The Mediator complex kinase module is necessary for fructose regulation of liver glycogen levels through induction of glucose-6-phosphatase catalytic subunit (G6pc).

OBJECTIVE: Liver glycogen levels are dynamic and highly regulated by nutrient availability as the levels decrease during fasting and are restored during the feeding cycle. However, feeding in the presence of fructose in water suppresses glycogen accumulation in the liver by upregulating the expression of the glucose-6-phosphatase catalytic subunit (G6pc) gene, although the exact mechanism is unknown. We generated liver-specific knockout MED13 mice that lacked the transcriptional Mediator complex kinase module to examine its effect on the transcriptional activation of inducible target gene expression, such as the ChREBP- and FOXO1-dependent control of the G6pc gene promoter. METHODS: The relative changes in liver expression of lipogenic and gluconeogenic genes as well as glycogen levels were examined in response to feeding standard low-fat laboratory chow supplemented with water or water containing sucrose or fructose in control (Med13fl/fl) and liver-specific MED13 knockout (MED13-LKO) mice. RESULTS: Although MED13 deficiency had no significant effect on constitutive gene expression, all the dietary inducible gene transcripts were significantly reduced despite the unchanged insulin sensitivity in the MED13-LKO mice compared to that in the control mice. G6pc gene transcription displayed the most significant difference between the Med13 fl/fl and MED13-LKO mice, particularly when fed fructose. Following fasting that depleted liver glycogen, feeding induced the restoration of glycogen levels except in the presence of fructose. MED13 deficiency rescued the glycogen accumulation defect in the presence of fructose. This resulted from the suppression of G6pc expression and thus G6PC enzymatic activity. Among two transcriptional factors that regulate G6pc gene expression, FOXO1 binding to the G6pc promoter was not affected, whereas ChREBP binding was dramatically reduced in MED13-LKO hepatocytes. In addition, there was a marked suppression of FOXO1 and ChREBP-ß transcriptional activities in MED13-LKO hepatocytes. CONCLUSIONS: Taken together, our data suggest that the kinase module of the Mediator complex is necessary for the transcriptional activation of metabolic genes such as G6pc and has an important role in regulating glycogen levels in the liver through altering transcription factor binding and activity at the G6pc promoter.

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Performance Effects of Carbohydrate Ingestion Between Bouts of Intense Aerobic Interval Exercise.

PURPOSE: This study tested whether CHO intake during a 2-h rest between exercise bouts improved performance in the subsequent bout. METHODS: In a randomized, single-blinded, crossover design, 10 recreationally-active participants (23 ± 4 yr, 70.8 ± 6.6 kg, VO2peak:47.0 ± 5.4 mL O2·min-1·kg body mass-1) arrived at the lab post-prandial and completed 2 exercise bouts separated by 2-h rest. Bouts included 5 x 4-min intervals at ~80% VO2peak separated by 2-min at ~40% VO2peak and ended with an endurance trial (ET) to voluntary exhaustion at ~90% VO2peak. During intervals 1 and 4 in each bout expired gases were collected and O2 deficit was estimated. Immediately following bout-1, either a CHO (1.2 g CHO·kg body mass-1) or placebo (PL) solution was consumed. RESULTS: ET duration decreased in bout-2 vs. 1 in both conditions (P<0.01) but was ~35% longer in bout-2 with CHO vs. PL (Interaction, P=0.03; post-hoc, P=0.03). VO2 increased during interval 4 vs. 1 in both bouts (P<0.01) but was unaffected by CHO (P≥0.58). O2 deficit was unaffected by CHO (P=0.93), bout or interval (P≥0.15). Perceived exertion was higher in bout-2 vs. 1 (P<0.001) and reduced in intervals 2 and 4 in CHO (P≤0.01). CONCLUSIONS: When rest between training sessions is 2 hours, athletes may improve subsequent performance by consuming CHO during recovery. Supported by NSERC, Canada.

PEPCK1 Antisense Oligonucleotide Prevents Adiposity and Impairs Hepatic Glycogen Synthesis in High-Fat Male Fed Rats.

The increased hepatic gluconeogenesis in type 2 diabetes mellitus has often been ascribed to increased transcription of phosphoenolpyruvate carboxykinase 1, cystolic form (PEPCK1), although recent evidence has questioned this attribution. To assess the metabolic role of PEPCK1, we treated regular chow fed and high-fat fed (HFF) male Sprague-Dawley rats with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) against PEPCK1 and compared them with control ASO-treated rats. PEPCK1 ASO effectively decreased PEPCK1 expression in the liver and white adipose tissue. In chow fed rats, PEPCK1 ASO did not alter adiposity, plasma glucose, or insulin. In contrast, PEPCK1 ASO decreased the white adipose tissue mass in HFF rats but without altering basal rates of lipolysis, de novo lipogenesis, or glyceroneogenesis in vivo. Despite the protection from adiposity, hepatic insulin sensitivity was impaired in HFF PEPCK1 ASO-treated rats. PEPCK1 ASO worsened hepatic steatosis, although without additional impairments in hepatic insulin signaling or activation of inflammatory signals in the liver. Instead, the development of hepatic insulin resistance and the decrease in hepatic glycogen synthesis during a hyperglycemic clamp was attributed to a decrease in hepatic glucokinase (GCK) expression and decreased synthesis of glycogen via the direct pathway. The decrease in GCK expression was associated with increased expression of activating transcription factor 3, a negative regulator of GCK transcription. These studies have demonstrated that PEPCK1 is integral to coordinating cellular metabolism in the liver and adipose tissue, although it does not directly effect hepatic glucose production or adipose glyceroneogenesis.

Melatonin attenuates smoking-induced hyperglycemia via preserving insulin secretion and hepatic glycogen synthesis in rats.

Epidemiology survey indicated that cigarette smoking is a risk factor of diabetes. However, the precise mechanisms remain to be clarified. In this study, we found that smoking caused metabolic malfunctions on pancreas and liver in experimental animal model. These were indicated by hyperglycemia, increased serum hemoglobin A1c level and decreased insulin secretion, inhibition of liver glycogen synthase (LGS), and hepatic glycogen synthesis. Mechanistic studies revealed that all these alterations were caused by the inflammatory reaction and reactive oxygen species (ROS) induced by the smoking. Melatonin treatment significantly preserved the functions of both pancreas and liver by reducing ß cell apoptosis, CD68-cell infiltration, ROS production, and caspase-3 expression. The siRNA-knockdown model identified that the protective effects of melatonin were mediated by melatonin receptor-2 (MT2). This study uncovered potentially underlying mechanisms related to the association between smoking and diabetes. In addition, it is, for first time, to report that melatonin effectively protects against smoking-induced glucose metabolic alterations and the signal transduction pathway of melatonin is mainly mediated by its MT2 receptor. These observations provide solid evidence for the clinically use of melatonin to reduce smoking-related diabetes, and the therapeutic regimens are absent currently.

Hepatic mitochondrial dysfunction is a feature of Glycogen Storage Disease Type Ia (GSDIa).

Glycogen storage disease type Ia (GSDIa, von Gierke disease) is the most common glycogen storage disorder. It is caused by the deficiency of glucose-6-phosphatase, an enzyme which catalyses the final step of gluconeogenesis and glycogenolysis. Clinically, GSDIa is characterized by fasting hypoglycaemia and hepatic glycogen and triglyceride overaccumulation. The latter leads to steatohepatitis, cirrhosis, and the formation of hepatic adenomas and carcinomas. Currently, little is known about the function of various organelles and their impact on metabolism in GSDIa. Accordingly, we investigated mitochondrial function in cell culture and mouse models of GSDIa. We found impairments in oxidative phosphorylation and changes in TCA cycle metabolites, as well as decreased mitochondrial membrane potential and deranged mitochondrial ultra-structure in these model systems. Mitochondrial content also was decreased, likely secondary to decreased mitochondrial biogenesis. These deleterious effects culminated in the activation of the mitochondrial apoptosis pathway. Taken together, our results demonstrate a role for mitochondrial dysfunction in the pathogenesis of GSDIa, and identify a new potential target for the treatment of this disease. They also provide new insight into the role of carbohydrate overload on mitochondrial function in other hepatic diseases, such as non-alcoholic fatty liver disease.

MicroRNA-20a-5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression.

Recently, it is implicated that aberrant expression of microRNAs (miRs) is associated with insulin resistance. However, the role of miR-17 family in hepatic insulin resistance and its underlying mechanisms remain unknown. In this study, we provided mechanistic insight into the effects of miR-20a-5p, a member of miR-17 family, on the regulation of AKT/GSK pathway and glycogenesis in hepatocytes. MiR-20a-5p was down-regulated in the liver of db/db mice, and NCTC1469 cells and Hep1-6 cells treated with high glucose, accompanied by reduced glycogen content and impaired insulin signalling. Notably, inhibition of miR-20a-5p significantly reduced glycogen synthesis and AKT/GSK activation, whereas overexpression of miR-20a-5p led to elevated glycogenesis and activated AKT/GSK signalling pathway. In addition, miR-20a-5p mimic could reverse high glucose-induced impaired glycogenesis and AKT/GSK activation in NCTC1469 and Hep1-6 cells. P63 was identified as a target of miR-20a-5p by bioinformatics analysis and luciferase reporter assay. Knockdown of p63 in the NCTC1469 cells and the Hep1-6 cells by transfecting with siRNA targeting p63 could increase glycogen content and reverse miR-20a-5p inhibition-induced reduced glycogenesis and activation of AKT and GSK, suggesting that p63 participated in miR-20a-5p-mediated glycogenesis in hepatocytes. Moreover, our results indicate that p63 might directly bind to p53, thereby regulating PTEN expression and in turn participating in glycogenesis. In conclusion, we found novel evidence suggesting that as a member of miR-17 family, miR-20a-5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression.

MicroRNA 152 regulates hepatic glycogenesis by targeting PTEN.

Hepatic insulin resistance, defined as a diminished ability of hepatocytes to respond to the action of insulin, plays an important role in the development of type 2 diabetes and metabolic syndrome. Aberrant expression of mmu-miR-152-3p (miR-152) is related to the pathogenesis of tumors such as hepatitis B virus related hepatocellular carcinoma. However, the role of miR-152 in hepatic insulin resistance remains unknown. In the present study, we identified the potential role of miR-152 in regulating hepatic glycogenesis. The expression of miR-152 and the level of glycogen were significantly downregulated in the liver of db/db mice and mice fed a high fat diet. In vivo and in vitro results suggest that inhibition of miR-152 expression induced impaired glycogenesis in hepatocytes. Interestingly, miR-152 expression, glycogen synthesis and protein kinase B/glycogen synthase kinase (AKT/GSK) pathway activation were significantly decreased in the liver of mice injected with 16 µg·mL(-1) interleukin 6 (IL-6) by pumps for 7 days and in NCTC 1469 cells treated with 10 ng·mL(-1) IL-6 for 24 h. Moreover, hepatic overexpression of miR-152 rescued IL-6-induced impaired glycogenesis. Finally, phosphatase and tensin homolog (PTEN) was identified as a direct target of miR-152 to mediate hepatic glycogen synthesis. Our findings provide mechanistic insight into the effects of miR-152 on the regulation of the AKT/GSK pathway and the synthesis of glycogen in hepatocytes. Downregulated miR-152 induced impaired hepatic glycogenesis by targeting PTEN. PTEN participated in miR-152-mediated glycogenesis in hepatocytes via regulation of the AKT/GSK pathway.

Contribution of dietary starch to hepatic and systemic carbohydrate fluxes in European seabass (Dicentrarchus labrax L.).

In the present study, the effects of partial substitution of dietary protein by digestible starch on endogenous glucose production were evaluated in European seabass (Dicentrarchus labrax). The fractional contribution of dietary carbohydrates v. gluconeogenesis to blood glucose appearance and hepatic glycogen synthesis was quantified in two groups of seabass fed with a diet containing 30% digestible starch (DS) or without a carbohydrate supplement as the control (CTRL). Measurements were performed by transferring the fish to a tank containing water enriched with 5% (2)H2O over the last six feeding days, and quantifying the incorporation of (2)H into blood glucose and hepatic glycogen by (2)H NMR. For CTRL fish, gluconeogenesis accounted for the majority of circulating glucose while for the DS fish, this contribution was significantly lower (CTRL 85 (SEM 4) % v. DS 54 (SEM 2) %; P < 0.001). Hepatic glycogen synthesis via gluconeogenesis (indirect pathway) was also significantly reduced in the DS fish, in both relative (CTRL 100 (SEM 1) % v. DS 72 (SEM 1) %; P < 0.001) and absolute terms (CTRL 28 (SEM 1) v. DS 17 (sem 1) µmol/kg per h; P < 0.001). A major fraction of the dietary carbohydrates that contributed to blood glucose appearance (33 (sem 1) % of the total 47 (SEM 2) %) had undergone exchange with hepatic glucose 6-phosphate. This indicated the simultaneous activity of hepatic glucokinase and glucose 6-phosphatase. In conclusion, supplementation of digestible starch resulted in a significant reduction of gluconeogenic contributions to systemic glucose appearance and hepatic glycogen synthesis.

Glycogenin-2 is dispensable for liver glycogen synthesis and glucagon-stimulated glucose release.

CONTEXT: The synthesis of glycogen is initiated by glycogenin. In humans, glycogenin-1 is expressed ubiquitously, whereas glycogenin-2 (GN2) is highly expressed in liver. It has therefore been suggested that GN2 is a liver isoform of glycogenin. In a search for possible copy number variations associated with monogenic diabetes, we identified a 102-kb deletion of the X chromosome involving the entire GYG2 gene (encoding GN2) in 2 families. OBJECTIVE: The purpose of this study was to test whether male GYG2 deletion carriers had abnormal glucose metabolism and/or glycogen synthesis. DESIGN, SETTING, AND PATIENTS: Two families with diabetes and a GYG2 deletion were investigated with medical history and examination, glucagon stimulation tests, and liver biopsies. RESULTS: We identified a GYG2 deletion in 3 members of family 1, 8 members of family 2, and 1 blood donor. The deletion showed no clear cosegregation with diabetes. Deletion carriers reported no symptoms related to fasting. Results of cardiac examination and abdominal ultrasound imaging were normal. A glucagon stimulation test in 4 male deletion carriers showed a mean rise in plasma glucose of 3.6 mmol/L (95% confidence interval, 2.9-4.2) compared with 2.8 mmol/L (95% confidence interval, 2.2-3.4) in control subjects. Liver biopsy specimens did not show clear morphologic changes by light microscopy and showed the presence of both α- and ß-glycogen by electron microscopy. We detected GYG1 but not GYG2 mRNA expression in the liver biopsy specimens. CONCLUSIONS: This is the first evaluation of humans without GN2 expression. Our data indicate that GN2 is not required for liver glycogen synthesis and glucagon-stimulated glucose release.

Roles of hepatic glucokinase in intertissue metabolic communication: Examination of novel liver-specific glucokinase knockout mice.

Glucokinase is expressed principally in pancreatic ß-cells and hepatocytes, and catalyzes the phosphorylation of glucose to glucose-6-phosphate, a rate-limiting step of glycolysis. To better understand the roles of hepatic glucokinase, we generated Gck knockout mice by ablating liver-specific exon 1b. The knockout mice exhibited impaired glucose tolerance, decreased hepatic glycogen content, and reduced Pklr and Fas gene expression in the liver, indicating that hepatic glucokinase plays important roles in glucose metabolism. It has also been reported that hepatic glucokinase regulates the expression of thermogenesis-related genes in brown adipose tissue (BAT) and insulin secretion in response to glucose. However, the liver-specific Gck knockout mice displayed neither altered expression of thermogenesis-related genes in BAT nor impaired insulin secretion by ß-cells under a normal chow diet. These results suggest that chronic suppression of hepatic glucokinase has a small influence on intertissue (liver-to-BAT as well as liver-to-ß-cell) metabolic communication.

A polysaccharide from Grifola frondosa relieves insulin resistance of HepG2 cell by Akt-GSK-3 pathway.

Grifola frondosa is an important fungal research resource. However, there was little report about hyperglycemic activity of Grifola frondosa polysaccharide on insulin resistance in vitro. In this study, the hypoglycemic activity of a polysaccharide obtained from Grifola frondosa (GFP) on HepG2 cell and hpyerglycemic mechanism were investigated. The purity of the isolated polysaccharides was examined by HPLC. In this research, it was found that GFP enhanced the absorption of glucose of HepG2 cells in a dose dependent manner at 24 h of 30 ugmL⁻¹. GC-MS and FT-IR spectroscopy analysis results showed that glucose and galactose were the dominant monosaccharides in GFP and the major component of GFP was ß-pyranoside. Western-blotting results showed that the HepG2 cell model treated with GFP activated the insulin receptor protein (IRS) in the cell membrane and increased phosphorylated-AktSer473 expression, which had an inhibition of glycogen synthase kinase (GSK-3). The down-regulation of GSK-3 stimulated synthesis of intracellular glycogen. The results above suggested that the GFP increased the metabolism of glucose and stimulated synthesis of intracellular glycogen through the Akt/GSK-3 pathway.

SIRT1-metabolite binding histone macroH2A1.1 protects hepatocytes against lipid accumulation.

Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development.

The glycogen synthase 2 gene (Gys2) displays parallel evolution between Old World and New World fruit bats.

Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism.

Regulation of glucose homeostasis and lipid metabolism by PPP1R3G-mediated hepatic glycogenesis.

Liver glycogen metabolism plays an important role in glucose homeostasis. Glycogen synthesis is mainly regulated by glycogen synthase that is dephosphorylated and activated by protein phosphatase 1 (PP1) in combination with glycogen-targeting subunits or G subunits. There are seven G subunits (PPP1R3A to G) that control glycogenesis in different organs. PPP1R3G is a recently discovered G subunit whose expression is changed along the fasting-feeding cycle and is proposed to play a role in postprandial glucose homeostasis. In this study, we analyzed the physiological function of PPP1R3G using a mouse model with liver-specific overexpression of PPP1R3G. PPP1R3G overexpression increases hepatic glycogen accumulation, stimulates glycogen synthase activity, elevates fasting blood glucose level, and accelerates postprandial blood glucose clearance. In addition, the transgenic mice have a reduced fat composition, together with decreased hepatic triglyceride level. Fasting-induced hepatic steatosis is relieved by PPP1R3G overexpression. In addition, PPP1R3G overexpression is able to elevate glycogenesis in primary hepatocytes. The glycogen-binding domain is indispensable for the physiological activities of PPP1R3G on glucose metabolism and triglyceride accumulation in the liver. Cumulatively, these data indicate that PPP1R3G plays a critical role in postprandial glucose homeostasis and liver triglyceride metabolism via its regulation on hepatic glycogenesis.

Glucose-6-phosphate-mediated activation of liver glycogen synthase plays a key role in hepatic glycogen synthesis.

The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through glycogen synthase kinase-3 and the glycogen-associated form of protein phosphatase 1. Here, we initially performed mutagenesis analysis and identified a key residue (Arg(582)) required for activation of GYS2 by G6P. We then used GYS2 Arg(582)Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation had been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P toward glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.

Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3ß.

The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P<0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3ß (GSK-3ß) (P<0.001). No differences in the protein levels for the insulin receptor ß (IRß), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P<0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P<0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3ß in the liver tissue was also decreased (P<0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggest that resistin reduces the levels of GSK-3ß phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin.

Activation of basal gluconeogenesis by coactivator p300 maintains hepatic glycogen storage.

Because hepatic glycogenolysis maintains euglycemia during early fasting, proper hepatic glycogen synthesis in the fed/postprandial states is critical. It has been known for decades that gluconeogenesis is essential for hepatic glycogen synthesis; however, the molecular mechanism remains unknown. In this report, we show that depletion of hepatic p300 reduces glycogen synthesis, decreases hepatic glycogen storage, and leads to relative hypoglycemia. We previously reported that insulin suppressed gluconeogenesis by phosphorylating cAMP response element binding protein-binding protein (CBP) at S436 and disassembling the cAMP response element-binding protein-CBP complex. However, p300, which is closely related to CBP, lacks the corresponding S436 phosphorylation site found on CBP. In a phosphorylation-competent p300G422S knock-in mouse model, we found that mutant mice exhibited reduced hepatic glycogen content and produced significantly less glycogen in a tracer incorporation assay in the postprandial state. Our study demonstrates the important and unique role of p300 in glycogen synthesis through maintaining basal gluconeogenesis.

The selective control of glycolysis, gluconeogenesis and glycogenesis by temporal insulin patterns.

Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin-signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLC(ex)). Based on the experimental results, we constructed a simple computational model that characterises response of insulin-signalling-dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLC(ex) was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin.

Portal vein glucose entry triggers a coordinated cellular response that potentiates hepatic glucose uptake and storage in normal but not high-fat/high-fructose-fed dogs.

The cellular events mediating the pleiotropic actions of portal vein glucose (PoG) delivery on hepatic glucose disposition have not been clearly defined. Likewise, the molecular defects associated with postprandial hyperglycemia and impaired hepatic glucose uptake (HGU) following consumption of a high-fat, high-fructose diet (HFFD) are unknown. Our goal was to identify hepatocellular changes elicited by hyperinsulinemia, hyperglycemia, and PoG signaling in normal chow-fed (CTR) and HFFD-fed dogs. In CTR dogs, we demonstrated that PoG infusion in the presence of hyperinsulinemia and hyperglycemia triggered an increase in the activity of hepatic glucokinase (GK) and glycogen synthase (GS), which occurred in association with further augmentation in HGU and glycogen synthesis (GSYN) in vivo. In contrast, 4 weeks of HFFD feeding markedly reduced GK protein content and impaired the activation of GS in association with diminished HGU and GSYN in vivo. Furthermore, the enzymatic changes associated with PoG sensing in chow-fed animals were abolished in HFFD-fed animals, consistent with loss of the stimulatory effects of PoG delivery. These data reveal new insight into the molecular physiology of the portal glucose signaling mechanism under normal conditions and to the pathophysiology of aberrant postprandial hepatic glucose disposition evident under a diet-induced glucose-intolerant condition.