type 2 diabetic mellitus KKA(y) mice. However, the molecular mechanism of these complexes is not fully understood. Thus, we attempted to reveal the intracellular mechanism of these complexes in 3T3-L1 adipocytes. Both zinc complexes induced Akt/protein kinase B (Akt/PKB) phosphorylation. The phosphorylation of Akt/PKB enhanced glucose transporter 4 translocation to the plasma membrane; this in turn enhanced the glucose utilization in a dose- and time-dependent manner. Glucose utilization by the complexes depended on the intracellular zinc concentration. Moreover, zinc complexes suppressed the cyclic AMP dependent protein kinase mediated phosphorylation of hormone-sensitive lipase (HSL), leading to the inhibition of free fatty acid release from the 3T3-L1 adipocytes. Such responses were inhibited by wortmannin, suggesting that the suppression of HSL by zinc complexes was dependent in the phosphoinositide 3-kinase-Akt/PKB signaling cascade. On the basis of these results, we proposed that both zinc complexes activated the Akt/PKB-mediated insulin-signaling pathway and improved both glucose utilization and lipid metabolism.     

IL-10 synergistically enhances GM-CSF-induced CCR1 expression in myelomonocytic cells

CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).     

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.     

Insulinomimetic Zn complex (Zn(opt)2) enhances insulin signaling pathway in 3T3-L1 adipocytes

Zinc (Zn) is an essential trace element with multiple regulatory functions, involving insulin synthesis, secretion, signaling and glucose transport. Since 2000, we have proposed that Zn complexes with different coordination environments exhibit high insulinomimetic and antidiabetic activities in type 2 diabetic animals. However, the molecular mechanism for the activities is still unsolved. The purpose of this study was to reveal the molecular mechanism of several types of Zn complexes in 3T3-L1 adipocytes, with respect to insulin signaling pathway. Obtained results shows that bis(1-oxy-2-pyridine-thiolato)Zn(II), Zn(opt)2, with S(2)O(2) coordination environment induced most strongly Akt/protein kinase B (Akt/PKB) phosphorylation, in which the optimal phosphorylation was achieved at a concentration of 25 microM, and this Zn(opt)2-induced Akt/PKB phosphorylation was inhibited by wortmannin at 100 nM. Further, the phosphorylation was maximal at 5-10 min stimulation, in agreement with the Zn uptake which was also maximal at 5-10 min stimulation. The Akt/PKB phosphorylation was in concentration- and time-dependent manners. Zn(opt)2 was also capable to translocate GLUT4 protein to the plasma membrane. We conclude that Zn(opt)2 was revealed to exhibit both insulinomimetic and antidiabetic activities by activating insulin signaling cascade through Akt/PKB phosphorylation, which in turn caused the GLUT4 translocation from the cytosol to the plasma membrane.     

Ketamine Affects the Neurogenesis of Rat Fetal Neural Stem Progenitor Cells via the PI3K/Akt‐p27 Signaling Pathway

Ketamine is widely used as an anesthetic, analgesic, or sedative in pediatric patients. We reported that ketamine alters the normal neurogenesis of rat fetal neural stem progenitor cells (NSPCs) in the developing brain, but the underlying mechanisms remain unknown. The PI3K‐PKB/Akt (phosphatidylinositide 3‐kinase/protein kinase B) signaling pathway plays many important roles in cell survival, apoptosis, and proliferation. We hypothesized that PI3K‐PKB/Akt signaling may be involved in ketamine‐altered neurogenesis of cultured NSPCs in vitro. NSPCs were isolated from Sprague‐Dawley rat fetuses on gestational day 17. 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation, Ki67 staining, and differentiation tests were utilized to identify primary cultured NSPCs. Immunofluorescent staining was used to detect Akt expression, whereas Western blots measured phosphorylated Akt and p27 expression in NSPCs exposed to different treatments. We report that cultured NSPCs had properties of neurogenesis: proliferation and neural differentiation. PKB/Akt was expressed in cultured rat fetal cortical NSPCs. Ketamine inhibited the phosphorylation of Akt and further enhanced p27 expression in cultured NSPCs. All ketamine‐induced PI3K/Akt signaling changes could be recovered by N‐methyl‐d ‐aspartate (NMDA) receptor agonist, NMDA. These data suggest that the inhibition of PI3K/Akt‐p27 signaling may be involved in ketamine‐induced neurotoxicity in the developing brain, whereas excitatory NMDA receptor activation may reverse these effects     

Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle

To determine the molecular mechanism underlying hyperglycemia-induced insulin resistance in skeletal muscles, postreceptor insulin-signaling events were assessed in skeletal muscles of neonatally streptozotocin-treated diabetic rats. In isolated soleus muscle of the diabetic rats, insulin-stimulated 2-deoxyglucose uptake, glucose oxidation, and lactate release were all significantly decreased compared with normal rats. Similarly, insulin-induced phosphorylation and activation of Akt/protein kinase B (PKB) and GLUT-4 translocation were severely impaired. However, the upstream signal, including phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and -2 and activity of phosphatidylinositol (PI) 3-kinase associated with IRS-1/2, was enhanced. The amelioration of hyperglycemia by T-1095, a Na(+)-glucose transporter inhibitor, normalized the reduced insulin sensitivity in the soleus muscle and the impaired insulin-stimulated Akt/PKB phosphorylation and activity. In addition, the enhanced PI 3-kinase activation and phosphorylation of IR and IRS-1 and -2 were reduced to normal levels. These results suggest that sustained hyperglycemia impairs the insulin-signaling steps between PI 3-kinase and Akt/PKB, and that impaired Akt/PKB activity underlies hyperglycemia-induced insulin resistance in skeletal muscle.     

Human vascular cell responses to the circulating bone hormone osteocalcin

The purpose of this study was to characterize the direct effects of uncarboxylated osteocalcin (ucOCN) on vascular cell biology in vitro, to assess its potential function in pathophysiological conditions such as atherosclerosis. Human aortic endothelial cells (HAECs) and smooth muscle cells (HASMCs) were treated with ucOCN (0.1–50 ng/ml) and changes in phosphorylation of intracellular signaling proteins, angiogenesis, proliferation, migration, monolayer permeability, and protein secretion were measured. In HAECs, phosphorylated JNK and CREB were decreased with ucOCN (p < 0.05). In HASMCs, phosphorylated p70S6K and NF-ΚB were increased by ucOCN (p < 0.05). Cell proliferation increased in both cell types dose dependently which was blocked by AKT and ERK pathway inhibitors. ucOCN did not affect cell permeability, angiogenesis, or migration. The direct activity of ucOCN on vascular cells is recognized, particularly its proliferative effects. However, at least in physiological settings, it does not appear that osteocalcin may directly promote atherogenesis based on the outcomes measured.