α1-肾上腺素受体激活大鼠心脏腺苷酸活化蛋白激酶

为了验证心脏腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）是否为肾上腺素受体（adrenergic receptor,AR）的下游信号分子,本实验在大鼠心室肌源细胞和大鼠心脏中观察了α-AR对AMPK的激活作用,利用Western blot检测了AMPK-α总蛋白表达量及其172位苏氨酸磷酸化水平。雄性Sprague-Dawley大鼠皮下植入去甲肾上腺素（norepinephrine,NE）,苯肾上腺素（phenylephrine,PE）或者溶剂载体[0．01％（W／V）维生素C]的缓释微泵（osmotic minipump）。NE或PE以每小时0．2 mg／kg的速率持续输注,7 d后用AMPK-α抗体免疫沉淀处理样本并测定AMPK的活性。结果显示,在细胞水平,NE引起的AMPK磷酸化水平增高具有时间依赖和剂量依赖特点, NE处理细胞10 min后AMPK磷酸化水平达到最高峰;NE引起的这种效应对β-AR的拮抗剂普萘洛尔（propranolol）不敏感,但是可以被α1-AR拮抗剂哌唑嗪（prazosin）所阻断。结果提示,α1-AR介导AMPK的磷酸化,但β-AR无此作用。AR激动剂持续灌注7 d后,AMPK的活性在NE（7．4倍）和PE（6．0倍）灌注组较对照组显著增高（P〈0．05,H=6）。PE持续灌注组大鼠与对照组相比无明显的心肌肥厚和组织纤维化变化。本文证明α1-AR激动剂可以增强AMPK的活性,揭示了心脏中α1-AR激动在调控AMPK活性方面的重要作用。深入了解α1-AR介导的AMPK激活可能在心衰治疗中具有重要的临床意义。     

Palmitate activates AMP-activated protein kinase and regulates insulin secretion from beta cells

AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Changes in AMPK activity contribute to the regulation of insulin secretion. Epidemiological evidence links the ingestion of saturated fatty acid with hyperinsulinemia. The aim of the present study was to examine the effects of palmitate on beta cell AMPK activity and insulin secretion. Isolated rat islets and MIN6 beta cells were treated acutely (5-60 min) or chronically (24 h) with palmitate. Insulin secretion, AMPK and acetyl CoA carboxylase phosphorylation were assessed. The acute effects of palmitate included AMPK activation and augmentation in insulin secretion. Activation of AMPK by 24h pretreatment with palmitate suppressed glucose-stimulated insulin secretion, but not the response of insulin secretion to combined stimuli of glucose and palmitate. This study demonstrated that palmitate availability affected beta cell AMPK activity. In beta cells, an increase in AMPK activity may be required for fatty acid-induced fatty acid oxidation and prevention of lipotoxicity.     

Troglitazone acutely activates AMP-activated protein kinase and inhibits insulin secretion from beta cells

Changes in AMP-activated protein kinase (AMPK) activity contribute to the regulation of insulin secretion. Troglitazone has been shown to lower serum insulin levels and protect beta cell function. The aim of the present study was to examine the effects of troglitazone on AMPK activity and insulin secretion in beta cells. Isolated rat islets and MIN6 cells were treated for a short (1 h) or a long time (20 h) with troglitazone. One-hour troglitazone treatment activated AMPK and inhibited both glucose-stimulated insulin secretion (GSIS) and the response of insulin secretion to combined stimuli of glucose and palmitate. Long (20 h) treatment with troglitazone caused a sustained phosphorylation of AMPK and acetyl-CoA carboxylase, and increased GSIS after withdrawal of the drug. This study provided evidence that troglitazone activated AMPK in beta cells. In addition to the insulin-sensitizing effects in peripheral tissues, troglitazone also directly inhibits insulin hypersecretion by the elevated glucose and fatty acids, and thus protects beta cells from glucolipotoxicity.     

An activating mutation in the γ1 subunit of the AMP-activated protein kinase

The AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic subunit and two regulatory subunits, β and γ. The γ subunit is essential for enzyme activity by virtue of its binding to the C-terminus of the subunit and appears to play some role in the determination of AMP sensitivity. We demonstrate that a γ1R70Q mutation causes a marked increase in AMPK activity and renders it largely AMP-independent. This activation is associated with increased phosphorylation of the subunit activation loop T172. These in vitro characteristics of AMPK are also reflected in increased intracellular phosphorylation of one of its major substrates, acetyl-CoA carboxylase. These data illustrate the importance of the γ1 subunit in the regulation of AMPK and its modulation by AMP.     

microRNA-141 is associated with hepatic steatosis by downregulating the sirtuin1/AMP-activated protein kinase pathway in hepatocytes

Sirtuin1 (SIRT1) is a crucial regulator of metabolism and it is implicated in the metabolic pathophysiology of several disorders inclusive of Type 2 diabetes and fatty liver disease (NAFLD). The aim of this study was to investigate the role of miR-141 in hepatic steatosis via regulation of SIRT1/AMP-activated protein kinase (AMPK) pathway in hepatocytes. Liver hepatocellular cells (HepG2) were treated with high concentration of glucose to be subsequently used for the assessment of miR-141 and SIRT1 levels in a model of hepatic steatosis. On the other hand, cells were transfected with miR-141 to investigate its effect on hepatocyte steatosis and viability as well as SIRT1 expression and activity along with AMPK phosphorylation. Targeting of SIRT1 by miR-141 was evaluated by bioinformatics tools and confirmed by luciferase reporter assay. Following the intracellular accumulation of lipids in HepG2 cells, the level of miR-141 was increased while SIRT1 mRNA and protein levels, as well as AMPK phosphorylation, was decreased. Transfection with miR-141 mimic significantly downregulated SIRT1 expression and activity while miR-141 inhibitor had the opposite effects. Additionally, modulation of miR-141 levels significantly influenced AMPK phosphorylation status. The results of luciferase reporter assay verified SIRT1 to be directly targeted by miR-141. miR-141 could effectively suppress SIRT1 and lead to decreased AMPK phosphorylation in HepG2 cells. Thus, miR-141/SIRT1/AMPK signaling pathway may be considered a potential target for the therapeutic management of NAFLD.     

The zinc transporter Slc39a5 controls glucose sensing and insulin secretion in pancreatic β-cells via Sirt1- and Pgc-1α-mediated regulation of Glut2

Zinc levels are high in pancreatic β-cells, and zinc is involved in the synthesis, processing and secretion of insulin in these cells. However, precisely how cellular zinc homeostasis is regulated in pancreatic β-cells is poorly understood. By screening the expression of 14 Slc39a metal importer family member genes, we found that the zinc transporter Slc39a5 is significantly downregulated in pancreatic β-cells in diabetic db/db mice, obese ob/ob mice and high-fat diet-fed mice. Moreover,β-cell-specific Slc39a5 knockout mice have impaired insulin secretion. In addition, Slc39a5-deficient pancreatic islets have reduced glucose tolerance accompanied by reduced expression of Pgc-1α and its downstream target gene Glut2. The down-regulation of Glut2 in Slc39a5-deficient islets was rescued using agonists of Sirt1, Pgc-1α and Ppar-γ. At the mechanistic level, we found that Slc39a5-mediated zinc influx induces Glut2 expression via Sirt1-mediated Pgc-1α activation. These findings suggest that Slc39a5 may serve as a possible therapeutic target for diabetes-related conditions.     

Stress‐induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration

Mitogen‐activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin‐rich vesicles upon stress. Stress‐induced DLK vesicle recruitment is essential for kinase activation; blocking DLK‐membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase

Activation of 5′-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5′-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase ce:sup>/ce:sup>/Mn²⁺-dependent (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the ce:sup>/ce:sup>/Mn²⁺-dependent protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggests that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae

The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomycescerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.     

蛋白激酶C-细胞外信号调节激酶1/2通路介导精氨酸升压素对成年大鼠心肌成纤维细胞的促增殖作用

精氨酸升压素(arginine vasopressin, AVP)是高血压和心力衰竭时激活的神经体液和血流动力学因子,同时,它还具有直接的生长刺激作用.我们以往的研究显示AVP可诱导新生大鼠心肌成纤维细胞(cardiac fibroblasts, CFs)增殖.本研究旨在进一步观察AVP是否对成年大鼠CFs具有促增殖作用,并探计其机制.采用组织块法培养成年大鼠CFs,用[3H]-TdR掺入法和流式细胞仪方法观察AVP作用下CFs的DNA合成和细胞周期分布.根据特异性底物髓磷脂基质蛋白(myelin basic protein, MBP)的磷酸化水平测定细胞外信号调节激酶1/2 (extracellular signal-regulated kinase 1/2, ERK1/2)的活性.用Western blot检测ERK1/2的磷酸化和p27Kip1、细胞周期蛋白D1、 A、 E的表达.结果显示,AVP(0.1μmol/L)可促进成年大鼠CFs的DNA合成,该作用可被V1受体拮抗剂d(CH2)5[Tyr2(Me),Arg8]-vasopressin (0.1μmol/L)阻断,而不受V2受体拮抗剂desglycinamide [d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin (0.1μmol/L)的影响.AVP可激活ERK1/2,用蛋白激酶C(protein kinase C, PKC)激动剂佛波酯(phorbol 12-myristate 13-acetate, PMA, 30nmol/L, 5min)急性刺激可模拟该作用,而PMA持续慢性作用(2.5μmol/L,24h)耗竭PKC后则抑制AVP对ERK1/2的激活.AVP可抑制p27Kip1的蛋白表达,升高细胞周期蛋白D1、 A和E的表达,同时促进细胞周期由G0/G1期进入S期.ERK1/2抑制剂PD98059 (30μmol/L)阻断AVP对DNA合成、p27Kip1、细胞周期蛋白D1、A和E蛋白表达的作用,并抑制细胞周期进程.以上结果表明,AVP可促进成年大鼠CFs增殖,该作用由V1受体和PKC-ERK1/2通路介导.AVP可通过ERK1/2调控p27Kip1、细胞周期蛋白D1、A和E的表达,从而促进成年大鼠CFs的细胞周期进程.     

Decreased AMP-activated protein kinase activity is associated with increased inflammation in visceral adipose tissue and with whole-body insulin resistance in morbidly obese humans

Inflammation and infiltration of immune cells in white adipose tissue have been implicated in the development of obesity-associated insulin resistance. Likewise, dysregulation of the fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been proposed as a pathogenetic factor for these abnormalities based on both its links to insulin action and its anti-inflammatory effects. In this study, we examined the relationships between AMPK activity, the expression of multiple inflammatory markers in visceral (mesenteric and omental) and abdominal subcutaneous adipose tissue, and whole-body insulin sensitivity in morbidly obese patients (BMI 48 ± 1.9 kg/m²) undergoing gastric bypass surgery. AMPK activity was assessed by Western-blots (P-AMPK/T-AMPK) and mRNA levels of various markers of inflammation by qRT-PCR. Patients were stratified as insulin sensitive obese or insulin-resistant obese according to their HOMA-IR values. The results indicate that AMPK activity is lower in visceral than in subcutaneous abdominal adipose tissue of these patients and that this is associated with an increased expression of multiple inflammatory genes. They also revealed that AMPK activity is lower in adipose tissue of obese patients who are insulin resistant (HOMA-IR > 2.3) than in BMI-matched insulin sensitive subjects. Furthermore, this difference was evident in all three fat depots. In conclusion, the data suggest that there are close links between reduced AMPK activity and inflammation in white adipose tissue, and whole-body insulin resistance in obese humans. Whether adipose tissue AMPK dysregulation is a causal factor for the development of the inflammation and insulin resistance remains to be determined.     

Extracellular ATP through P2 receptors activates AMP-activated protein kinase and suppresses superoxide generation in cultured mouse podocytes

Podocytes are an important constituent of the glomerular filtration barrier. The function of these glomerular cells is affected by extracellular nucleotides through P2 receptors. The activation of P2 receptors may lead to the activation of NAD(P)H oxidase, the key enzyme in oxidative stress, with the intracellular pathways leading to intracellular ATP depletion associated with an increase in the intracellular AMP:ATP ratio. This deregulation of the energy balance activates AMP-activated protein kinase (AMPK) to restore energy homeostasis. We investigated whether P2 receptor activation influences NAD(P)H oxidase-dependent rate of superoxide anion (O₂^•−) generation and AMPK activity in cultured mouse podocytes. The rate of O₂^•− generation was measured by chemiluminescence and changes in AMPK activity were determined by immunoblotting against AMPKα-Thr¹⁷²-P. The addition of 100 μM ATP induced a rapid and transient decrease in rate of O₂^•− generation and increased AMPK phosphorylation with maximal effects in the first minute (2.44 ± 0.09 versus 1.62 ± 0.06 nmol/mg protein/min, P < 0.05 and 0.64 ± 0.04 versus 0.97 ± 0.07, P < 0.05, respectively). Both parameters returned to control levels at 10 min. Suramin (300 μM, P2 receptor antagonist) and compound C (100 μM, AMPK inhibitor) completely, and STO-609 (25 μM, CaMKK-β inhibitor) partially, prevented ATP action in rate of O₂^•− generation and AMPK phosphorylation. Various ATP analogues (10 μM) mimicked the effects of ATP on rate of O₂^•− generation and AMPK phosphorylation. The data indicate that extracellular ATP, acting through P2 receptors upstream of CaMKK-β, modulates podocyte function through simultaneous effects on AMPK and NAD(P)H oxidase activities. This mechanism may play a role in restoring energy homeostasis after oxidative stress.     

AMP-activated protein kinase and metabolic regulation in cold-hardy insects

Winter survival for many insects depends on cold hardiness adaptations as well as entry into a hypometabolic diapause state that minimizes energy expenditure. We investigated whether AMP-activated protein kinase (AMPK) could be involved in this adaptation in larvae of two cold-hardy insects, Eurosta solidaginis that is freeze tolerant and Epiblema scudderiana that uses a freeze avoidance strategy. AMPK activity was almost 2-fold higher in winter larvae (February) compared with animals collected in September. Immunoblotting revealed that phosphorylation of AMPK in the activation loop and phosphorylation of acetyl-CoA carboxylase (ACC), a key target of AMPK, were higher in Epiblema during midwinter whereas no seasonal change was seen in Eurosta. Immunoblotting also revealed a significant increase in ribosomal protein S6 phosphorylation in overwintering Epiblema larvae, and in both Eurosta and Epiblema, phosphorylation of eukaryotic initiation factor 4E-binding protein-1 dramatically increased in the winter. Pyruvate dehydrogenase (PDH) E1α subunit site 1 phosphorylation was 2-fold higher in extracts of Eurosta larvae collected in February versus September while PDH activity decreased by about 50% in Eurosta and 80% in February Eurosta larvae compared with animals collected in September. Glycogen phosphorylase phosphorylation was 3-fold higher in Epiblema larvae collected in February compared with September and also in these animals, triglyceride lipase activity increased by 70% during winter. Overall, our study suggests a re-sculpting of metabolism during insect diapause, which shifted to a more catabolic poise in freeze-avoiding overwintering Epiblema larvae, possibly involving AMPK.