Effect of adenosine monophosphate-activated protein kinase–p53–Krüppel-like factor 2a pathway in hyperglycemia-induced cardiac remodeling in adult zebrafish

Aims/IntroductionDiabetic cardiomyopathy is a type of myocardial disease. It causes left ventricular hypertrophy, followed by diastolic and systolic dysfunction, eventually leading to congestive heart failure. However, the underlying mechanism still requires further elucidation.Materials and MethodsA high‐glucose zebrafish model was constructed by administering streptozocin intraperitoneally to enhance the development of cardiomyopathy and then treated with adenosine monophosphate‐activated protein kinase (AMPK) activator. Cardiac structure and function, and protein and gene expression were then analyzed. Cardiomyocytes (CMs) culture in vitro using lentivirus were used for detection of AMPK, p53 and Krüppel‐like factor 2a (klf2a) gene expression.ResultsIn the hyperglycemia group, electrocardiogram findings showed arrhythmia, echocardiography results showed heart enlargement and dysfunction, and many differences, such as increased apoptosis and myocardial fiber loss, were observed. The phospho‐AMPK and klf2a expression were downregulated, and p53 expression was upregulated. Activation of phospho‐AMPK reduced p53 and increased klf2a expression, alleviated apoptosis in CMs and improved cardiac function in the hyperglycemic zebrafish. In vitro knockdown system of AMPK, p53 and klf2a using lentivirus illustrated an increased p53 expression and decreased klf2a expression in CMs by inhibiting AMPK. Repression of p53 and upregulation of klf2a expression were observed, but no changes in the expression of AMPK and its phosphorylated type.ConclusionsIn the model of streptozocin‐induced hyperglycemia zebrafish, the reduction of phosphorylated AMPK increased p53, which led to KLF2a decrease to facilitate apoptosis of CMs, inducing the cardiac remodeling and cardiac dysfunction. These results can be reversed by AMPK activator, which means the AMPK–p53–klf2a pathway might be a potential target for diabetic cardiomyopathy intervention.     

Testicular orphan receptor 4 induced hepatic stellate cells activation via the regulation of TGF-β receptor Ⅰ/Smad2/3 signaling pathway

Introduction and ObjectivesLiver fibrosis is a common pathological change in many chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the core event in liver fibrosis. This study aimed to investigate the role of testicular orphan receptor 4 (TR4) in the activation of HSCs.Materials and MethodsIn vivo, bile duct ligation (BDL)-induced rat liver fibrosis model was established, and the expressions of TR4 and α-smooth muscle actin (α-SMA) in liver tissues were detected. In vitro, TR4 knockdown and overexpression in JS-1 cells using lentiviral vectors were constructed, and the expressions of TR4, α-SMA, Col-I, and TGF-β1/smads and retinoid X receptor (RXR) pathway-related genes were detected.ResultsTR4 was highly expressed in BDL-induced fibrotic liver, accompanied by increased expression of α-SMA. Knockdown of TR4 significantly inhibited the expressions of α-SMA, Col-I, p-TβRI, and p-Smad2/3, and up-regulated the expression of RXRα in HSCs in vitro. In contrast, TR4 overexpression significantly increased the expressions of α-SMA, Col-I, p-TβRI, and p-Smad2/3, and inhibited the expression of RXRα.ConclusionsTR4 may promote the activation of HSCs by up-regulating TβR I/Smad2/3 signaling pathway and down-regulating RXRα signaling, thereby promoting the progression of liver fibrosis. Our findings may provide a new therapeutic target against hepatic fibrosis.     

Correction to: Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium

Journal of Gastroenterology - In the original publication of the article, the following errors were noted and corrected in this correction.     

Inhibitory effect of angiotensinⅡreceptor antagonist on hepatic stellate cell activation in non-alcoholic steatohepatitis

AIM: To investigate the efficacy of angiotensinⅡreceptor antagonist on hepatic stellate cells (HSCs) activation in the patients with non-alcoholic steatohepatitis (NASH). METHODS: Seven patients with NASH were prescribed losartan, a selective angiotensinⅡtype 1 receptor antagonist (50 mg/d) for 48 wk. Liver biopsies were performed both at the entry and end of the study in all patients. Quiescent and activated HSCs were identified by double immunostaining using anti-p75 andα-smooth muscle actin antibodies, and the number of each phenotype was counted. Similarly, the liver specimens obtained from the eight patients with non-alcoholic fatty liver (NAFL) were also examined as controls. RESULTS: In NASH hepatic tissues, activated HSCs were dominantly distributed as compared with those in NAFL. The 48-wk losartan treatment induced a remarkable decrease in activated HSCs and a mild increase in quiescent phenotypes. CONCLUSION: Our data suggest the crucial involvement of HSCs in anti-fibrotic effect of angiotensinⅡreceptor antagonist on patients with NASH.     

Inhibitory effect of angiotensin Ⅱ receptor antagonist on hepatic stellate cell activation in non-alcoholic steatohepatitis

AIM: To investigate the efficacy of angiotensin II receptor antagonist on hepatic stellate cells (HSCs) activation in the patients with non-alcoholic steatohepatitis (NASH). METHODS: Seven patients with NASH were prescribed losartan, a selective angiotensin II type 1 receptor antagonist (50 mg/d) for 48 wk. Liver biopsies were performed both at the entry and end of the study in all patients. Quiescent and activated HSCs were identified by double immunostaining using anti-p75 and -smooth muscle actin antibodies, and the number of each phenotype was counted. Similarly, the liver specimens obtained from the eight patients with non-alcoholic fatty liver (NAFL) were also examined as controls. RESULTS: In NASH hepatic tissues, activated HSCs were dominantly distributed as compared with those in NAFL. The 48-wk losartan treatment induced a remarkable decrease in activated HSCs and a mild increase in quiescent phenotypes. CONCLUSION: Our data suggest the crucial involvement of HSCs in anti-fibrotic effect of angiotensin II receptor antagonist on patients with NASH.     

MCP-1-induced protein attenuates endotoxin-induced myocardial dysfunction by suppressing cardiac NF-кB activation via inhibition of IкB kinase activation

Myocardial contractile dysfunction is a major consequence of septic shock, which is mainly mediated by nuclear factor-kappa B (NF-кB)-dependent production of inflammatory mediators in the heart. A novel zinc-finger protein, MCP-1-induced protein (MCPIP), is thought to have NF-кB inhibitory activity in certain cell cultures, but its pathophysiological consequence in vivo remains undefined. This study aims to clarify whether the anti-inflammatory potency of MCPIP contribute to amelioration of septic myocardial inflammation and dysfunction in vivo. Transgenic mice (TG) with cardiac-specific expression of MCPIP and their littermate wild-type (WT) controls were challenged with Escherichia coli LPS (10 mg/kg ip) and myocardial function was assessed 18 h later using echocardiography. LPS administration markedly deteriorated myocardial contractile function evidenced by reduction of the percentage of left ventricular fractional shortening, which was significantly attenuated by myocardial expression of MCPIP. MCPIP TG mice exhibited a markedly reduced myocardial inflammatory cytokines, less of iNOS expression and peroxynitrite formation, decreased caspase-3/7 activities and apoptotic cell death compared with LPS-treated WT mice. Activation of cardiac NF-кB observed in LPS-challenged WT mice was suppressed by the presence of MCPIP, as evidenced by decreased phosphorylation of IкB kinase (IKKα/β), reduced degradation of the cytosolic IкBα, and decreased nuclear translocation of NF-кB p65 subunit and its target DNA-binding activity. These results suggest that MCPIP has therapeutic values to protect heart from inflammatory pathologies, possibly through inhibition of IкB kinase complex, leading to blockade of NF-кB activation, and subsequently, attenuation of the proinflammatory state and nitrosative stress in the myocardium.     

Expression of gamma-aminobutyric acid A receptor subunits α1，β1，γ2 mRNA in rats with hepatic encephalopathy

AIM: To investigate the mRNA expression of gammaaminobutyric acid A (GABAA) receptor subunitsα1,β1,γ2 in different parts of the brain of rats with hepatic encephalopathy.METHODS: Twelve adult male Sprague-Dawley rats were randomly divided into two groups: (1) hepatic encephalopabhy model group (n = 6), which was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg/kg) for three consecutive days; (2) control group (n = 6), in which the rats were treated with same dose of normal saline solution. After the freeze slice of cerebrum was made, in situ hybridization was used to detect the mRNA of GABAA receptor subunits α1,β1,, and γ2 in rat cerebral cortex,basal nuclei, substantia nigra and hippocampi. Image data were collected and analyzed quantitatively by QWin550CW model image signal gather and analysis system.RESULTS: In rats with hepatic encephalopathy, mRNA expression levels of GABAA receptor subunits α1,β1 increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis and hippocampi (144.7±15.67/184.14±4.41, 60.61±33.66/113.07±32.44,87.71± 21.25/128.40±18.85, 122.34±5.56/161.60±4.56,123.29±5.21/140.65±4.15, 123.40±4.42/140.09±4.52,124.76±4.18/140.09±4.12, 141.62±15.09/182.80±5.20,69.13±30.74/134.21±43.76, 87.87±25.16/151.01±19.49,122.14±6.30/162.33±3.92, 122.81±5.09/137.19±7.12,123.00±4.63/138.11±5.92, 125.75 ±2.43/138.81±6.10,P<0.01), but did not change in the cerebral cortex compared to the control group. Similar changes were found in the mRNA expression levels of GABAA receptor subunit γ2,which increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis (136.81±26.41/167.97±16.23, 51.00±36.14/113.18±36.52,86.35±20.30/126.90±19.74, P<0.01), CA1 of hippocampal(162.15:1:9.05/178.62:1:6.45, P<0.05), and no changes were found in the cerebral cortex and CA2, CA3, CA4 of hippocampi.CONCLUSION: In rats with hepatic encephalopathy,mRNA expression levels of GABAA receptor subunits α1,β1,γ2 increase significantly in basal nuclei, substantia nigra and hippocampi, suggesting that the changes of mRNA expression levels in GABAA receptor subunits may contribute to the pathogenesis of hepatic encephalopathy.     

Protein kinase C-dependent activation of P44／42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning

AIM: To investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (MAPKs) and heat shock protein (HSP)70 signal transduction during hepatocyte ischemic preconditioning. METHODS: In this study we used an in vitro ischemic preconditioning (IP) model for hepatocytes and an in vivo model for rat liver to investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (P44/42 MAPKs) and heat shock protein 70 (HSP70) signal transduction in IP. Through a normal liver cell hypoxic preconditioning (HP) model in which cultured normal liver cells were subjected to 3 cycles of 5 rain of incubation under hypoxic conditions followed by 5 rain of reoxygenation and subsequently exposed to hypoxia and reoxygenation for 6 h and 9 h respectively. PKC inhibitor, activator and MEK inhibitor were utilized to analyze the phosphorylation of PKC, the expression of P44/42 MAPKs and HSP70. Viability and cellular ultrastructure were also observed. By using rat liver as an in vivo model of liver preconditioning (3 cycles of 10-min occlusion and 10-min reperfusion), in vivo phosphorylation of PKC and P44/42MAPKs, HSP70 expression were further analyzed. AST/ALT concentration, cellular structure and ultrastruture were also observed. All the data were statistically analyzed. RESULTS: Similar results were obtained in both in vivo and in vitro IP models. Compared with the control withouts IP (or HP), the phosphorylation of PKC and P44/42 MAPKs and the expression of HSP70 were obviously increased in IP (or HP) treated model in which cytoprotection could be found. The effects of preconditioning were mimicked by stimulating PKC with 4β phorobol-12-myristate 13-acetate (PMA). Conversely, inhibiting PKC with chelerythrine abolished the protection given by preconditioning. PD98059, inhibitor of MEK (the upstream kinase of P44/42MAPKs), also reverted the cytoprotection exerted by preconditioning. CONCLUSION: The results demonstrate that preconditioning induces a rapid activation of P44/421VlAPKs and PKC activation plays a pivotal role in the activation of P44/42 MAPKs pathway that participates in the preservation of liver cells. HSP expression is regulated by signals in PKC dependent P44/42 MAPKs pathway.     

Protein kinase C-dependent activation of P44/42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning

AIM:To investigate the significance of protein kinase C(PKC),P44/42 mitogen-activated protein kinase (MAPKs)and heat shock protein (HSP)70 signal transduction duringhepatocyte ischemic preconditioning.METHODS:In this study we used an in vitro ischemicpreconditioning (IP) model for hepatocytes and an in vivomodel for rat liver to investigate the significance of proteinkinase C (PKC),P44/42 mitogen-activated protein kinase(P44/42 MAPKs) and heat shock protein 70 (HSP70) signaltransduction in IP.Through a normal liver cell hypoxicpreconditioning (HP) model in which cultured normal livercells were subjected to 3 cycles of 5 min of incubationunder hypoxic conditions followed by 5 min of reoxygenationand subsequently exposed to hypoxia and reoxygenationfor 6 h and 9 h respectively.PKC inhibitor,activator andMEK inhibitor were utilized to analyze the phosphorylationof PKC,the expression of P44/42 MAPKs and HSP70.Viability and cellular ultrastructure were also observed.Byusing rat liver as an in vivo model of liver preconditioning(3 cycles of 10-min occlusion and 10-min reperfusion),invivo phosphorylation of PKC and P44/42MAPKs,HSP70expression were further analyzed.AST/ALT concentration,cellular structure and ultrastruture were also observed.All the data were statistically analyzed.RESULTS:Similar results were obtained in both in vivoand in vitro IP models.Compared with the control withoutIP (or HP),the phosphorylation of PKC and P44/42 MAPKsand the expression of HSP70 were obviously increased inIP (or HP) treated model in which cytoprotection could befound.The effects of preconditioning were mimicked bystimulating PKC with 4β phorobol-12-myristate13-acetate(PMA).Conversely,inhibiting PKC with chelerythrineabolished the protection given by preconditioning.PD98059,inhibitor of MEK (the upstream kinase of P44/42MAPKs),also reverted the cytoprotection exerted by preconditioning.CONCLUSION:The results demonstrate that preconditioninginduces a rapid activation of P44/42MAPKs and PKC activationplays a pivotal role in the activation of P44/42 MAPKs pathway that participates in the preservation of liver cells.HSPexpression is regulated by signals in PKC dependent P44/42 MAPKs pathway.     

Enhanced expression of insulin receptor substrate–2 and activation of protein kinase B/Akt in regenerating pancreatic duct epithelium of 60 %-partial pancreatectomy rats

Aims/hypothesis: Early compensatory mechanisms of regeneration following partial pancreatectomy involve ductal proliferation and, subsequently, differentiation into acinar and endocrine cell types, although it is not clear how these processes are regulated. We investigated the expression and roles of insulin receptor substrate-2 (IRS-2) and protein kinase B/Akt (Akt) in pancreatic regeneration that starts with the common duct epithelium using a non-diabetic model of beta cell adaptation and mass expansion, 60 %-pancreatectomy rats. Methods: We used confocal immunofluorescence microscopy to study IRS-2 and Akt expression and activation in pancreatic common ducts at intervals after surgery. These proteins were studied in relation to proliferation markers and insulin immunostaining. Results: In pancreatectomized rats, a short-term increase in proliferation was observed in the common duct epithelial lining ( ∼ 4-fold) compared with sham-operated control rats which correlated with about a 1.8-fold increase in IRS-2 immunoreactivity 2 days after surgery. Interspersed with proliferating cells of the common duct, evaginations were rare single and clustered insulin immunopositive cells which expressed high levels of IRS-2 immunoreactivity. Epithelium of duct evaginations from 2-day post-Px rats exhibited striking phospho-Akt staining ( ∼ 3.5-fold above control rats) without any detectable changes in total Akt staining. Conclusion/interpretation: Our data suggest that IRS-2 plays an important role in pancreatic regeneration and growth by mediating duct proliferation and by maintaining the differentiated beta cell. The restricted staining pattern of phospho-Akt to cells of the common duct evaginations suggests that it has a role in regulating post-mitotic events related to cell-specific gene expression or survival or both. [Diabetologia (2001) 44: 2056–2065] Received: 13 April 2001 and in revised form: 20 June 2001     

α-Lipoic acid regulates lipid metabolism through induction of sirtuin 1 (SIRT1) and activation of AMP-activated protein kinase

Aims/hypothesis  

Sirtuin 1 (SIRT1) is a longevity-associated protein, which regulates energy metabolism and lifespan in response to nutrient deprivation. It has been proposed to be a therapeutic target for obesity and metabolic syndrome. We investigated whether α-lipoic acid (ALA) exerts a lipid-lowering effect through regulation of SIRT1 activation and production in C₂C₁₂ myotubes.     

Ca(2+) /calmodulin- dependent protein kinase II mediates transforming growth factor-β-induced hepatic stellate cells proliferation but not in collagen α1(I) production

Aim: Hepatic stellate cells (HSC) are the major players in hepatic fibrosis. As a most potent mitogen, transforming growth factor-β (TGF-β) strongly activates HSC and increases intracellular Ca(2+) concentration. Here, we assessed the potential role of Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), a main downstream effector of the Ca(2+) signal in liver fibrogenesis cascade. Methods: A human immortal HSC cell line, LX-2, and primary rat hepatic stellate cells were used in current study. CaMKII blockage and Akt inhibition were performed by KN-93/CaMKIIα siRNA and LY294002, respectively. HSC proliferation was detected by 5-bromodeoxyuridine incorporation assay. Real-time polymerase chain reaction, western blot and enzyme-linked immunosorbent assay were used to measure mRNA, cellular protein and protein in medium, respectively. Procollagen α1(I) expression was detected by immunocytochemistry. The role of CaMKII on TGF-β/Smad-induced collagen α1(I) expression was determined by (CAGA)(12) -MLP luciferase activity assay. Results: TGF-β dramatically increased CaMKII mRNA, and total and phosphorylated CaMKII expression. KN-93 and CaMKIIα siRNA suppressed TGF-β-mediated HSC proliferation. CaMKII interruption blocked TGF-β-elicited Akt activation. LY294002 arrested HSC proliferation and collagen α1(I) production but had no effect on CaMKII. Furthermore, CaMKII led to increased p21 and p27 expression. KN-93 and CaMKIIα siRNA inhibited TGF-β-induced and basal collagen α1(I) production but had no effect on the activity of (CAGA)(12) -MLP luciferase in response to TGF-β stimulation. Conclusion: CaMKII is a pivotal signal in TGF-β-induced fibrogenic cascades by means of stimulating HSC proliferation, and involved in a basal collagen production. Therefore, CaMKII will be a potentially effective target in the development of therapeutic intervention strategies to attenuate hepatic fibrosis.     

Short-term adenosine monophosphate-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside treatment increases the sirtuin 1 protein expression in skeletal muscle

Adenosine monophosphate-activated protein kinase (AMPK) has been proposed to stimulate mitochondrial biogenesis and fat and glucose metabolism in skeletal muscle. Nicotinamide adenine dinucleotide-dependent histone deacetylase sirtuin 1 (SIRT1) is also thought to play a pivotal role for such metabolic adaptations. The purpose of the present study was to examine the effect of AMPK activation with the administration of AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) to rats on skeletal muscle SIRT1 protein expression as well as peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) and glucose transporter 4 (GLUT4) protein expression and hexokinase activity. The AICAR promoted the phosphorylation of AMPK α-subunit (Thr¹⁷²) and acetyl-coenzyme A carboxylase (Ser⁷⁹) without any change of total AMPK α-subunit or acetyl-coenzyme A carboxylase protein levels in both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. The SIRT1 protein expression increased at 24 hours after administration of AICAR in the EDL muscle but not in the soleus muscle. The PGC-1α protein expression increased in both the soleus and EDL muscles and GLUT4 did in the EDL muscle at 24 hours after an administration of AICAR. The hexokinase activity increased at 18 and 24 hours in the soleus and at 12, 18, and 24 hours in the EDL after an AICAR treatment. These results suggest that short-term AICAR treatment to rats promotes skeletal muscle AMPK phosphorylation and then coincidently increases the SIRT1 protein expression. In addition, such treatment also enhances the PGC-1α and GLUT4 protein contents and hexokinase activity in skeletal muscle.     

Roles of prostaglandin production and mitogen-activated protein kinase activation in hepatocyte growth factor—mediated rat hepatocyte proliferation

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF)-stimulated DNA synthesis in primary cultured rat hepatocytes. HGF-induced DNA synthesis was concentration-dependently inhibited by a cyclooxygenase inhibitor, indomethacin. BW755C, a dual inhibitor for cyclooxygenase and lipoxygenase activities, also inhibited hepatocyte growth. Prostaglandin E₁ (PGE₁), PGE₂, and PGF_2α induced DNA synthesis even at such a low concentration as 5 nmol/L and potentiated [³H] thymidine incorporation induced by HGF in an additive manner. HGF caused arachidonic acid (AA) release and eicosanoid production. These events were detectable within 10 minutes after stimulation and lasted for at least 60 minutes. Furthermore, two proteins with approximately 40 kd were tyrosine phosphorylated by HGF. These proteins were identified as p42/p44 mitogenactivated protein (MAP) kinases by anti-MAP kinase immunoblots, which were known to activate cytosolic phospholipase A₂ (cPLA₂), a key enzyme in AA release. Activation of MAP kinases was detectable within 5 minutes after stimulation with HGF and lasted for at least 60 minutes. EGF-mediated DNA synthesis was also inhibited by the above cyclooxygenase inhibitors. EGF caused AA release and tyrosine phosphorylation of MAP kinases. These results suggest that HGF as well as EGF causes AA release, probably through activation of cPLA₂ mediated by MAP kinases, and that PGs, metabolites of AA, might play a pivotal role in hepatocyte proliferation in an autocrine mechanism.     

Reduction of ischemia reperfusion injury after liver resection and hepatic inflow occlusion by α-Iipoic acid in humans

AIM: To evaluate the protective effects of preconditioning by alpha-lipoic acid (LA) in patients undergoing hepatic resection under inflow occlusion of the liver. METHODS: Twenty-four patients undergoing liver resection for various reasons either received 600 mg LA or NaCl 15 min before transection performed under inflow occlusion of the liver. Blood samples and liver wedge biopsy samples were obtained after opening of the abdomen immediately after inflow occlusion of the liver, and 30 min after the end of inflow occlusion of the liver. RESULTS: Serum levels of aspartate transferase and alanine transferase were reduced at all time points in patients who received LA in comparison to those who received NaCL. This was accompanied by reduced histomorphological features of oncosis. We observed TUNEL-positive hepatocytes in the livers of the untreated patients, especially after 30 min of ischemia. LA attenuated this increase of TUNEL-positive hepatocytes. Under preconditioning with LA, ATP content was significantly enhanced after 30 min of ischemia and after 30 min of reperfusion. CONCLUSION: This is the first report on the potential for LA reducing ischemia/reperfusion injury (IRI) of the liver in humans who were undergoing liver surgery. Beside its simple and rapid application, side effects did not occur. LA might therefore represent a new strategy against hepatic IRI in humans.     

Reduction of ischemia reperfusion injury after liver resection and hepatic inflow occlusion by α-lipoic acid in humans

INTRODUCTION Excessive blood loss during surgery and the need for transfusion have been shown to hinder the post-operative course of patients[1]. In order to minimize blood loss, clamping of the portal triad (inflow occlusion), also called pringle maneuvr…