All techniques were performed in guidelines which were accepted by the Sungkyunkwan University College of Medicine Institutional Pet Treatment And Use Committee. American Blot Immunoprecipitation and Evaluation. effect is normally blunted by S171A CRTC2, which is normally refractory to salt-inducible kinase (SIK)-reliant inhibition. Taken jointly, we would suggest that mammalian SMEK/PP4C protein get excited about the legislation of hepatic blood sugar fat burning capacity through dephosphorylation of CRTC2. SMK-1, a homolog of the suppressor of MEK null (SMEK), was been shown to be responsible for tension level U-104 of resistance phenotypes as well as for life expectancy extension observed with minimal insulin/insulin-like growth aspect (IGF)-1 receptor signaling and with diet plan limitation (10, 11). Later, it was shown that Psy2, an ortholog of SMK-1, is usually a unique regulatory subunit for phosphatase family 4 (PP4), and is involved in the cisplatin-based anticancer therapeutical resistance in this organism (12). In mammalians, two PP4R3 subunits were identified, termed PP4R3a (SMEK1) and PP4R3b (SMEK2). The resulting active PP4 complex includes protein phosphatase 4 catalytic subunit (PP4C), PP4R3, and PP4R2, and belongs to the type 2A family of phosphatases (13). In this study, we show that mammalian orthologs for SMK-1, SMEK1 and SMEK2, function as PP4R3s for a PP4 complex that enhances hepatic glucose production. We observed increased expression of both PP4R3 isoforms under fasting conditions or in mouse models of insulin resistance. In vitro phosphatase assay revealed that SMEK/PP4C is able to dephosphorylate phosphoserine 171 directly in CRTC2. Overexpression of SMEK promotes elevations in plasma glucose with increased hepatic gluconeogenic gene expression, whereas knockdown of hepatic SMEK proteins improves hyperglycemia by reducing hepatic glucose production via promotion of CRTC2 phosphorylation. These data support the proposal that SMEK/PP4C regulates hepatic glucose production by controlling CRTC2-dependent transcriptional events. Results SMEK Expression Is usually Induced on Fasting or by Insulin Resistance in the Liver. In and Fig. S1and Fig. S1and and and S2 0.05 and ** 0.01, test; = 8). (mice under ad libitum conditions (** 0.01, test; = 5). ( 0.05, test; = 4). ( 0.01 and * 0.05, test; = 10). The area under the curve U-104 (AUC) during the perfusion period for each condition is usually indicated. ( 0.01 and * 0.05, test; = 3). pcf, pcDNA-flag vacant vector. Data in represent the mean SD, and data in and represent the mean SEM. SMEK Enhances Hepatic Gluconeogenesis in Vivo. To assess the functional role of SMEK1/2 in hepatic glucose metabolism, we generated adenoviruses for expression of SMEK1 and SMEK2 and used them to infect hepatocytes. SMEK1/2 overexpression enhances mRNA levels for phosphoenol pyruvate carboxykinase (PEPCK) and glucose-6-phosphatase catalytic subunit (G6Pase) (Fig. S2and Fig. S3 and and Fig. S3and to confirm the in vivo conversation with SMEK1 in mouse liver. (and and and and Fig. S4 and 0.01 and * 0.05, test; = 10). ( 0.01 and * 0.05, test; = 10). (= 10). U-104 Six-week-old mice were fed a high-fat diet for 6 wk, and hyperinsulinemic-euglycemic clamp studies were performed (** 0.01 and * 0.05, test; = 13C14). Basal hepatic glucose output (basal HGO) (and represent the mean SEM, and data in represent the mean SD. SMEK Regulates CRTC2-Dependent Hepatic Gluconeogenesis in Vivo. Increased activation of CRTC2-dependent transcription has been linked to the hyperglycemic phenotype in rodent models U-104 of type 2 diabetes (9). Enhanced expression of SMEK proteins is observed in mice or WT mice feed a high-fat diet, which led us to test whether knockdown of SMEK also normalizes elevated blood glucose levels in these settings. Indeed, depletion of hepatic SMEK1/2 reduces hyperglycemia, with a slight decline in plasma insulin levels in mice (Fig. 4and Fig. S5mice (Fig. 4mice (Fig. U-104 S5mice (* 0.05, test; = 5). (mice (** 0.01 and * 0.05, test; = 7). (mice. HSP90, heat shock protein 90. (mice infected with Ad-SMEK RNAi (** 0.01, test; = 5). (mice infected with Ad-SMEK RNAi (** 0.01 and * 0.05, test; = 5). (represent the mean SEM, and data in represent the mean SD. As in the case of WT mice, SMEK deficiency greatly reduces gluconeogenic gene expression and CRE activity in livers of mice or mice fed a high-fat diet (Fig. S6 mice (Fig. S7mice does not further affect blood glucose levels or hepatic gluconeogenic gene expression (Fig. S7 and and Fig. S7and Cd33 and Fig. S1 and diabetic mice were purchased from Charles River Laboratories. Recombinant adenovirus (0.5 109 plaque-forming unit/mice) was delivered by tail vein injection to mice. Fasting blood glucose levels were measured from animals that were fasted for 16 or 4 h with free access to water. Plasma insulin levels were measured using insulin assay ELISA kits (SHIBAYAGI). For the glucose tolerance test or pyruvate challenge, mice were injected i.p. with glucose or pyruvate (2 g/kg.
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