Phosphoenolpyruvate Carboxykinase Gene Research Articles

Metabolic balancing acts of vitamin A in type-2 diabetes and obesity

Using mice that lack retinaldehyde dehydrogenase 1 gene (Raldh1-/- mice), Kierfer et al demonstrated that retinoids (metabolites of Vitamin A) play an important role in the regulation of cellular metabolisms and energetics. The Aldh1a1-/- mice were leaner and less prone to accumulate subcutaneous and visceral fat, and to acquire insulin resistance on high fat diet. Their lower fasting glucose levels concomitant with reduced hepatic expression of glucose 6-phosphatase and phosphoenol pyruvate carboxy kinase genes indicated that Aldh1a1-/- mice were defective in gluconeogenesis. These mice also had lower plasma levels of triglycerides, very low-density lipoprotein and low-density lipoprotein-triacylglycerol, while their skeletal muscles elicited higher expression of carnitine palmatoyl transferase, medium chain acyl-A dehydrogenase, peroxisome proliferation activated receptor (PPARα and PPARδ. Thus, the improved lipid and lipoprotein profiles of Raldh1a1-/- mice resulted from a combination of reduced lipogenesis and enhanced fatty acid oxidation by retinoids. The mechanistic details of how retinoids integrate fasting glucose, hepatic gluconeogenesis and adaptive thermogenesis independent of body mass deserve further study.

Molecular Mechanism of Hepatitis C Virus-Induced Glucose Metabolic Disorders

Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including metabolic disorders. Chronic HCV infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Glucose is transported into hepatocytes via glucose transporter 2 (GLUT2). Hepatocytes play a crucial role in maintaining plasma glucose homeostasis via the gluconeogenic and glycolytic pathways. We have been investigating the molecular mechanism of HCV-related type 2 diabetes using HCV RNA replicon cells and HCV J6/JFH1 system. We found that HCV replication down-regulates cell surface expression of GLUT2 at the transcriptional level. We also found that HCV infection promotes hepatic gluconeogenesis in HCV J6/JFH1-infected Huh-7.5 cells. HCV infection transcriptionally up-regulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. Gene expression of PEPCK and G6Pase was regulated by the transcription factor forkhead box O1 (FoxO1) in HCV-infected cells. Phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells, resulting in increased nuclear accumulation of FoxO1. HCV NS5A protein was directly linked with the FoxO1-dependent increased gluconeogenesis. This paper will discuss the current model of HCV-induced glucose metabolic disorders.

Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris

We have identified a methanol- and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P. pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucose-ammonium (Bio(-)) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC). Deletion of ROP results in a strain (ΔROP) that can grow under biotin-deficient conditions due to derepression of a biotin- and PC-independent pathway of anaplerotic synthesis of oxaloacetate. Northern analysis as well as microarray expression profiling of RNA isolated from WT and ΔROP strains cultured in Bio(-) medium indicate that expression of the phosphoenolpyruvate carboxykinase gene (PEPCK) is induced in ΔROP during biotin- or PC-deficiency even under glucose-abundant conditions. There is an excellent correlation between PEPCK expression and growth of ΔROP in Bio(-) medium, suggesting that ROP-mediated regulation of PEPCK may have a crucial role in the biotin- and PC-independent growth of the ΔROP strain. To our knowledge, ROP is the first example of a zinc finger transcription factor involved in the catabolite repression of PEPCK in yeast cells cultured under biotin- or PC-deficient and glucose-abundant conditions.

DNA damage induces down-regulation of <italic>PEPCK</italic> and <italic>G6P</italic> gene expression through degradation of PGC-1α

Hepatic gluconeogenesis plays a crucial role in glucose homeostasis. Although it is well established that various cellular processes are modulated by DNA damage, whether the DNA damage signaling pathway regulates gluconeogenesis has not yet been studied. In this study, we found that mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P), key enzymes for gluconeogenesis, were dramatically decreased upon IR- and UV-irradiation. PEPCK and G6P promoter activities were also suppressed by IR- and UV-irradiation, suggesting that PEPCK and G6P gene transcription are down-regulated upon DNA damage. We also found that the protein level of PGC-1α, which is a critical transcription factor for PEPCK gene expression, is decreased upon UV-irradiation. The decreased PGC-1α protein level was abolished by MG132, a potent proteasome inhibitor, suggesting that PGC-1α is degraded through the ubiquitin-proteasome pathway upon UV-irradiation. These results reveal a novel link between glucose metabolism and the DNA damage signaling pathway and suggest a possible role for PEPCK and G6P in the DNA damage response.

Rubratoxin B induces signs of fatty acid oxidation disorders (FAODs) in mice

Rubratoxin B is a mycotoxin that causes hypoglycemia and fatty liver. We investigated the effect of rubratoxin B on hepatic glycogen content and regulation, because blood glucose levels are associated with hepatic glycogen storage. Mice were treated with 1.5mg/kg rubratoxin B for 24h. Stomachs of treated mice became extremely swollen, and the contents were significantly heavier than those of controls. Hypoglycemia stimulates appetite; therefore, rubratoxin B may perturb satiation. Rubratoxin B evidently depleted hepatic glycogen stores. Phosphoenolpyruvate carboxykinase (PEPCK) activity and mRNA levels in treated mice were reduced, indicating that rubratoxin B caused hepatic glycogen depletion by inhibiting PEPCK. PEPCK activity and mRNA levels were reduced to similar degrees; it appears that PEPCK activity is regulated transcriptionally. Levels of the PEPCK gene trans-activators phospho-CREB (active form) and C/EBPα were significantly reduced in the livers of treated mice, suggesting that these factors are important for PEPCK gene transcription. Rubratoxicosis and fatty acid oxidation disorders (FAODs) share characteristic signs, such as robust appetite, hypoglycemia, hepatic glycogen depletion, and fatty liver. Although FAODs are generally considered genetic deficiencies, our results indicate that a chemical can also cause FAOD-like signs in mice.

Fatty liver is associated with impaired activity of PPARγ-coactivator 1α (PGC1α) and mitochondrial biogenesis in mice

Accumulating evidence indicates that mitochondria have a key role in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Histological studies demonstrated accumulation of fat vacuoles in up to 90% of hepatocytes in mice fed the CDE diet for 14 days. In addition, a decrease in mitochondrial levels, together with an increase in superoxide radicals' levels were observed, indicating elevation of oxidative stress in hepatocytes. ATP levels were decreased in livers from CDE-fed mice after overnight fasting. This was accompanied by a compensative and significant increase in peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α) mRNA levels in comparison to control livers. However, there was a reduction in PGC1α protein levels in CDE-treated mice. Moreover, the expression of mitochondrial biogenesis genes nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M) and mitochondrial transcription factor B2 (TFB2M), which are all regulated by PGC1α activity, remained unchanged in fasted CDE-treated mice. These results indicate impaired activity of PGC1α. The impaired activity was further confirmed by chromatin immunoprecipitation analysis, which demonstrated decreased interaction of PGC1α with promoters containing NRF-1 and NRF-2 response elements in mice fed the CDE diet. A decrease in PGC1α ability to activate the expression of the gluconeogenic gene phosphoenol-pyruvate carboxykinase was also observed. This study demonstrates, for the first time, that attenuated mitochondrial biogenesis in steatotic livers is associated with impaired biological activity of PGC1α.

Hepatitis C Virus Infection Promotes Hepatic Gluconeogenesis through an NS5A-Mediated, FoxO1-Dependent Pathway

Chronic hepatitis C virus (HCV) infection is often associated with type 2 diabetes. However, the precise mechanism underlying this association is still unclear. Here, using Huh-7.5 cells either harboring HCV-1b RNA replicons or infected with HCV-2a, we showed that HCV transcriptionally upregulated the genes for phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), the rate-limiting enzymes for hepatic gluconeogenesis. In this way, HCV enhanced the cellular production of glucose 6-phosphate (G6P) and glucose. PEPCK and G6Pase gene expressions are controlled by the transcription factor forkhead box O1 (FoxO1). We observed that although neither the mRNA levels nor the protein levels of FoxO1 expression were affected by HCV, the level of phosphorylation of FoxO1 at Ser319 was markedly diminished in HCV-infected cells compared to the control cells, resulting in an increased nuclear accumulation of FoxO1, which is essential for sustaining its transcriptional activity. It was unlikely that the decreased level of FoxO1 phosphorylation was mediated through Akt inactivation, as we observed an increased phosphorylation of Akt at Ser473 in HCV-infected cells compared to control cells. By using specific inhibitors of c-Jun N-terminal kinase (JNK) and reactive oxygen species (ROS), we demonstrated that HCV infection induced JNK activation via increased mitochondrial ROS production, resulting in decreased FoxO1 phosphorylation, FoxO1 nuclear accumulation, and, eventually, increased glucose production. We also found that HCV NS5A mediated increased ROS production and JNK activation, which is directly linked with the FoxO1-dependent increased gluconeogenesis. Taken together, these observations suggest that HCV promotes hepatic gluconeogenesis through an NS5A-mediated, FoxO1-dependent pathway.

Pharmacological evaluation of insulin mimetic novel suppressors of PEPCK gene transcription from Paeoniae Rubra Radix

Paeoniae Rubra Radix (root of Paeonia lactiflora) has been frequently employed in Traditional Chinese Medicine (TCM) as and anti-diabetic therapy to enhance blood circulation and dissipate stasis. Previously, we identified a novel hypoglycemic action of a crude extract from Paeoniae Rubra Radix, which also suppressed phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Therefore, the current investigation intended to elucidate potential active bio-constituents of this herb and mechanisms of action. Glucocorticoid receptor (GR) nuclear localization, the PEPCK messenger (m)RNA level, pregnane X receptor (PXR) mRNA expression, cAMP-responsive element-binding protein (CREB) serine phosphorylation and DNA binding were evaluated in dexamethasone (Dex) and 8-bromo-cAMP (CA)-stimulated H4IIE cells, while efficacy of agents was assessed in a stable cell line containing a green fluorescent protein (GFP) reporter driven by the PEPCK promoter. HPLC profiling, colorimetric assays, and NMR analysis were employed for chemical characterization purpose. An extract of Paeoniae Rubra Radix lacking the insulin mimetic compound, 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG), and termed the non-PGG fraction (NPF), consisting of tannin polymers, suppressed PEPCK expression in the presence of an insulin receptor antagonist (HNMPA-AM(3)), suggesting the action of this fraction is independent of the insulin receptor. Furthermore, Dex-stimulated GR nuclear localization and transactivation were prevented by the NPF. Similarly, CA-stimulated CREB serine phosphorylation and DNA binding were also inhibited by the NPF in H4IIE cells. Hence NPF antagonizes both signaling pathways that induce PEPCK gene transcription. In conclusion, the current study proposes that the potent suppressive activity on PEPCK gene transcription observed with Paeoniae Rubra Radix extract, can be attributed to at least two distinct components, namely PGG and NPF.

Gestational high fat diet programs hepatic phosphoenolpyruvate carboxykinase gene expression and histone modification in neonatal offspring rats

In insulin resistance and type II diabetes, there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycaemia. Studies in experimental animals have provided evidence that consumption of high fat (HF) diets by female rats programs the progeny for glucose intolerance in adulthood, but the mechanisms behind the in utero programming remain poorly understood. The present study analysed the effect of a maternal HF diet on fetal gluconeogenic gene expression and potential regulation mechanism related to histone modifications. Dams were fed either a Control (C, 16% kcal fat) or a high-fat (HF, 45% kcal fat) diet throughout gestation. Livers of the offspring were collected on gestational day 21 and analysed to determine the consequences of a maternal HF diet on molecular markers of fetal liver gluconeogenesis. We demonstrated that offspring of HF-fed dams were significantly heavier and had significantly higher blood glucose levels at the time of delivery than offspring of dams fed the C diet. While maternal gluconeogenesis and plasma glucose were not affected by the HF diet, offspring of HF-fed dams had significantly higher mRNA contents of gluconeogenic genes in addition to the elevated plasma glucose. In addition to increased transcription rate, a gestational HF diet resulted in modifications of the Pck1 histone code in livers of offspring. Our results demonstrate that in utero exposure to HF diet has the potential to program the gluconeogenic capacity of offspring through epigenetic modifications, which could potentially lead to excessive glucose production and altered insulin sensitivity in adulthood.

Allogalathea (Decapoda: Galatheidae): a monospecific genus of squat lobster?

The genus Allogalathea was established by Baba in 1969 to include the well-known species Galathea elegans. This species is widely distributed across the Indo-West Pacific Ocean, and is characterized by living in close association with crinoids, and by its conspicuous coloration. Although the genus is considered monospecific, different colour patterns and discrete morphological variations mainly associated with the rostrum and chelipeds have been reported. These differences could point to cryptic species, thereby questioning Allogalathea as a monotypic taxon. To address this issue, we sequenced the mitochondrial cytochrome oxidase I (COI; 658 bp) and 16S rRNA (882 bp) genes and the nuclear gene phosphoenolpyruvate carboxykinase (PEPCK; 598 bp) in numerous specimens from eight different localities, and also examined their morphological characters. DNA sequences were analysed using maximum-parsimony, maximum-likelihood, and Bayesian approaches of phylogenetic inference. The resulting trees were combined with morphological evidence to test species boundaries. Our molecular data revealed four deeply divergent clades, which can be distinguished by subtle morphological differences in the spinulation and length : breadth ratio of the P1 carpus, spinulation of the walking legs, and shape of the rostrum. Our findings indicated that Allogalathea elegans is in fact a species complex comprising four different species, which, although genetically very distinct, are morphologically very similar. We provide morphological descriptions and a key to these four species of the genus.

Retinoids induced Pck1 expression and attenuated insulin-mediated suppression of its expression via activation of retinoic acid receptor in primary rat hepatocytes

Insulin regulates the expression of genes involved in hepatic glucose and lipid metabolism, such as the cytosolic form of phosphoenolpyruvate carboxykinase gene (Pck1). We have reported that lipophilic molecules from rat livers induced Pck1 transcription and attenuated insulin-mediated suppression of its expression in primary rat hepatocytes. After identification of retinol and retinal as the active molecules, the present study was aimed to determine the effects of retinoids on insulin-mediated suppression of Pck1 expression in primary rat hepatocytes. Real-time PCR and reporter gene assays were designed to determine retinoid effects in the absence or presence of insulin on the expression levels of Pck1 mRNA and activation of its promoter constructs, respectively. The lipophilic extract from rat livers specifically induced the expression of Pck1, but not that of two other insulin-suppressed genes, glucose 6-phosphatase catalytic subunit and insulin-like growth factor-binding protein 1. Retinol, retinal, and retinoic acid (RA) induced Pck1 expression dose-dependently in primary hepatocytes. Specific activation of retinoic acid receptor (RAR), but not retinoid X receptor, attenuated insulin-mediated suppression of Pck1 expression. RARα antagonist (Ro41-5253) abolished the retinal-mediated induction of Pck1 expression and attenuation of insulin-mediated suppression of its expression. Disruption of the proximal, but not the distal, RA responsive element in the Pck1 promoter eliminated the RA response of Pck1 promoter reporter constructs in primary hepatocytes. The results of this study demonstrated for the first time that retinoid treatment attenuated insulin-mediated suppression of Pck1 expression in primary rat hepatocytes. It suggests that retinoid metabolism in hepatocytes may modulate hepatic insulin action.

Suppression of phosphoenolpyruvate carboxykinase gene expression by secoisolariciresinol diglucoside (SDG), a new antidiabetic agent

Secoisolariciresinol diglucoside (SDG) from flaxseed has been shown to be effective in preventing/delaying the development of type-1 and type-2 diabetes. The hypoglycemic effect of SDG in type-2 diabetes has been suggested to be due to its antioxidant activity. Hyperglycemia in type-2 diabetes could be due to an increase in the expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in the gluconeogenesis in the liver. It is possible that the hypoglycemic effect of SDG in type-2 diabetes is due to suppression of expression of PEPCK gene. An investigation, therefore, was made on the effect of SDG on the PEPCK gene expression. Primary hepatocyte culture was treated with insulin, a physiological inhibitor of PEPCK gene expression, or with SDG for 12 hours. After RNA extractions, Northern blot analysis was done. SDG in the concentration of 100 µM completely suppressed the expression of PEPCK gene. Insulin in the concentration of 10 nM almost completely suppressed the PEPCK gene expression. The results suggest that SDG suppresses the expression of PEPCK gene and that its known hypoglycemic effect may be due to suppression of PEPCK gene expression.

1162 ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN INTERFERON STIMULATED GENES WITH CHRONIC HEPATITIS C TREATMENT OUTCOME

insulin resistance and hepatic steatosis. Steatosis may increase susceptibility to apoptosis, inflammation and fibrosis by triggering hepatocytes to up-regulate CD95/Fas. We investigated this hypothesis and potential role of adipocytokines in modulating the progression of liver disease in patients with HCV-4. Methods: In 147 HCV patients and 89 controls we measured serum adiponectin, HMW adiponectin, leptin, TNF-a, IL-6 and CK-18. Liver biopsies were evaluated for steatosis/inflammation/ fibrosis, adiponectin mRNA/protein, AdipoR1/-R2 mRNA and phosphoenolpyruvate carboxykinase (PEPCK) gene expression; and adiponectin and CD95 immunoreactivity. The potential associations with hepatic steatosis and fibrosis were analyzed. Results: CD95 immunoreactivity and adiponectin immunoreactivity were readily detected in all biopsies examined and scored as grade 3 in 24 (16.3%) of patients, and as bright in 20 (13.6%) of patients, respectively. Adiponectin immunostaining within the liver correlated positively with the intensity of hepatic CD95/Fas immunostaining within the liver (r = 424; p =0.001). A significant association between high serum adiponectin and HMW adiponectin levels with CD95/Fas immunoreactivity (r = −0.16, p = 0.04, r = 0.21, p = 0.001; respectively), but not with adiponectin immunoreactivity in the liver was also identified. Adiponectin and HMW adiponectin were negatively correlated with the expression of AdipoR1, but positively correlated with the expression of AdipoR2. Hepatocyte CD95/Fas up-regulation correlated with steatosis, inflammation and fibrosis (p = 0.001). Significant correlation of serum adiponectin, HMW adiponectin and AdipoR1 and 2 mRNA expression, as well as liver adiponectin immunostaining within the liver were identified with steatosis. mRNA transcription for PEPCK was also significantly correlated with the amount of steatosis. A positive association between adiponectin and HMW adiponectin and hepatic inflammation was identified. This correlation remained significant even after following adjusting for age, gender and BMI (r = 0.17; p = 0.03; r = 0.45; p = 0.0001) respectively. Factors independently associated with the stage of fibrosis were HOMA-IR, inflammation score and age. Conclusions: Our findings in HCV-4 infection shows that adiponectin correlates with the different stages of liver injury. Steatosis up-regulates hepatocyte CD95/Fas and thus increases apoptosis, which facilitates inflammation and fibrosis. These findings may offer potential clues for future therapeutic intervention.

Maternal protein restriction during pregnancy induces CCAAT/enhancer-binding protein (C/EBPβ) expression through the regulation of histone modification at its promoter region in female offspring rat skeletal muscle

Maternal nutrition during pregnancy is an important intrauterine environmental factor that can cause persistent alterations of the offspring genome and is associated with potential disease risk later in life. In the present study, we investigated the impact of a maternal low protein diet (LP) on the expression of the transcription factor CCAAT/enhancer-binding protein (C/EBPβ) in offspring skeletal muscle. C/EBPβ belongs to a family of transcription factors that regulates the expression of genes involved in energy homeostasis and muscle development. We investigated C/EBPβ transcriptional regulation from an epigenetic aspect. We observed sex-dependent differences in C/EBPβ expression in offspring skeletal muscle subjected to a maternal protein-restricted diet. In female offspring skeletal muscle, both C/EBPβ mRNA and protein levels were increased by maternal protein restriction. However, C/EBPβ expression was not altered in other tissues or male offspring. Analysis of transcriptional and epigenetic regulation showed acetylated histone 3 and acetylated histone 4 at significantly increased levels at the C/EBPβ promoter region in female LP pup's muscle. Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription was also up-regulated in female LP pups through the increased binding of C/EBPβ at its promoter. The induction of C/EBPβ expression in female offspring skeletal muscle by maternal protein restriction during pregnancy may indicate C/EBPβ involvement in signaling response in energy metabolism to a low maternal protein diet.

ʟ-Leucine, ʟ-Methionine, and ʟ-Lysine Are Involved in the Regulation of Intermediary Metabolism-Related Gene Expression in Rainbow Trout Hepatocytes ,

Using rainbow trout hepatocytes stimulated with ʟ-leucine, ʟ-methionine, or ʟ-lysine in the presence or absence of bovine insulin, we investigated the ability of these amino acids to mimic the effects of a pool of amino acids on protein kinase B (Akt)/target of rapamycin (TOR) signaling pathways and expression of 6 genes known to be subjected to insulin and/or amino acid regulation [glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GK), pyruvate kinase (PK), fatty acid synthase (FAS), and serine dehydratase (SDH)]. Emphasis was placed on leucine, known to be a signaling molecule in mammals, and methionine and lysine that are essential amino acids limiting in plant-based diets for fish. In the presence of insulin, leucine (but not methionine or lysine) phosphorylated Akt and ribosomal protein S6 as previously observed with a pool of amino acids, suggesting that leucine might participate in the activation of the TOR pathway by amino acids in fish, as in mammals. G6Pase, PEPCK, GK, and SDH gene expression were higher in leucine-treated cells compared with control cells. Leucine combined with insulin reduced G6Pase gene expression by 90% and increased FAS gene expression > 4-fold compared with the control treatment. Methionine weakly decreased G6Pase, GK, and SDH gene expression and lysine weakly but significantly decreased the mRNA level of PEPCK. Thus, leucine regulated gluconeogenesis and lipogenesis, but not glycolysis, in the same way as a pool of amino acids. Methionine appeared to be involved in the regulation of specific genes, whereas lysine only had limited effects. These findings are particularly relevant regarding the involvement of amino acids in the regulation of metabolism-related gene expression.

Peroxisome Proliferator-activated Receptor α Is Responsible for the Up-regulation of Hepatic Glucose-6-phosphatase Gene Expression in Fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.

Ginseng Leaf Extract Prevents High Fat Diet-Induced Hyperglycemia and Hyperlipidemia through AMPK Activation

This study evaluated the protective effects of ginseng leaf extract (GLE) against high fat-diet-induced hyperglycemia and hyperlipidemia, and explored the potential mechanism underlying these effects in C57BL/6J mice. The mice were randomly divided into four groups: normal control, high fat diet control (HFD), GLE-treated at 250 ㎎/㎏, and GLE-treated at 500 ㎎/㎏. To induce hyperglycemic and hyperlipidemic states, mice were fed a high fat diet for 6 weeks and then administered GLE once daily for 8 weeks. At the end of the treatment, we examined the effects of GLE on plasma glucose, lipid levels, and the expression of genes related to lipogenesis, lipolysis, and gluconeogenesis. Both GLE groups lowered levels of plasma glucose, insulin, triglycerides, total cholesterol, and non-esterified fatty acids when compared to those in HFD group. Histological analysis revealed significantly fewer lipid droplets in the livers of GLE-treated mice compared with HFD mice. To elucidate the mechanism, Western blots and RT-PCR were performed using liver tissue. Compared with HFD mice, GLE-treated mice showed higher levels of phosphorylation of AMP-activated protein kinase (AMPK) and its substrate, acetyl-CoA carboxylase, but no differences in the expression of lipogenic genes such as sterol regulatory element-binding protein 1a, fatty acid synthase, sterol-CoA desaturase 1 and glycerol-3-phosphate acyltransferase. However, the expression levels of lipolysis and fatty acid uptake genes such as peroxisome proliferator-activated receptor-α and CD36 were increased. In addition, phospho-α and CD36 were increased. In addition, phosphoenolpyruvate carboxykinase gene expression was decreased. These results suggest that GLE ameliorates hyperglycemia and hyperlipidemia by inhibiting gluconeogenesis and stimulating lipolysis, respectively, via AMPK activation.

Gluconeogenesis in dairy cows: The secret of making sweet milk from sour dough

Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short-chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl-CoA carboxylase, methylmalonyl-CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6-bisphosphatase and glucose 6-phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high-producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An improved understanding of the complex mechanisms underlying gluconeogenesis may further improve our options to enhance the postpartum health status of dairy cows.

AMPK-dependent Repression of Hepatic Gluconeogenesis via Disruption of CREB·CRTC2 Complex by Orphan Nuclear Receptor Small Heterodimer Partner

Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of gluconeogenic enzyme gene expression. Here, we show that SHP inhibited protein kinase A-mediated transcriptional activity of cAMP-response element-binding protein (CREB), a major regulator of glucose metabolism, to modulate hepatic gluconeogenic gene expression. Deletion analysis of phosphoenolpyruvate carboxykinase (PEPCK) promoter demonstrated that SHP inhibited forskolin-mediated induction of PEPCK gene transcription via inhibition of CREB transcriptional activity. In vivo imaging demonstrated that SHP inhibited CREB-regulated transcription coactivator 2 (CRTC2)-mediated cAMP-response element-driven promoter activity. Furthermore, overexpression of SHP using adenovirus SHP decreased CRTC2-dependent elevations in blood glucose levels and PEPCK or glucose-6-phosphatase (G6Pase) expression in mice. SHP and CREB physically interacted and were co-localized in vivo. Importantly, SHP inhibited both wild type CRTC2 and S171A (constitutively active form of CRTC2) coactivator activity and disrupted CRTC2 recruitment on the PEPCK gene promoter. In addition, metformin or overexpression of a constitutively active form of AMPK (Ad-CA-AMPK) inhibited S171A-mediated PEPCK and G6Pase gene expression, and hepatic glucose production and knockdown of SHP partially relieved the metformin- and Ad-CA-AMPK-mediated repression of hepatic gluconeogenic enzyme gene expression in primary rat hepatocytes. In conclusion, our results suggest that a delayed effect of metformin-mediated induction of SHP gene expression inhibits CREB-dependent hepatic gluconeogenesis.

Lack of Association Between PCK1 Polymorphisms and Obesity, Physical Activity, and Fitness in European Youth Heart Study (EYHS)

Phosphoenolpyruvate carboxykinase-1 (PCK1) is the rate-limiting enzyme in the hepatic gluconeogenic pathway. Studies have shown that overexpression of Pck1 in mice results in obesity-related traits and higher levels of physical activity (PA). Therefore, our aims were to investigate whether common genetic variation in the PCK1 gene influences obesity-related traits, PA, and fitness, and to examine whether PA and fitness attenuate the influence of the PCK1 polymorphisms on obesity in children. Analyses were undertaken on data from Danish and Estonian children (958 boys and 1,104 girls) from the European Youth Heart Study (EYHS), a school-based, cross-sectional study of children (mean ± s.d. age: 9.6 ± 0.4 years) and adolescents (15.5 ± 0.5 years). We genotyped eight polymorphisms that captured the common genetic variations in the PCK1 gene. The association between the PCK1 polymorphisms and BMI, waist circumference (WC), sum of four skinfolds, PA, and fitness was tested using an additive model adjusted for age, age-group, gender, maturity, and country. Interactions were tested by including interaction terms in the model. None of the polymorphisms were significantly associated with BMI, WC, sum of four skinfolds, PA, and fitness, and also with the risk of being overweight or obese (P > 0.05). The interactions between the polymorphisms and age-group, gender, PA, and fitness were not statistically significant. This is the first study to comprehensively examine the association of PCK1 polymorphisms with obesity, PA, and fitness. Despite strong evidence from animal studies, our study in the EYHS cohort failed to identify an association of PCK1 polymorphisms with obesity, PA, and fitness.