O-GlcNAcylation of Orphan Nuclear Receptor Estrogen-Related Receptor γ Promotes Hepatic Gluconeogenesis.

Estrogen-related receptor γ (ERRγ) is a major positive regulator of hepatic gluconeogenesis. Its transcriptional activity is suppressed by phosphorylation signaled by insulin in the fed state, but whether posttranslational modification alters its gluconeogenic activity in the fasted state is not known. Metabolically active hepatocytes direct a small amount of glucose into the hexosamine biosynthetic pathway, leading to protein O-GlcNAcylation. In this study, we demonstrate that ERRγ is O-GlcNAcylated by O-GlcNAc transferase in the fasted state. This stabilizes the protein by inhibiting proteasome-mediated protein degradation, increasing ERRγ recruitment to gluconeogenic gene promoters. Mass spectrometry identifies two serine residues (S317, S319) present in the ERRγ ligand-binding domain that are O-GlcNAcylated. Mutation of these residues destabilizes ERRγ protein and blocks the ability of ERRγ to induce gluconeogenesis in vivo. The impact of this pathway on gluconeogenesis in vivo was confirmed by the observation that decreasing the amount of O-GlcNAcylated ERRγ by overexpressing the deglycosylating enzyme O-GlcNAcase decreases ERRγ-dependent glucose production in fasted mice. We conclude that O-GlcNAcylation of ERRγ serves as a major signal to promote hepatic gluconeogenesis.

Propofol-ketamine or propofol-remifentanil for deep sedation and analgesia in pediatric patients undergoing burn dressing changes: a randomized clinical trial.

OBJECTIVE: In this study, we compared the propofol-ketamine and propofol-remifentanil combinations for deep sedation and analgesia during pediatric burn wound dressing changes. METHODS: Fifty pediatric patients aged 12-36 months, undergoing burn wound dressing changes, were randomly assigned to receive propofol-remifentanil (group PR) or propofol-ketamine (group PK) for deep sedation and analgesia. Patients in the group PR received 2 mg·kg(-1) propofol and 0.1 µg·kg(-1) remifentanil, and 0.05 µg·kg(-1) ·min(-1) remifentanil was infused continuously until the end of the procedure. Patients in the group PK received 2 mg·kg(-1) propofol and 1 mg·kg(-1) ketamine, and the same volume of isotonic saline was infused continuously until the end of the procedure. Additional propofol with remifentanil or ketamine was administered when required. Hemodynamic variables, drug requirements, occurrence of patient movement, surgeon's satisfaction score, recovery time, and the incidence of adverse events were recorded throughout the procedure and recovery. RESULTS: Recovery time was significantly shorter in the group PR compared to that in the group PK (10.3 [9.1-11.5] min vs 22.5 [20.3-25.6] min, median [interquartile range], respectively; P < 0.001). No significant hypotension or bradycardia occurred throughout the procedure. No significant differences were observed in terms of drug requirements, occurrence of patient movement, surgeon's satisfaction, incidence of respiratory depression, hypoxia, or nausea and vomiting CONCLUSIONS: The combinations of propofol-ketamine and propofol-remifentanil were effective for sedation and analgesia in pediatric patients undergoing burn dressing changes, but the propofol-remifentanil combination provided faster recovery compared to the propofol-ketamine combination.

Effect of ramosetron on the QT interval during sevoflurane anaesthesia in children: a prospective observational study.

BACKGROUND: We investigated the effects of concomitant administration of sevoflurane and ramosetron on the QT interval, the interval between the peak and end of the T wave (Tpe) and Tpe/QT ratio in children. OBJECTIVES: To compare the effects of concomitant administration of ramosetron and sevoflurane on heart rate corrected interval with Bazett's formula (QTc), Tpe interval and Tpe/QT ratio. DESIGN: A prospective observational study. SETTING: Elective orthopaedic surgery with patient-controlled analgesia. PATIENTS: Forty children aged between 3 and 12 years. INTERVENTION: ECG recordings were collected before induction (BASE), before sevoflurane administration (SEVO) and after the administration of ramosetron (SEVO and R). MAIN OUTCOME MEASURES: The heart rate corrected interval with Bazett's formula (QTc), Tpe interval and Tpe/QT ratio were calculated and the changes were analysed using repeated-measures analysis of variance (ANOVA). RESULTS: The QTc interval at BASE was 388.5 ±â€Š29.3 ms. It increased with sevoflurane anaesthesia to 414.9 ±â€Š21.4 ms and did not change with the administration of ramosetron (418.2 ±â€Š23.0 ms). The Tpe interval and Tpe/QT ratio did not differ between measurements. No ventricular arrhythmias occurred during the study. CONCLUSION: Ramosetron was not associated with prolongation of the QTc interval when it was given concomitantly with sevoflurane in children. No ventricular arrhythmias or other adverse effects occurred during the study.

PKB/Akt phosphorylation of ERRγ contributes to insulin-mediated inhibition of hepatic gluconeogenesis.

AIMS/HYPOTHESIS: Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. METHODS: We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) ß-deficient (Pkbß (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. RESULTS: We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbß (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBß-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. CONCLUSIONS/INTERPRETATION: These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.

Regulation of acetylation of histone deacetylase 2 by p300/CBP-associated factor/histone deacetylase 5 in the development of cardiac hypertrophy.

RATIONALE: Histone deacetylases (HDACs) are closely involved in cardiac reprogramming. Although the functional roles of class I and class IIa HDACs are well established, the significance of interclass crosstalk in the development of cardiac hypertrophy remains unclear. OBJECTIVE: Recently, we suggested that casein kinase 2α1-dependent phosphorylation of HDAC2 leads to enzymatic activation, which in turn induces cardiac hypertrophy. Here we report an alternative post-translational activation mechanism of HDAC2 that involves acetylation of HDAC2 mediated by p300/CBP-associated factor/HDAC5. METHODS AND RESULTS: Hdac2 was acetylated in response to hypertrophic stresses in both cardiomyocytes and a mouse model. Acetylation was reduced by a histone acetyltransferase inhibitor but was increased by a nonspecific HDAC inhibitor. The enzymatic activity of Hdac2 was positively correlated with its acetylation status. p300/CBP-associated factor bound to Hdac2 and induced acetylation. The HDAC2 K75 residue was responsible for hypertrophic stress-induced acetylation. The acetylation-resistant Hdac2 K75R showed a significant decrease in phosphorylation on S394, which led to the loss of intrinsic activity. Hdac5, one of class IIa HDACs, directly deacetylated Hdac2. Acetylation of Hdac2 was increased in Hdac5-null mice. When an acetylation-mimicking mutant of Hdac2 was infected into cardiomyocytes, the antihypertrophic effect of either nuclear tethering of Hdac5 with leptomycin B or Hdac5 overexpression was reduced. CONCLUSIONS: Taken together, our results suggest a novel mechanism by which the balance of HDAC2 acetylation is regulated by p300/CBP-associated factor and HDAC5 in the development of cardiac hypertrophy.

Clusterin decreases hepatic SREBP-1c expression and lipid accumulation.

Hepatic steatosis is emerging as the most important cause of chronic liver disease and is associated with the increasing incidence of obesity with insulin resistance. Sterol regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic gene expression in the liver. Hyperinsulinemia induces SREBP-1c transcription through liver X receptor (LXR), specificity protein 1, and SREBP-1c itself. Clusterin, an 80-kDa disulfide-linked heterodimeric protein, has been functionally implicated in several physiological processes including lipid transport; however, little is known about its effect on hepatic lipogenesis. The present study examined whether clusterin regulates SREBP-1c expression and lipid accumulation in the liver. Adenovirus-mediated overexpression of clusterin inhibited insulin- or LXR agonist-stimulated SREBP-1c expression in cultured liver cells. In reporter assays, clusterin inhibited SREBP-1c promoter activity. Moreover, adenovirus-mediated overexpression of clusterin in the livers of mice fed a high-fat diet inhibited hepatic steatosis through the inhibition of SREBP-1c expression. Reporter and gel shift assays showed that clusterin inhibits SREBP-1c expression via the repression of LXR and specificity protein 1 activity. This study shows that clusterin inhibits hepatic lipid accumulation through the inhibition of SREBP-1c expression and suggests that clusterin is a negative regulator of SREBP-1c expression and hepatic lipogenesis.

Protective role of clusterin/apolipoprotein J against neointimal hyperplasia via antiproliferative effect on vascular smooth muscle cells and cytoprotective effect on endothelial cells.

OBJECTIVE: Clusterin is induced in vascular smooth muscle cells (VSMCs) during atherosclerosis and injury-induced neointimal hyperplasia. However, its functional roles in VSMCs and endothelial cells remain controversial and elusive. This study was undertaken to clarify the role of clusterin in neointimal hyperplasia and elucidate its mechanism of action. METHODS AND RESULTS: Adenovirus-mediated overexpression of clusterin (Ad-Clu) repressed TNF-alpha-stimulated expression of MCP-1, fractalkine, ICAM-1, VCAM-1, and MMP-9, leading to inhibition of VSMC migration. Both Ad-Clu and secreted clusterin suppressed VSMC proliferation by inhibiting DNA synthesis, but not by inducing apoptosis. Ad-Clu upregulated p53 and p21(cip1/waf1) but downregulated cyclins D and E, leading to suppression of pRb phosphorylation and subsequent induction of G1 arrest in VSMCs. Clusterin deficiency augmented VSMC proliferation in vitro and accelerated neointimal hyperplasia in vivo, but concomitantly impaired reendothelialization in wire-injured murine femoral arteries. Moreover, Ad-Clu significantly reduced neointimal thickening in balloon-injured rat carotid arteries. Clusterin also diminished TNF-alpha-induced apoptosis of human umbilical vein endothelial cells and restored endothelial nitric oxide synthase expression suppressed by TNF-alpha. CONCLUSIONS: These results suggest that upregulation of clusterin during vascular injury may be a protective response against, rather than a causative response to, the development of neointimal hyperplasia.

Effects of PGC-1alpha on TNF-alpha-induced MCP-1 and VCAM-1 expression and NF-kappaB activation in human aortic smooth muscle and endothelial cells.

Increased oxidative stress in vascular cells is implicated in the pathogenesis of atherosclerosis. Reactive oxygen species (ROS) induce vascular inflammation via the proinflammatory cytokine/NF-kappaB pathway. Several lines of evidence suggest that peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1alpha) is an important regulator of intracellular ROS levels. However, no studies have examined the effects of PGC-1alpha on this process. We investigated the effects of PGC-1alpha on inflammatory molecule expression and activity of the redox-sensitive transcription factor, NF-kappaB, in vascular cells. PGC-1alpha expressed in human aortic smooth (HASMCs) and endothelial cells (HAECs) is upregulated by AMP-activated protein kinase activators, including metformin, rosiglitazone and alpha-lipoic acid. Tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory factor in the development of vascular inflammation, stimulates intracellular ROS production through an increase in both mitochondrial ROS and NAD(P)H oxidase activity. Adenovirus-mediated overexpression of the PGC-1alpha gene in HASMCs and HAECs leads to a significant reduction in intracellular and mitochondrial ROS production as well as NAD(P)H oxidase activity. Consequently, NF-kappaB activity and MCP-1 and VCAM-1 induced by TNF-alpha are suppressed. Our data support the possibility that agents stimulating PGC-1alpha expression in the vasculature aid in preventing the development of atherosclerosis.

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase 'Synoviolin'.

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Predominance of the group A killer Ig-like receptor haplotypes in Korean patients with T1D.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which pancreatic beta cells are selectively destroyed. Although autoimmune diseases are driven by inappropriate adaptive immunity, innate immunity may play a role in the development of T1D. To study the potential involvement of innate immunity in the pathogenesis of autoimmune disease, we investigated associations of the genes for 14 different killer Ig-like receptors (KIRs), the well-characterized receptors in natural killer cells, with Korean T1D patients. Genetic association analyses revealed that some of the KIR genes were associated with T1D. KIR2DL5 and 2DS2 genes were present at significantly low frequency in Korean T1D patients (P < 10(-4)). We did not detect any influence of ligand distribution on KIR association. With the haplotype assignments, 53% of the KIR haplotypes in the control are of type A. Compared with the control (P < 10(-3)) and autoantibody-negative patients (P < 10(-2)), the group A haplotype predominates in Korean patients with T1D. The KIR gene is associated with T1D and distribution differences between T1D and controls were not influenced by the HLA genes (DR-DQ-A-C). T1D, at least in Koreans, is associated with KIR genes, especially in the group A KIR haplotypes. There is a close relationship between innate and adaptive immunity.

Association of the polymorphism for Toll-like receptor 2 with type 1 diabetes susceptibility.

Type 1 diabetes mellitus (T1DM) is a T cell-mediated autoimmune disease in which pancreatic beta cells are selectively destroyed. Although autoimmune diseases are driven by inappropriate adaptive immunity, innate immunity may play a role in the development of T1DM. We investigated the association of the genes for toll-like receptor 2 (TLR2), one of the key surface receptors on innate effectors, with T1DM in Korean patients. Genetic association analyses revealed that the genotype composed of the rare allele (CC) of TLR2 1350 showed weak and protective association with T1DM (OR = 1.7, 95% CI: 1.1-2.7; P < .05) irrespective of the duration of disease, age, and autoantibody status. One of the TLR2 SNP haplotypes, TLR2-Ht4, was strongly associated with T1DM in that those subjects having more than one copy of Ht4 showed strong protection from developing T1DM (OR = 90.5; 95% CI: 13.8-235.7; P < 10(-5)). The TLR2 polymorphisms are associated with T1DM, and distribution differences between T1DM versus controls were not influenced by the HLA genes. There is a close relationship between innate and adaptive immunity.

SOX13 autoantibodies are likely to be a supplementary marker for type 1 diabetes in Korea.

The SOX13, one of the family of transcription factors that play key roles in organ development, is reported to be a diabetes autoantigen, islet cell antigen 12 (ICA12). Recently, a study of antibodies to SOX13 was conducted in patients with type 1 diabetes mellitus (T1DM) indicating that these antibodies potentially identified patients without antibodies to the major T1DM-associated autoantigens, insulin, GAD, or IA-2. We know that the prevalence of islet-specific autoantibodies (GAD, IA-2) in Korean patients is much lower than that in white patients. It may be possible that other autoantibodies that could be directed to as yet unknown antigen may play a role in Korean T1DM patients. To investigate this, we measured SOX13 autoantibodies applying a radioligand binding assay using in vitro transcribed and translated antigen in 188 T1DM patients (mean duration, 4.2 years) and 64 T2DM patients and compared the results with those of 101 healthy control subjects. SOX13 autoantibodies occurred at a significantly higher frequency among T1DM patients (55/188, 29.3%) than among T2DM patients (4/64, 6.2%) or healthy adult controls (1/101, 1%). The 55 patients with positive SOX13 antibodies had significantly shorter duration of diabetes than SOX13 antibody-negative patients (3.6 +/- 2.8 vs. 4.5 +/- 3.9 years; p < 0.05). We could detect a prevalence similar to control in patients with Hashimoto's thyroiditis (4.9%, n = 101) and rheumatoid arthritis (6.7%, n = 89). As a whole, 44 of the 55 patients with SOX13 antibodies had at least one or more other autoantibodies to the major T1DM-associated autoantigens. However, SOX13 antibodies were the only antibodies detected as positive in 1 of the 11 new-onset patients. We conclude, therefore, that these antibodies are likely to be one of several epitope-spreading responses to islet- or nonislet-specific autoantigens seen in the development of T1DM, and they may be used as a supplementary marker for investigating T1DM in Korea.