Glycogen Synthesis In Hepatocytes Research Articles

Resveratrol Ameliorates High-Fat-Diet-Induced Abnormalities in Hepatic Glucose Metabolism in Mice via the AMP-Activated Protein Kinase Pathway.

Diabetes mellitus is highly prevalent worldwide. High-fat-diet (HFD) consumption can lead to liver fat accumulation, impair hepatic glycometabolism, and cause insulin resistance and the development of diabetes. Resveratrol has been shown to improve the blood glucose concentration of diabetic mice, but its effect on the abnormal hepatic glycometabolism induced by HFD-feeding and the mechanism involved are unknown. In this study, we determined the effects of resveratrol on the insulin resistance of high-fat-diet-fed mice and a hepatocyte model by measuring serum biochemical indexes, key indicators of glycometabolism, glucose uptake, and glycogen synthesis in hepatocytes. We found that resveratrol treatment significantly ameliorated the HFD-induced abnormalities in glucose metabolism in mice, increased glucose absorption and glycogen synthesis, downregulated protein phosphatase 2A (PP2A) and activated Ca2+/CaM-dependent protein kinase kinase β (CaMKKβ), and increased the phosphorylation of AMP-activated protein kinase (AMPK). In insulin-resistant HepG2 cells, the administration of a PP2A activator or CaMKKβ inhibitor attenuated the effects of resveratrol, but the administration of an AMPK inhibitor abolished the effects of resveratrol. Resveratrol significantly ameliorates abnormalities in glycometabolism induced by HFD-feeding and increases glucose uptake and glycogen synthesis in hepatocytes. These effects are mediated through the activation of AMPK by PP2A and CaMKKβ.

Saturated fatty acids-induced miR-424–5p aggravates insulin resistance via targeting insulin receptor in hepatocytes

The excessive intake of saturated fatty acids (SFA) causes obesity and liver steatosis, which are major risk factors for insulin resistance and type 2 diabetes. Although the expression of certain microRNAs (miRNAs) targeting the insulin signaling molecules are regulated aberrantly in SFA-induced obesity, their implications on hepatic insulin resistance are largely unknown. This study examined the associations of miR-424–5p, which is induced by SFA, with the development of insulin resistance. SFA palmitate (PA)-treated HepG2 cells and high fat diet (HFD)-induced obese mouse livers showed an impairment of insulin signaling due to a significant decrease in INSR and IRS-1 expression. Based on expression profiling and qRT-PCR analysis, miR-424–5p, which presumably targets the 3′UTR of INSR, was upregulated in both PA-treated HepG2 cells and the liver of HFD-fed mice. miR-424–5p was found to target the 3′UTR of INSR directly and downregulated INSR expression at the post-transcriptional step. Furthermore, the overexpression of miR-424–5p suppressed INSR expression significantly, leading to impaired insulin signaling and glycogen synthesis in hepatocytes. A novel mechanism for how SFA-induced miR-424–5p impairs insulin signaling through the targeting of INSR is reported. In addition, the crucial role and underlying mechanism of miR-424–5p in the obesity-induced hepatic insulin resistance is explained.

Induction of miR-96 by Dietary Saturated Fatty Acids Exacerbates Hepatic Insulin Resistance through the Suppression of INSR and IRS-1.

Obesity is defined as the excessive accumulation of body fat that ultimately leads to chronic metabolic diseases. Diets rich in saturated fatty acids (SFA) exacerbate obesity and hepatic steatosis, which increase the risk of hepatic insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play an important role in a range of biological processes, the implications of SFA-induced miRNAs in metabolic dysregulation, particularly in the pathogenesis of hepatic insulin resistance, are not well understood. This study investigated the implications of miR-96, which is induced strongly by SFA, in the development of hepatic insulin resistance. The liver of HFD mice and the palmitate-treated hepatocytes exhibited an impairment of insulin signaling due to the significant decrease in INSR and IRS-1 expression. According to expression profiling and qRT-PCR analysis of the miRNAs, the expression level of miR-96 was higher in hepatocytes treated with palmitate. Moreover, miR-96 was also upregulated in the liver of HFD mice. Interestingly, miR-96 targeted the 3’UTRs of INSR and IRS-1 directly, and repressed the expression of INSR and IRS-1 at the post-transcriptional level. Accordingly, the overexpression of miR-96 was found to cause a significant decrease in INSR and IRS-1 expression, thereby leading to an impairment of insulin signaling and glycogen synthesis in hepatocytes. These results reveal a novel mechanism whereby miR-96 promotes the pathogenesis of hepatic insulin resistance resulted from SFA or obesity.

Potential Role of Glycogen Synthase Kinase-3β in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3β inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3β inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3β inhibitors were mimicked by transfection with GSK-3β siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3β mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3β reduces myocardin transcriptional activity, suggesting a role for GSK-3β in myocardin transcriptional activity and smooth muscle differentiation.

Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes.

Obesity increases intracellular lipid accumulation in key tissues or organs, which often leads to metabolic dysfunction and insulin resistance. Diets rich in saturated fatty acid (SFA) exacerbate obesity and hepatic steatosis, which accentuate the risk of insulin resistance and type 2 diabetes (T2DM). Although microRNAs (miRNAs) play a critical role in the regulation of gene expression, the implication of obesity-induced miRNAs in metabolic disorders particularly in the development of insulin resistance is largely unknown. Here, we investigated the implication of miR-15b, which is induced by SFA palmitate or obesity, in hepatic insulin resistance. Diet-induced obesity (DIO) in mice developed hyperglycemia and insulin resistance, accompanying with a reduction of insulin receptor (INSR) expression. Palmitate impaired insulin signaling as well as a decrease of INSR in hepatocytes. The expression of miR-15b was upregulated by DIO or palmitate in hepatocytes. Furthermore, the overexpression of miR-15b suppressed the protein expression of INSR through targeting INSR 3' untranslated region directly, resulting in an impairment of the insulin signaling and glycogen synthesis in hepatocytes. These results unveil a novel mechanism whereby miR-15b is linked causally to the pathogenesis of hepatic insulin resistance in SFA-induced obesity.

MiR-19a regulates PTEN expression to mediate glycogen synthesis in hepatocytes.

MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1–6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1–6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression.

Protective effect of pretreatment with propofol against tumor necrosis factor-α-induced hepatic insulin resistance

Insulin resistance is common in critically ill patients and seriously affects their prognosis. The anesthetic propofol (2,6-diisopropylphenol) has been shown to cause insulin resistance in rats; however, the specific mechanism underlying this phenomenon remains unknown. Thus, the aim of the present study was to determine the molecular mechanism through which propofol influences insulin resistance in the liver. The current study assessed the effects of propofol on the phosphorylation level of key enzymes involved in the insulin signaling pathway, as well as the glycogen content in primary mouse hepatocytes. Propofol administration was demonstrated to considerably reduce the phosphorylation levels of Akt (Ser473) and glycogen synthase kinase (GSK)-3β (Ser9) in the primary mouse hepatocytes. In addition, propofol was shown to downregulate the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3β signaling pathway and inhibit glycogen synthesis in hepatocytes. Thus, the present results indicated that propofol induced insulin resistance in primary mouse hepatocytes. Notably, pretreatment with propofol in tumor necrosis factor (TNF)-α-induced primary mouse hepatocytes with insulin resistance was demonstrated to alleviate the inhibitory effects of TNF-α on the PI3K/Akt/GSK-3β signaling pathway and glycogen synthesis. These results indicated that propofol exerts a protective effect against insulin resistance in primary mouse hepatocytes induced by TNF-α, indicating that propofol therapy may be clinically feasible to alleviate insulin resistance in critically ill patients.

S 50131 and S 51434, two novel small molecule glucokinase activators, lack chronic efficacy despite potent acute antihyperglycaemic activity in diabetic mice

Small molecule glucokinase activators (GKAs) have been associated with potent antidiabetic efficacy and hepatic steatosis in rodents. This study reports the discovery of S 50131 and S 51434, two novel GKAs with an original scaffold and an atypical pharmacological profile. Activity of the compounds was assessed in vitro by measuring activation of recombinant glucokinase, stimulation of glycogen synthesis in rat hepatocytes and increased insulin secretion from rat pancreatic islets of Langerhans. Efficacy and safety in vivo were evaluated after oral administration in db/db mice by measuring glycaemia, HbA1c and dyslipidaemia-associated events. S 50131 and S 51434 activated GK and stimulated glycogen synthesis in hepatocytes and insulin secretion from pancreatic islets. Unexpectedly, while both compounds effectively lowered glycaemia after acute oral administration, they did not decrease HbA1c after a 4-week treatment in db/db mice. This lack of antidiabetic efficacy was associated with increased plasma free fatty acids (FFAs), contrasting with the effect of GKA50 and N00236460, two GKAs with sustained HbA1c lowering activity but neutral regarding plasma FFAs. S 50131, but not S 51434, also induced hepatic steatosis, as did GKA50 and N00236460. However, a shorter, 4-day treatment resulted in increased hepatic triglycerides without changing the plasma FFA levels, demonstrating dynamic alterations in the lipid profile over time. In addition to confirming the occurrence of dyslipidaemia with GKAs, these findings provide new insights into understanding how such compounds may sustain or lose efficacy over time.

Protein kinase Cδ but not PKCα is involved in insulin‐induced glucose metabolism in hepatocytes

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

A Novel Mechanism for Regulating Hepatic Glycogen Synthesis Involving Serotonin and Cyclin-Dependent Kinase-5

Hepatic autonomic nerves regulate postprandial hepatic glucose uptake, but the signaling pathways remain unknown. We tested the hypothesis that serotonin (5-hydroxytryptamine [5-HT]) exerts stimulatory and inhibitory effects on hepatic glucose disposal. Ligands of diverse 5-HT receptors were used to identify signaling pathway(s) regulating glucose metabolism in hepatocytes. 5-HT had stimulatory and inhibitory effects on glycogen synthesis in hepatocytes mediated by 5-HT1/2A and 5-HT2B receptors, respectively. Agonists of 5-HT1/2A receptors lowered blood glucose and increased hepatic glycogen after oral glucose loading and also stimulated glycogen synthesis in freshly isolated hepatocytes with greater efficacy than 5-HT. This effect was blocked by olanzapine, an antagonist of 5-HT1/2A receptors. It was mediated by activation of phosphorylase phosphatase, inactivation of glycogen phosphorylase, and activation of glycogen synthase. Unlike insulin action, it was not associated with stimulation of glycolysis and was counteracted by cyclin-dependent kinase (cdk) inhibitors. A role for cdk5 was supported by adaptive changes in the coactivator protein p35 and by elevated glycogen synthesis during overexpression of p35/cdk5. These results support a novel mechanism for serotonin stimulation of hepatic glycogenesis involving cdk5. The opposing effects of serotonin, mediated by distinct 5-HT receptors, could explain why drugs targeting serotonin function can cause either diabetes or hypoglycemia in humans.

Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Attenuates the Action of Insulin in Hepatocytes

TNF-like weak inducer of apoptosis (TWEAK), a relatively new member of the TNF superfamily, is an important immune/inflammatory regulator that has different functional properties from that of other members of this superfamily. We report herein that TWEAK induces cellular insulin resistance in both human hepatocellular carcinoma cell lines (Huh7 and HepG2) and primary rat hepatocytes by inhibiting both early insulin receptor (IR) signaling events and the downstream actions of insulin. TWEAK profoundly inhibited insulin-induced Akt phosphorylation in both a concentration- and time-dependent manner. This inhibitory effect occurred via mechanisms that involved the TWEAK receptor Fn14 and the activation of the canonical and noncanonical nuclear factor-kappaB signaling pathways. Furthermore, TWEAK significantly inhibited IRbeta autophosphorylation and IR substrate-1 activation, with concomitant increases in serine phosphorylation of IR substrate-1. Moreover, insulin-induced reduction of gluconeogenic enzyme gene expression and increases in glycogen synthesis in hepatocytes were significantly attenuated by TWEAK treatment. Therefore, these findings not only reveal a novel pathophysiological function of TWEAK/Fn14 but also uncover a new player that may contribute to the development of cellular insulin resistance in hepatocytes.

Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes

To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.

Acetylcholine exerts additive and permissive but not synergistic effects with insulin on glycogen synthesis in hepatocytes

Parasympathetic (cholinergic) innervation is implicated in the stimulation of hepatic glucose uptake by portal vein hyperglycaemia. We determined the direct effects of acetylcholine on hepatocytes. Acute exposure to acetylcholine mimicked insulin action on inactivation of phosphorylase, stimulation of glycogen synthesis and suppression of phosphoenolpyruvate carboxykinase mRNA levels but with lower efficacy and without synergy. Pre-exposure to acetylcholine had a permissive effect on insulin action similar to glucocorticoids and associated with increased glucokinase activity. It is concluded that acetylcholine has a permissive effect on insulin action but cannot fully account for the rapid stimulation of glucose uptake by the portal signal.

Stimulation of glycogen synthesis and inactivation of phosphorylase in hepatocytes by serotonergic mechanisms, and counter-regulation by atypical antipsychotic drugs

Intraportal infusion of serotonin (5-hydroxytryptamine, 5-HT) or inhibitors of its cellular uptake stimulate hepatic glucose uptake in vivo by either direct or indirect mechanisms. The aims of this study were to determine the direct effects of 5-HT in hepatocytes and to test the hypothesis that atypical antipsychotic drugs that predispose to type 2 diabetes counter-regulate the effects of 5-HT. Rat hepatocytes were studied in short-term primary culture. Serotonin (5-HT) stimulated glycogen synthesis at nanomolar concentrations but inhibited it at micromolar concentrations. The stimulatory effect was mimicked by alpha-methyl-5-HT, a mixed 5-HT1/5-HT2 receptor agonist, whereas the inhibition was counteracted by a 5-HT2B/2C receptor antagonist. alpha-Methyl-5-HT stimulated glycogen synthesis additively with insulin, but unlike insulin, did not stimulate glucose phosphorylation and glycolysis, nor did it cause Akt (protein kinase B) phosphorylation. Stimulation of glycogen synthesis by alpha-methyl-5-HT correlated with depletion of phosphorylase a. This effect could not be explained by elevated levels of glucose 6-phosphate, which causes inactivation of phosphorylase, but was explained, at least in part, by decreased phosphorylase kinase activity in situ. The antipsychotic drugs clozapine and olanzapine, which bind to 5-HT receptors, counteracted the effect of alpha-methyl-5-HT on phosphorylase inactivation. This study provides evidence for both stimulation and inhibition of glycogen synthesis in hepatocytes by serotonergic mechanisms. The former effects are associated with the inactivation of phosphorylase and are counteracted by atypical antipsychotic drugs that cause hepatic insulin resistance. Antagonism of hepatic serotonergic mechanisms may be a component of the hepatic dysregulation caused by antipsychotic drugs that predispose to type 2 diabetes.

Increased sensitivity of glycogen synthesis to phosphorylase‐a and impaired expression of the glycogen‐targeting protein R6 in hepatocytes from insulin‐resistant Zucker fa/fa rats

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

Reduced Expression of Focal Adhesion Kinase Disrupts Insulin Action in Skeletal Muscle Cells

Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-(14)C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr(397) phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.

Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt

Previous studies showed an insulin-"desensitizing" action of IL-6 on glycogen synthesis in hepatocytes. We recently found no inhibition of the proximal steps of the insulin signal cascade in human skeletal muscle cells. Because these data indicate a possible tissue-specific effect of IL-6, we investigated the influence of IL-6 on insulin-stimulated glycogen synthesis in these cells. At first, we found that incubation of the cells with 20 ng/ml IL-6 alone induced phosphorylation of Ser473 of Akt, but not of Thr308 time dependently and we observed that IL-6 augments insulin-induced Ser473 and Thr308 phosphorylation in the low nanomolar range of insulin. Moreover, IL-6 increased insulin-stimulated phosphorylation of glycogen synthase kinase-3. Accordingly, IL-6 enhanced glycogen synthesis in the presence of 3 and 10 nM insulin, whereas IL-6 alone had only a marginal effect. IL-6 treatment of C57Bl/6 mice readily stimulated phosphorylation of Ser473 in skeletal muscle. Our result that IL-6 did not induce Ser473 phosphorylation in the liver of these mice suggests a tissue-specific effect. Together, our data demonstrate a novel insulin-sensitizing function of IL-6 on glycogen synthesis in skeletal muscle cells and indicate that IL-6 exerts cell/tissue-specific effects on insulin action.

Effects of Glucagon-like Peptide 1 on the Hepatic Glucose Metabolism

Glucagon-like peptide 1 has important actions in lowering blood glucose, both through its incretin action and by regulating other systems affecting glucose metabolism. There is good evidence that the coordinate regulation of islet hormones by GLP-1 has significant effects on hepatic glucose metabolism, and this likely contributes to the potent effect of GLP-1 on fasting hyperglycemia in diabetic patients. More controversial are potential effects of GLP-1 on hepatic glucose production or storage independent of insulin and glucagon. There are data from in vitro studies supporting an effect of GLP-1 to promote glycogen synthesis in hepatocytes, and several in vivo studies suggesting that GLP-1 has independent effects on hepatic glucose uptake and/or production. However, these findings must be considered against a backdrop of studies that have not demonstrated islet-independent actions of GLP-1. This paper will review the current literature addressing hepatic effects of GLP-1 and identify important gaps in the knowledge base for this topic.

Focal Adhesion Kinase (FAK) Regulates Insulin-stimulated Glycogen Synthesis in Hepatocytes

Experimental data support a role for FAK, an important component of the integrin signaling pathway, in insulin action. To test the hypothesis that FAK plays a regulatory role in hepatic insulin action, we overexpressed wild type (WT), a kinase inactive (KR), or a COOH-terminal focal adhesion targeting (FAT) sequence-truncated mutant of FAK in HepG2 hepatoma cells. In control untransfected (NON) and vector (CMV2)- and WT-transfected cells, insulin stimulated an expected 54 +/- 13, 37 +/- 4, and 47 +/- 12 increase in [U-(14)C]glucose incorporation into glycogen, respectively. This was entirely abolished in the presence of either KR (-1 +/- 7%) or FAT mutants (0 +/- 8%, n = 5, p < 0.05 for KR or FAT versus other groups), and this was associated with a significant attenuation of incremental insulin-stimulated glycogen synthase (GS) activity. Insulin-stimulated serine phosphorylation of Akt/protein kinase B was significantly impaired in mutant-transfected cells. Moreover, the ability of insulin to inactivate GS kinase-3beta (GSK-3beta), the regulatory enzyme immediately upstream of GS, by serine phosphorylation (308 +/- 16, 321 +/- 41, and 458 +/- 34 optical densitometric units (odu) in NON, CMV2, and WT, respectively, p < 0.02 for WT versus CMV2) was attenuated in the presence of either FAT (205 +/- 14, p < 0.01) or KR (189 +/- 4, p < 0.005) mutants. FAK co-immunoprecipitated with GSK-3beta, but only in cells overexpressing the KR (374 +/- 254 odu) and FAT (555 +/- 308) mutants was this association stimulated by insulin compared with NON (-209 +/- 92), CMV2 (-47 +/- 70), and WT (-39 +/- 31 odu). This suggests that FAK and GSK-3beta form both a constitutive association and a transient complex upon insulin stimulation, the dissociation of which requires normal function and localization of FAK. We conclude that FAK regulates the activity of Akt/protein kinase B and GSK-3beta and the association of GSK-3beta with FAK to influence insulin-stimulated glycogen synthesis in hepatocytes. Insulin action may be subject to regulation by the integrin signaling pathway, ensuring that these growth and differentiation-promoting pathways act in a coordinated and/or complementary manner.

Hepatic Glycogen Synthesis Is Highly Sensitive to Phosphorylase Activity: EVIDENCE FROM METABOLIC CONTROL ANALYSIS

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.