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Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation., Competing Interests: Declaration of competing interest The author have declared that no competing interest exists., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Whole-grain food (WGF) is well known for its anti-diabetic effect, and alleviation of obesity-induced insulin resistance (IR) might be one of the underlying mechanisms. In the current study, the effects of starch, as the main component in WGF, and β-glucan in a whole-grain-like structural form (WGLSF) on hepatic IR and glucose homeostasis were investigated using high-fat (HF)-induced obese C57BL/6J mice. After treatment for 8 weeks, the body weight gain and IR of the mice were significantly reduced. The hepatic Akt, the key component in insulin signaling, was activated, and the hepatic expression and protein levels of glucose-6-phosphatase (G-6-P) and phosphoenolpyruvate carboxykinase (PEPCK) were reduced. Moreover, WGLSF effectively reduced the hepatic levels of free fatty acids and the pro-inflammatory cytokines TNF-α, IL-6, and NF-κB. Additionally, the reduced level of the phosphorylated c-Jun-N-terminal kinases (JNK) indicated that WGLSF treatment might inactivate the JNK signaling, leading to improved hepatic IR. These results demonstrated that starch and β-glucan in a whole grain-like structural form have the potential as a dietary strategy to combat obesity-induced hepatic IR for improved health.

FoxO proteins are major targets of insulin action, and FoxO1 mediates the effects of insulin on hepatic glucose metabolism. We reported previously that serpinB1 is a liver-secreted factor (hepatokine) that promotes adaptive β-cell proliferation in response to insulin resistance in the liver-specific insulin receptor knockout (LIRKO) mouse. Here we report that FoxO1 plays a critical role in promoting serpinB1 expression in hepatic insulin resistance in a non-cell-autonomous manner. Mice lacking both the insulin receptor and FoxO1 (LIRFKO) exhibit reduced β-cell mass compared with LIRKO mice because of attenuation of β-cell proliferation. Although hepatic expression of serpinB1 mRNA and protein levels was increased in LIRKO mice, both the mRNA and protein levels returned to control levels in LIRFKO mice. Furthermore, liver-specific expression of constitutively active FoxO1 in transgenic mice induced an increase in hepatic serpinB1 mRNA and protein levels in refed mice. Conversely, serpinB1 mRNA and protein levels were reduced in mice lacking FoxO proteins in the liver. ChIP studies demonstrated that FoxO1 binds to three distinct sites located ∼9 kb upstream of the serpinb1 gene in primary mouse hepatocytes and that this binding is enhanced in hepatocytes from LIRKO mice. However, adenoviral expression of WT or constitutively active FoxO1 and insulin treatment are sufficient to regulate other FoxO1 target genes (IGFBP-1 and PEPCK) but not serpinB1 expression in mouse primary hepatocytes. These results indicate that liver FoxO1 promotes serpinB1 expression in hepatic insulin resistance and that non-cell-autonomous factors contribute to FoxO1-dependent effects on serpinB1 expression in the liver., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Red pepper seed, a by-product of red pepper, has been reported to have antioxidant and antiobesity activities. However, its role in diabetes has not yet been highly investigated. Glucose homeostasis is mainly maintained by insulin, which suppresses glucose production in the liver and enhances glucose uptake in peripheral tissues. In this study, we investigated the underlying mechanisms through which red pepper seed extract (RPSE) affects glucose production in AML12 hepatocytes and glucose uptake in C2C12 myotubes. RPSE reduced glucose production in a dose-dependent manner in AML12 cells. The levels of glucose 6 phosphatase, phosphoenolpyruvate carboxykinase, and critical enzymes for hepatic gluconeogenesis were decreased by RPSE. Gluconeogenesis regulating proteins, Akt and forkhead box protein O1, were also activated by RPSE. In addition, RPSE increased glucose uptake in C2C12 via inducing translocation of glucose transporter type 4 from cytosol to plasma membrane. Analysis of the insulin-dependent pathway showed that the activities of insulin receptor substrate 1, phosphatidylinositol 3-kinase, and Akt were significantly stimulated by RPSE. In conclusion, RPSE might improve glucose homeostasis by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. Results obtained also suggest that RPSE can be a compelling antidiabetic nutraceutical.

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (G L ) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion ( Ppp1r3b ) or liver-specific overexpression of Δ hep ) or liver-specific overexpression of Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b Δ hep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3b Δ hep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic-free, conventional wild-type mice. We found that transferring obese-mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non-inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese-mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC-fed mice then fed with a high-fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD-increased hepatic gluconeogenesis compared to non-inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean-mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

MicroRNAs (miRNAs) are a new class of regulatory molecules implicated in type 2 diabetes, which is characterized by insulin resistance and hepatic glucose overproduction. We show that miRNA-451 (miR-451) is elevated in the liver tissues of dietary and genetic mouse models of diabetes. Through an adenovirus-mediated gain- and loss-of-function study, we found that miR-451 negatively regulates hepatic gluconeogenesis and blood glucose levels in normal mice and identified glycerol kinase (Gyk) as a direct target of miR-451. We demonstrate that miR-451 and Gyk regulate hepatic glucose production, the glycerol gluconeogenesis axis, and the AKT-FOXO1-PEPCK/G6Pase pathway in an opposite manner; Gyk could reverse the effect of miR-451 on hepatic gluconeogenesis and AKT-FOXO1-PEPCK/G6Pase pathway. Moreover, overexpression of miR-451 or knockdown of Gyk in diabetic mice significantly inhibited hepatic gluconeogenesis, alleviated hyperglycemia, and improved glucose tolerance. Further studies showed that miR-451 is upregulated by glucose and insulin in hepatocytes; the elevation of hepatic miR-451 in diabetic mice may contribute to inhibiting Gyk expression. This study provides the first evidence that miR-451 and Gyk regulate the AKT-FOXO1-PEPCK/G6Pase pathway and play critical roles in hepatic gluconeogenesis and glucose homeostasis and identifies miR-451 and Gyk as potential therapeutic targets against hyperglycemia in diabetes., (© 2016 by the American Diabetes Association.)

Both epidemiologic and experimental animal studies demonstrate that chronic psychological stress exerts adverse effects on the initiation and/or progression of many diseases. However, intergenerational effects of this environmental information remains poorly understood. Here, using a C57BL/6 mouse model of restraint stress, we show that offspring of stressed fathers exhibit hyperglycemia due to enhanced hepatic gluconeogenesis and elevated expression of PEPCK. Mechanistically, we identify an epigenetic alteration at the promoter region of the Sfmbt2 gene, a maternally imprinted polycomb gene, leading to a downregulation of intronic microRNA-466b-3p, which post-transcriptionally inhibits PEPCK expression. Importantly, hyperglycemia in F1 mice is reversed by RU486 treatment in fathers, and dexamethasone administration in F0 mice phenocopies the roles of restraint stress. Thus, we provide evidence showing the effects of paternal psychological stress on the regulation of glucose metabolism in offspring, which may have profound implications for our understanding of health and disease risk inherited from fathers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nutritional status and glucose precursors are known regulators of gluconeogenic gene expression. Glycerol can replace corn in diets fed to dairy cows and use of glycerol is linked to increased rumen propionate production. The effect of dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The objective of this study was to examine the effect of glycerol on expression of pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six multiparous Holstein cows were fed either a control diet or a diet where high-moisture corn was replaced by glycerol from -28 through +56 d relative to calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at -28, -14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased 6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall, dietary glycerol increased PEPCK-C expression compared with that of cows fed control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC compared with precalving and tended to be decreased in cows fed glycerol. We detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and there were no interactions of dietary treatment and DRTC for any transcript measured. Substituting corn with glycerol increased the expression of PEPCK-C mRNA during transition to lactation and suggests that dietary energy source alters hepatic expression. The observed increase in PEPCK-C expression with glycerol feeding may indicate regulation of hepatic gene expression by changes in rumen propionate production., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Class IIa histone deacetylases (HDACs), such as HDAC4, HDAC5, and HDAC7, provide critical mechanisms for regulating glucose homeostasis. Here we report that HDAC9, another class IIa HDAC, regulates hepatic gluconeogenesis via deacetylation of a Forkhead box O (FoxO) family transcription factor, FoxO1, together with HDAC3. Specifically, HDAC9 expression can be strongly induced upon hepatitis C virus (HCV) infection. HCV-induced HDAC9 upregulation enhances gluconeogenesis by promoting the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, indicating a major role for HDAC9 in the development of HCV-associated exaggerated gluconeogenic responses. Moreover, HDAC9 expression levels and gluconeogenic activities were elevated in livers from HCV-infected patients and persistent HCV-infected mice, emphasizing the clinical relevance of these results. Our results suggest HDAC9 is involved in glucose metabolism, HCV-induced abnormal glucose homeostasis, and type 2 diabetes., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and glucagon in calves receiving propionate were not different than controls. Calves receiving propionate had increased PCK1 mRNA, tended to have increased G6PC mRNA, and had similar PC mRNA compared with saline controls. These data indicate a tendency for in vivo effects of propionate to alter hepatic gene expression in mid-lactation cows and neonatal calves, which are consistent with a feed-forward effect of propionate to regulate its own metabolism toward gluconeogenesis through changes in hepatic PCK1 mRNA., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Statin therapy is known to increase blood glucose levels in humans. Statins utilize pregnane X receptor (PXR) and serum/glucocorticoid regulated kinase 2 (SGK2) to activate phosphoenolpyruvate carboxykinase 1 (PEPCK1) and glucose-6-phosphatase (G6Pase) genes, thereby increasing glucose production in human liver cells. Here, the novel statin/PXR/SGK2-mediated signaling pathway has now been characterized for hepatic gluconeogenesis. Statin-activated PXR scaffolds the protein phosphatase 2C (PP2C) and SGK2 to stimulate PP2C to dephosphorylate SGK2 at threonine 193. Non-phosphorylated SGK2 co-activates PXR-mediated trans-activation of promoters of gluconeogenic genes in human liver cells, thereby enhancing gluconeogenesis. This gluconeogenic statin-PXR-SGK2 signal is not present in mice, in which statin treatment suppresses hepatic gluconeogenesis. These findings provide the basis for statin-associated side effects such as an increased risk for Type 2 diabetes.

Objective: Inhibition of intestinal NPC1L1 by ezetimibe has been demonstrated to improve glucose metabolism in rodent models; however, the role of hepatic NPC1L1 in glucose metabolism has not been elucidated. In this study, we analyzed the effects of hepatic NPC1L1 on glucose metabolism., Material and Methods: We overexpressed NPC1L1 in the livers of lean wild type mice, diet-induced obesity mice and db/db mice with adenoviral gene transfer., Results: We found that in all three mouse models, hepatic NPC1L1 overexpression lowered fasting blood glucose levels as well as blood glucose levels on ad libitum; in db/db mice, hepatic NPC1L1 overexpression improved blood glucose levels to almost the same as those found in lean wild type mice. A pyruvate tolerance test revealed that gluconeogenesis was suppressed by hepatic NPC1L1 overexpression. Further analyses revealed that hepatic NPC1L1 overexpression decreased the expression of FoxO1, resulting in the reduced expression of G6Pase and PEPCK, key enzymes in gluconeogenesis., Conclusions: These results indicate that hepatic NPC1L1 might have distinct properties of suppressing gluconeogenesis via inhibition of FoxO1 pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Activation of Liver X receptors (LXRs), key transcriptional regulators of glucose metabolism, normalizes glycemia and improves insulin sensitivity in rodent models with insulin resistance. However, the molecular mechanism is unclear. This study is aimed to elucidate the mechanism of LXRs-mediated liver glucose metabolic regulation in vitro and in vivo. Db/db mice were used as an in vivo model of diabetes; palmitate (PA)-stimulated HepG2 cells were used as an in vitro cell model with impairment of insulin signaling. TO901317 (TO) was chosen as the LXRs agonist. We demonstrated that TO treatment for 14 days potently improved the hepatic glucose metabolism in db/db mice, including fasting blood glucose, fasting insulin level, and HOMA-IR. TO had no effect on the glucose metabolism in normal WT mice. TO-mediated activation of hepatic LXRs led to strong inhibition of ROS production accompanied by inactivation of JNK pathway and re-activation of Akt pathway. TO also suppressed the expression of gluconeogenic genes such as PEPCK and G-6-pase in db/db mice, but not in WT mice. In HepG2 cells, TO almost completely restored PA-induced Akt inactivation, and suppressed PA-stimulated ROS production and JNK activation. Interestingly, basal level of ROS was also inhibited by TO in HepG2 cells. TO significantly inhibited PA-stimulated expressions of gluconeogenic genes. Finally, we found that anti-oxidative genes, such as Nrf2, were up-regulated after LXRs activation by TO. These results strongly support the notion that activation of LXRs is critical in suppression of liver gluconeogenesis and improvement of insulin sensitivity in diabetic individuals. At molecular levels, the mode of action appears to be as fellows: under diabetic condition, ROS production is increased, JNK is activated, and Akt activity is inhibited; TO-mediated LXR activation potently inhibits ROS production, increases anti-oxidative gene expressions, suppresses JNK activation, and restores Akt activity. Our data provide new evidence to support LXRs as promising therapeutic targets for anti-diabetic drug development.

Different animal models have been used to study the effects of prenatal protein undernutrition and the mechanisms by which these occur. In mammals, the maternal diet is manipulated, exerting both direct nutritional and indirect hormonal effects. Chicken embryos develop independent from the hen in the egg. Therefore, in the chicken, the direct effects of protein deficiency by albumen removal early during incubation can be examined. Prenatal protein undernutrition was established in layer-type eggs by the partial replacement of albumen by saline at embryonic day 1 (albumen-deprived group), compared to a mock-treated sham and a non-treated control group. At hatch, survival of the albumen-deprived group was lower compared to the control and sham group due to increased early mortality by the manipulation. No treatment differences in yolk-free body weight or yolk weight could be detected. The water content of the yolk was reduced, whereas the water content of the carcass was increased in the albumen-deprived group, compared to the control group, indicating less uptake of nutrients from the yolk. At embryonic day 16, 20 and at hatch, plasma triiodothyronine (T3), corticosterone, lactate or glucose concentrations and hepatic glycogen content were not affected by treatment. At embryonic day 20, the plasma thyroxine (T4) concentrations of the albumen-deprived embryos was reduced compared to the control group, indicating a decreased metabolic rate. Screening for differential protein expression in the liver at hatch using two-dimensional difference gel electrophoresis revealed not only changed abundance of proteins important for amino acid metabolism, but also of enzymes related to energy and glucose metabolism. Interestingly, GLUT1, a glucose transporter, and PCK2 and FBP1, two out of three regulatory enzymes of the gluconeogenesis were dysregulated. No parallel differences in gene expressions causing the differences in protein abundance could be detected pointing to post-transcriptional or post-translational regulation of the observed differences.

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.

Background: Exposure to maternal obesity or hyperglycemia increases the risk of obesity and poor glucose tolerance in the offspring. We hypothesized that maternal overnutrition in late pregnancy would result in (i) lower methylation in the promoter region of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C; PCK1) and (ii) higher expression of hepatic gluconeogenic factors in the fetal and postnatal lamb., Methods: Ewes were fed 100% (n = 18) or ~155% (n = 17) of energy requirements from 115 d gestation, and livers were collected at ~140 d gestation or 30 d postnatal age., Results: Maternal overnutrition resulted in a decrease in hepatic expression of the mitochondrial form of PEPCK (PEPCK-M; PCK2) but not of PEPCK-C or glucose-6-phosphatase (G6PHOS) before and after birth. Hepatic expression of peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), peroxisome proliferator-activated receptor α (PPARα), PEPCK-C, G6PHOS, and 11β hydroxysteroid dehydrogenase type 1 (11βHSD1), but not PEPCK-M, was higher in the postnatal lamb compared with that in the fetal lamb. The level of PCK1 methylation was paradoxically approximately twofold higher in the postnatal liver compared with that in the fetal liver., Conclusion: Maternal overnutrition programs a decrease in hepatic PEPCK-M in the offspring and as ~50% of total hepatic PEPCK is PEPCK-M, the longer-term consequences of this decrease may be significant.

Sodium meta-arsenite (SA) is implicated in the regulation of hepatic gluconeogenesis-related genes in vitro; however, the effects in vivo have not been studied. We investigated whether SA has antidiabetic effects in a type 2 diabetic mouse model. Diabetic db/db mice were orally intubated with SA (10 mg kg(-1) body weight/day) for 8 weeks. We examined hemoglobin A1c (HbA1c), blood glucose levels, food intake, and body weight. We performed glucose, insulin, and pyruvate tolerance tests and analyzed glucose production and the expression of gluconeogenesis-related genes in hepatocytes. We analyzed energy metabolism using a comprehensive animal metabolic monitoring system. SA-treated diabetic db/db mice had reduced concentrations of HbA1c and blood glucose levels. Exogenous glucose was quickly cleared in glucose tolerance tests. The mRNA expressions of genes for gluconeogenesis-related enzymes, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly reduced in the liver of SA-treated diabetic db/db mice. In primary hepatocytes, SA treatment decreased glucose production and the expression of G6Pase, PEPCK, and hepatocyte nuclear factor 4 alpha (HNF-4α) mRNA. Small heterodimer partner (SHP) mRNA expression was increased in hepatocytes dependent upon the SA concentration. The expression of Sirt1 mRNA and protein was reduced, and acetylated forkhead box protein O1 (FoxO1) was induced by SA treatment in hepatocytes. In addition, SA-treated diabetic db/db mice showed reduced energy expenditure. Oral intubation of SA ameliorates hyperglycemia in db/db mice by reducing hepatic gluconeogenesis through the decrease of Sirt1 expression and increase in acetylated FoxO1.

Background & Aims: Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver-specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice., Methods: The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using (2)H and (13)C tracers. Gluconeogenic potential, together with metabolic profiling, was investigated in vivo and in primary hepatocytes., Results: PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ∼10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms., Conclusions: We conclude that PEPCK-M has gluconeogenic potential per se, and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in the human liver., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The development of insulin resistance is tightly linked to fatty liver disease and is considered a major health concern worldwide, although their mechanistic relationship remains controversial. Activin has emerging roles in nutrient homeostasis, but its metabolic effects on hepatocytes remain unknown. In this study, we investigated the effects of increased endogenous activin bioactivity on hepatic nutrient homeostasis by creating mice with inactivating mutations that deplete the circulating activin antagonists follistatin-like-3 (FSTL3) or the follistatin 315 isoform (FST315; FST288-only mice). We investigated liver histology and lipid content, hepatic insulin sensitivity, and metabolic gene expression including the HepG2 cell and primary hepatocyte response to activin treatment. Both FSTL3-knockout and FST288-only mice had extensive hepatic steatosis and elevated hepatic triglyceride content. Unexpectedly, insulin signaling, as assessed by phospho-Akt (a.k.a. protein kinase B), was enhanced in both mouse models. Pretreatment of HepG2 cells with activin A increased their response to subsequent insulin challenge. Gene expression analysis suggests that increased lipid uptake, enhanced de novo lipid synthesis, decreased lipolysis, and/or enhanced glucose uptake contribute to increased hepatic triglyceride content in these models. However, activin treatment recapitulated only some of these gene changes, suggesting that increased activin bioactivity may be only partially responsible for this phenotype. Nevertheless, our results indicate that activin enhances hepatocyte insulin response, which ultimately leads to hepatic steatosis despite the increased insulin sensitivity. Thus, regulation of activin bioactivity is critical for maintaining normal liver lipid homeostasis and response to insulin, whereas activin agonists may be useful for increasing liver insulin sensitivity.

Background: Hepatic 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity, which converts cortisone (inactive) to cortisol, is downregulated in obesity. However, this compensation fails in obese with metabolic abnormalities, such as diabetes. To further characterize the tissue-specific cortisol regeneration in obesity, we have investigated the mRNA expression of genes related to local cortisol production, i.e., 11β-HSD1, hexose-6-phosphate dehydrogenase (H6PDH) and cortisol action, glucocorticoid receptor (GR) and a cortisol target gene, phosphoenolpyruvate carboxykinase (PEPCK) in the liver, and visceral (VAT) and subcutaneous (SAT) adipose tissues from morbidly obese patients with and without metabolic syndrome (MS)., Methods: Fifty morbidly obese patients undergoing bariatric surgery, 14 men (mean age, 41.3 ± 3.5 years; BMI, 48.0 ± 3.6 kg/m(2)) and 36 women (mean age, 44.6 ± 1.9 years; BMI, 44.9 ± 1.2 kg/m(2)), were classified as having MS (MS+, n = 20) or not (MS-, n = 30). Tissue mRNA levels were measured by real-time polymerase chain reaction., Results: Hepatic mRNA levels of these genes were higher in obese patients with MS (11β-HSD1, P = 0.002; H6PDH, P = 0.043; GR, P = 0.033; PEPCK, P = 0.032) and positively correlated with the number of clinical characteristics that define the MS. The expression of the four genes positively correlated among them. In contrast to the liver, these genes were not differently expressed in VAT or SAT, when MS+ and MS- obese patients were compared., Conclusions: Coordinated liver-specific upregulation of genes involved in local cortisol regeneration and action support the concept that local hepatic hypercortisolism contributes to development of MS in morbidly obese patients.

Many biological activities of green tea have been attributed to a major constituent, (minus;)-epigallocatechin gallate (EGCG). We previously reported that EGCG and an EGCG-free fraction derived from green tea modulated the gene expression of gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, in the mouse liver. EGCG is also known to affect the gene expression of enzymes related to lipid metabolism. However, it remains to be examined whether or not a constituent other than EGCG contributes to the change in gene expression of these enzymes. In this study, we prepared an EGCG-free water-soluble fraction (GT-W), and examined its effects on the hepatic gene expression of lipogenic enzymes in mice. The results of quantitative real-time PCR assays indicated that the dietary administration of GT-W for 4 weeks reduced the hepatic gene expression of lipogenic enzymes: fatty acid synthase, hydroxymethylglutaryl coenzyme A reductase, and acetyl-coenzyme A carboxylase alpha. Also, the gene expression of sterol regulatory element-binding transcription factor (Srebf)1 and/or Srebf2 was reduced, suggesting that the reduction of Srebfs contributed to the down-regulation of the lipogenic enzymes, since these transcription factors bind the promoter region to enhance their expression. The plasma levels of triglycerides and cholesterol were reduced with statistical significance in the group given a diet containing GT-W. These results suggest that in addition to EGCG, green tea contains some component(s) which may help to prevent arteriosclerosis and obesity.

Tea has many beneficial effects. We have previously reported that green tea and a catechin-rich green tea beverage modulated the gene expression of the gluconeogenic enzymes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the normal murine liver. In the present study, we examined the effects of oral administration of oolong tea on the hepatic expression of gluconeogenesis-related genes in the mouse. The intake of oolong tea for 4 weeks reduced the hepatic expression of G6Pase and PEPCK together with that of the transcription factor hepatocyte nuclear factor (HNF) 4α. When rat hepatoma H4IIE cells were incubated in the presence of oolong tea, the expression of these genes was repressed in accordance with the findings in vivo. The reduced protein expression of PEPCK and HNF4α was also demonstrated. We then fractionated oolong tea by sequential extraction with three organic solvents to give three fractions and the residual fraction (Fraction IV). In addition to organic fractions, Fraction IV, which was devoid of low-molecular-weight catechins such as (-)-epigallocatechin gallate (EGCG), had effects similar to those of oolong tea on H4IIE cells. Fraction IV repressed the gene expression of insulin-like growth factor binding protein 1, as insulin did. This activity was different from that of EGCG. The present findings suggest that drinking oolong tea may help to prevent diabetes and that oolong tea contains a component or components with insulin-like activity distinguishable from EGCG. Identification of such component(s) may open the way to developing a new drug for diabetes.

Many biological activities of green tea have been attributed to a major constituent, (-)-epigallocatechin gallate (EGCG). We previously reported that EGCG and a catechin-rich green tea beverage modulated the gene expression of gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), in the mouse liver. However, it remains to be examined whether or not a constituent other than EGCG contributes to the change in gene expression of these enzymes. In this study, we separated the hot water infusion of green tea leaves (GT) into an ethanol-soluble fraction (GT-E) and an EGCG-free water-soluble fraction (GT-W), and examined their effects using rat hepatoma H4IIE cells. The inclusion of GT, GT-E, and GT-W in the culture medium reduced the gene expression of G6Pase and PEPCK. GT-W caused a decrease in expression of the transcription factor HNF4α. Reduced levels of PEPCK and HNF4α proteins were demonstrated in the cells treated with GT-W. GT-W showed an activity similar to insulin, but different from EGCG. Administration of GT-W to mice for 4 weeks reduced the hepatic expression of G6Pase, PEPCK, and HNF4α. These results suggest that green tea contains some component(s) with insulin-like activity distinguishable from EGCG and that drinking green tea may help to prevent diabetes.

There is considerable evidence for non-genomic transmission between generations of phenotypes induced by environmental exposures during development, although the mechanism is poorly understood. We investigated whether alterations in expression of the liver transcriptome induced in F1 offspring by feeding F0 dams a protein-restricted (PR) diet during pregnancy were passed with or without further change to two subsequent generations. The number of genes that differed between adult female offspring of F0 protein-restricted (PR) and protein-sufficient (PS) dams was F1 1,684 genes, F2 1,680 and F3 2,062. 63/113 genes that were altered in all three generations showed directionally opposite differences between generations. There was a trend toward increased proportions of up-regulated genes in F3 compared to F1. KEGG analysis showed that only the Adherens Junctions pathway was altered in all three generations. PR offspring showed altered fasting glucose homeostasis and changes in phosphoenolpyruvate carboxykinase promoter methylation and expression in all three generations. These findings show that dietary challenge during F0 pregnancy induced altered gene expression in all three generations, but relatively few genes showed transmission of altered expression between generations. For the majority of altered genes, these changes were not found in all generations, including some genes that were changed in F3 but not F1, or the direction and magnitude of difference between PR and PS differed between generations. Such variation may reflect differences between generations in the signals received by the fetus from the mother as a consequence of changes in the interaction between her phenotype and the environment.

Background: Many epidemiologic studies have reported a link between calcium (Ca) deficiency and metabolic syndrome. In this study, we examine Ca deficiency in rats and whether changes in glucocorticoid metabolism are induced., Methods: Twelve-week-old female Wistar rats were weaned onto a very-low-Ca diet (low-Ca group) or a control diet (control group) for 2 weeks. Quantitative real-time PCR was used to assess mRNA for 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1), 11β-HSD2, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor-α, and glucocorticoid receptor in the liver. Concentrations of adiponectin, leptin, corticosterone, intact parathyroid hormone, asymmetrical dimethylarginine and insulin in fasting serum were determined using a rat-specific enzyme-linked immunosorbent assay. Glucose concentrations were measured using a glucose oxidase system. Serum ionized Ca levels were measured with an automatic ion-selective electrode analyzer. Serum nitrite/nitrate levels were measured using a colorimetric assay kit., Results: After 2 weeks, no differences in serum glucose, corticosterone or insulin levels were observed. The low-Ca group rats showed higher homeostasis model assessment insulin resistance, lower adiponectin and higher intact parathyroid hormone levels. Serum nitrite/nitrate and asymmetrical dimethylarginine were significantly higher in the low-Ca group than in the control group. The expression of hepatic 11β-HSD1 mRNA was upregulated, while hepatic phosphoenolpyruvate carboxykinase expression was downregulated in the low-Ca group. Glucocorticoid receptor, peroxisome proliferator-activated receptor-α and 11β-HSD2 expression levels showed a similar tendency., Conclusion: A low-Ca diet alters glucocorticoid metabolism, which leads to hepatic upregulation of 11β-HSD1, and is possibly a key mechanism inducing the metabolic complications of Ca deficiency., (Copyright © 2011 S. Karger AG, Basel.)

Aims/hypothesis: Fasting and exercise are strong physiological stimuli for hepatic glucose production. IL-6 has been implicated in the regulation of gluconeogenic genes, but the results are contradictory and the relevance of IL-6 for fasting- and exercise-induced hepatic glucose production is not clear., Methods: Investigations were performed in rat hepatoma cells, and on C57Bl6 and Il6(-/-) mice under the following conditions: IL-6 stimulation/injection, non-exhaustive exercise (60 min run on a treadmill) and fasting for 16 h. Metabolite analysis, quantitative real-time PCR and immunoblotting were performed., Results: IL-6 stimulation of rat hepatoma cells led to higher glucose production. Injection of IL-6 in mice slightly increased hepatic Pepck (also known as Pck1) expression. Fasting of Il6(-/-) mice for 16 h did not alter glucose production compared with wild-type mice, since plasma glucose concentrations were similar and upregulation of phosphoenolpyruvate carboxykinase (PEPCK) and Pgc-1alpha (also known as Ppargc1a) expression was comparable. In the non-fasting state, Il6(-/-) mice showed a mild metabolic alteration including higher plasma glucose and insulin levels, lower NEFA concentrations and slightly increased hepatic PEPCK content. Moderately intense exercise resulted in elevated IL-6 plasma levels in wild-type mice. Despite that, plasma glucose, insulin, NEFA levels and hepatic glycogen content were not different in Il6(-/-) mice immediately after running, while expression of hepatic G6pc, Pgc-1alpha, Irs2 and Igfbp1 mRNA was similarly increased., Conclusions/interpretation: These data suggest that in mice IL-6 is not essential for physiologically increased glucose production during fasting or non-exhaustive exercise.

Metformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMP-activated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK- and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARgamma coactivator 1alpha (PGC-1alpha) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state.

Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis.

Ethnopharmacological Relevance: Fructus Corni, the fruits of Cornus officinalis Sieb. et Zucc., is one important ingredient in Quei Fu Di Huang Wan, a Chinese herbal mixture., Aim of the Study: In the present study, additional anti-diabetic actions of Fructus Corni on transcriptional regulation of hepatic gluconeogenesis or beta-cell functions were investigated., Materials and Methods: Insulin mimetic action of Fructus Corni on dexamethasone and 8-bromo-cAMP induced phosphoenolpyruvate carboxykinase (PEPCK) expression in H4IIE cells was investigated. Besides, BRIN-BD11 cells were used to evaluate both insulinotropic and beta-cell protective effect of Fructus Corni., Results: Firstly, both methanol extract (CO-W-M) and fraction (CO-W-M2) had potent insulin mimic activity on PEPCK expression. Secondly, possibility of both loganin and ursolic acid as the responsible compounds was excluded. Moreover, indication of the existence of phenolic compounds in CO-W-M2 was noticed. In the presence of CO-W-M2, not only was the viability of BRIN-BD11 cells treated with alloxan, streptozotcin, or cytokine mix all significantly increased but also glucose-stimulated insulin secretion was potentiated., Conclusions: The ability of CO-W-M2 to reduce gene expression for hepatic gluconeogenesis, to protect beta-cell against toxic challenge, and to enhance insulin secretion strengthen the role of Fructus Corni in diabetes therapy.

Objective: To observe the effect of calorie restriction on the high fat diet rats mRNA expressions of liver forkhead box O1(FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-P) and to explore the possible mechanisms., Methods: 24 normal 6-week-old male Wistar rats were randomly divided into three groups: normal chow group (NC, n = 7), high fat diet group (HF, n = 9) and calorie restriction group (CR, n = 8). They were fed for 12 weeks. At the end of the experiment, the rats were sacrificed and their fasting blood glucose (FBG), insulin (INS), triglycerides (TG), total cholesterol (TC) were measured. Their visceral fat (VF) and body weight (BW) were also measured and VF/BW was calculated. Gene expression was investigated by using semi-quantitative RT-PCR methods. Liver histology was studied with HE stained slides., Results: Compared with the NC group, HF group rats developed visceral obesity which was accompanied by higher FBG, plasma INS, TG, and TC. The levels of FoxO1, PEPCK, and G-6-P increased by 18.9%, 33.8%, and 24.6%, respectively (P less than 0.01). Liver steatosis was observed with microscopy. The BW, VF FBG, INS, TG and TC of the CR group rats were lower in comparison to those of the HF group. The levels of FoxO1, PEPCK and G-6-P were lower by 26.6%, 35.0%, 34.3% (P less than 0.01). Meanwhile, liver steatosis was also milder., Conclusion: Calorie restriction can inhibit the expressions of FoxO1, PEPCK and G-6-P, strengthen insulin signal conduction, suppress gluconeogenesis and thus regulate glycometabolism.

Individuals born with a low birth weight (LBW) have an increased prevalence of type 2 diabetes, but the mechanisms responsible for this association are unknown. Given the important role of insulin resistance in the pathogenesis of type 2 diabetes, we examined insulin sensitivity in a rat model of LBW due to intrauterine fetal stress. During the last 7 days of gestation, rat dams were treated with dexamethasone and insulin sensitivity was assessed in the LBW offspring by a hyperinsulinemic euglycemic clamp. The LBW group had liver-specific insulin resistance associated with increased levels of PEPCK expression. These changes were associated with pituitary hyperplasia of the ACTH-secreting cells, increased morning plasma ACTH concentrations, elevated corticosterone secretion during restraint stress, and an approximately 70% increase in 24-h urine corticosterone excretion. These data support the hypothesis that prenatal stress can result in chronic hyperactivity of the hypothalamic-pituitary-adrenal axis, resulting in increased plasma corticosterone concentrations, upregulation of hepatic gluconeogenesis, and hepatic insulin resistance.

Proopiomelanocortin (POMC) deficiency causes severe obesity through hyperphagia of hypothalamic origin. However, low glucocorticoid levels caused by adrenal insufficiency mitigate against insulin resistance, hyperphagia and fat accretion in Pomc-/- mice. Upon exogenous glucocorticoid replacement, corticosterone-supplemented (CORT) Pomc-/- mice show exaggerated responses, including excessive fat accumulation, hyperleptinaemia and insulin resistance. To investigate the peripheral mechanisms underlying this glucocorticoid hypersensitivity, we examined the expression levels of key determinants and targets of glucocorticoid action in adipose tissue and liver. Despite lower basal expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which generates active glucocorticoids within cells, CORT-mediated induction of 11beta-HSD1 mRNA levels was more pronounced in adipose tissues of Pomc-/- mice. Similarly, CORT treatment increased lipoprotein lipase mRNA levels in all fat depots in Pomc-/- mice, consistent with exaggerated fat accumulation. Glucocorticoid receptor (GR) mRNA levels were selectively elevated in liver and retroperitoneal fat of Pomc-/- mice but were corrected by CORT in the latter depot. In liver, CORT increased phosphoenolpyruvate carboxykinase mRNA levels specifically in Pomc-/- mice, consistent with their insulin-resistant phenotype. Furthermore, CORT induced hypertension in Pomc-/- mice, independently of adipose or liver renin-angiotensin system activation. These data suggest that CORT-inducible 11beta-HSD1 expression in fat contributes to the adverse cardiometabolic effects of CORT in POMC deficiency, whereas higher GR levels may be more important in liver.

Isoleucine, a branched chain amino acid, plays an important role in the improvement of glucose metabolism as evidenced by the increase of insulin-independent glucose uptake in vitro. This study evaluated the effect of isoleucine on glucose uptake and oxidation in fasted rats and on gluconeogenesis in vivo and in vitro. Oral administration of isoleucine decreased the plasma glucose level by 20% and significantly increased muscle glucose uptake by 71% without significant elevation of the plasma insulin level compared with controls at 60 min after administration. Furthermore, expiratory excretion of 14CO2 from [U-14C]glucose in isoleucine-administered rats was increased by 19% compared with controls. Meanwhile, isoleucine decreased AMP levels in the liver but did not affect hepatic glycogen synthesis. Under insulin-free conditions, isoleucine significantly inhibited glucose production when alanine was used as a glucogenic substrate in isolated hepatocytes. This inhibition by isoleucine was also associated with a decline in mRNA levels for phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) and a decreased activity of G6Pase in isolated hepatocytes. These findings suggest that a reduction of gluconeogenesis in liver, along with an increase of glucose uptake in the muscle, is also involved in the hypoglycemic effect of isoleucine. In conclusion, isoleucine administration stimulates both glucose uptake in the muscle and whole body glucose oxidation, in addition to depressing gluconeogenesis in the liver, thereby leading to the hypoglycemic effect in rats.

It has been shown previously that maternal low protein diet (LPD) throughout rat gestation altered hepatic gene expression and enzyme activities in offspring. Here, we investigate the effect of maternal LPD (9% casein vs. 18% control) exclusively during the preimplantation period (switched diet group) or provided throughout gestation on hepatic gene expression in day 20 fetuses. Using quantitative competitive PCR, we found that switched diet induced a two-fold increase (P = 0.008) in hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK, a rate limiting enzyme for gluconeogenesis) in male fetuses and a 17% increase (P = 0.005) in 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1, acts primarily as a reductase to produce active glucocorticoid) in female liver compared with control fetuses. Maternal LPD administered throughout gestation increased 11beta-HSD1 expression in male fetal liver by 27% (P = 0.042) compared with controls. However, maternal LPD fed for either period did not affect fetal hepatic insulin receptor (IR), glucocorticoid receptor (GR), glycogen synthase (GS) nor placental glucose transporter 1 (Glut1) and 3 (Glut3) transcript levels. The alteration in fetal hepatic gene expression could not be attributed specifically to known regulators including insulin or glucose concentrations in fetal blood nor alteration in cAMP in fetal liver, although a combination of these regulatory factors may be responsible. Fetal hepatic glycogen level was unaffected by maternal diet. The present findings show that the long term potential of the preimplantation embryo is sensitive to maternal LPD such that basal levels of hepatic gene expression in day 20 fetuses are altered in a gender-specific manner.

Blood pH controls the activity of important regulatory enzymes in the metabolism. Serine dehydratase (SerDH) transforms l-serine into pyruvate and ammonium and is involved in the regulation of gluconeogenesis from serine in the rat liver. In this work, we investigate the effect of chronic metabolic acidosis on the kinetics, specific protein level, tissue location, and mRNA levels of rat liver SerDH. Experimental acidosis was induced in rats by ingestion of 0.28 M ammonium chloride solution for 10 days. Acidosis significantly (P<0.05) decreased SerDH activity at all substrate concentrations assayed. Moreover, the Vmax value was 38.50+/-3.51 mU/mg (n=7) of mitochondrial protein in the acidotic rats and 92.49+/-6.79 mU/mg (n=7) in the control rats. Western blot analysis revealed a significant reduction (14%) in the level of SerDH protein content in the rat liver during acidosis. Immunohistochemical analysis showed that SerDH location did not change in response to chronic metabolic acidosis and confirmed previous results on SerDH protein levels. Moreover, the SerDH mRNA level, estimated by RT-PCR, was also significantly 33.8% lower than in control. These results suggest that during experimental acidosis a specific repression of rat-liver SerDH gene transcription could result, lowering the amount and activity of this enzyme. The changes found in SerDH expression are part of an overall metabolic response of liver to maintain acid-base homeostasis during acidosis.

Human livers contain two pluripotent hepatic progenitors, hepatic stem cells and hepatoblasts, with size, morphology, and gene expression profiles distinct from that of mature hepatocytes. Hepatic stem cells, the precursors to hepatoblasts, persist in stable numbers throughout life, and those isolated from the livers of all age donors from fetal to adult are essentially identical in their gene and protein expression profiles. The gene expression profile of hepatic stem cells throughout life consists of high levels of expression of cytokeratin 19 (CK19), neuronal cell adhesion molecule (NCAM), epithelial cell adhesion molecule (EpCAM), and claudin-3 (CLDN-3); low levels of albumin; and a complete absence of expression of alpha-fetoprotein (AFP) and adult liver-specific proteins. By contrast, hepatoblasts, the dominant cell population in fetal and neonatal livers, decline in numbers with age and are found as <0.1% of normal adult livers. They express high levels of AFP, elevated levels of albumin, low levels of expression of adult liver-specific proteins, low levels of CK19, and a loss of NCAM and CLDN-3. Mature hepatocytes lack expression altogether of EpCAM, NCAM, AFP, CLDN-3, cytokeratin 19, and have acquired the well-known adult-specific profile that includes expression of high levels of albumin, cytochrome P4503A4, connexins, phosphoenolpyruvate carboxykinase, and transferrin. Thus, hepatic stem cells have a unique stem cell phenotype, whereas hepatoblasts have low levels of expression of both stem cell genes and genes expressed in high levels in mature hepatocytes.

Vitamin A deficiency decreases hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression in mice, and expression is restored with retinoic acid (RA) treatment in vivo. In the studies reported here, we examined changes in histone modification and coregulator association with the regulatory domains of the PEPCK gene in response to alterations in vitamin A status. We identified nuclear receptors that bind to retinoic acid response elements (RAREs) in the PEPCK promoter by electrophoretic mobility shift assay and verified these in vivo using chromatin immunoprecipitation in mouse liver. Hypothetically, nuclear receptors at PEPCK RAREs recruit specific coactivator molecules that contribute to the acetylation of core histones and/or serve as bridging molecules between nuclear receptors and basal transcription factors at the transcription start site. We identified 3 coactivator molecules, cAMP-response element binding protein (CBP), steroid receptor coactivator (SRC)-1, and peroxisome-proliferator activated receptor (PPAR)-gamma-coactivator (PGC)-1alpha, that bound in association with the PEPCK RAREs in vivo. Furthermore, there was differential binding of these coactivators in vitamin A-deficient mice. Related to this, specific lysine residues were acetylated on histones H3 and H4 at the 3 RAREs of the PEPCK promoter, consistent with the action of the above coactivators, and acetylation of certain lysines was significantly decreased with vitamin A deficiency. These results demonstrate the associated changes that occur in nuclear receptor binding, coactivator recruitment, and histone acetylation in response to vitamin A status, identified at specific RAREs in the PEPCK gene in vivo.

Until now, it is known that hypoxia increases the glycolytic enzyme expression at the transcriptional level. Here, we show evidence that hypoxia increases hepatic glucose output and HIF-1 and ATF-2-mediated transactivation of phosphoenolpyruvate carboxykinase (PEPCK), which plays a critical role as a rate-limiting enzyme in gluconeogenesis, gene in liver. HIF-1 directly bound to the specific PEPCK promoter region through its cognate binding element and found as an active complex with coactivator CBP. Additionally, ATF-2 was also involved to regulate hypoxia-dependent PEPCK transcription in the transcriptional complex with HIF-1 and CBP. Interestingly, retinoic acid (RA) signaling induced the recruitment of HIF-1 on the PEPCK promoter, resulting from the functional interaction of HIF-1 and ATF-2 with coactivator CBP. Taken together, these results suggest that hypoxia signaling leads the hepatic glucose production and release via the increased gene expression of gluconeogenic enzymes, possibly playing a role in providing glucose to other tissues, such as endothelial, brain and muscle cells.

We have previously characterized transcription factor LZIP to be a growth suppressor targeted by hepatitis C virus oncoprotein. In search of proteins closely related to LZIP, we have identified a liver-enriched transcription factor CREB-H. LZIP and CREB-H represent a new subfamily of bZIP factors. CREB-H activates transcription by binding to cAMP responsive element, box B, and ATF6-binding element. Interestingly, CREB-H has a putative transmembrane (TM) domain and it localizes ambiently to the endoplasmic reticulum. Proteolytic cleavage that removes the TM domain leads to nuclear translocation and activation of CREB-H. CREB-H activates the promoter of hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase. This activation can be further stimulated by cAMP and protein kinase A. CREB-H transcript is exclusively abundant in adult liver. In contrast, the expression of CREB-H mRNA is aberrantly reduced in hepatoma tissues and cells. The enforced expression of CREB-H suppresses the proliferation of cultured hepatoma cells. Taken together, our findings suggest that the liver-enriched bZIP transcription factor CREB-H is a growth suppressor that plays a role in hepatic physiology and pathology.

The amount of peroxisome proliferator-activated receptor-alpha (PPARalpha) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARalpha abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARalpha and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARalpha and ACO was seen to increase when the level of FFA in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARalpha gene in adult rats seems to be less sensitive to nutritional changes than in neonates.

Rats of different ages (3 to 15-wk-old) were fed on a 25% casein diet for one week, and the nitrogen balance and liver serine dehydratase (SDH, EC 4.2.1.13) activity were then determined. The value for nitrogen balance decreased with the age of the rats, while the liver SDH activity increased. A statistical analysis showed clear inverse correlation between the two factors (R(2) = 0.7372, p < 0.01). This result suggests that SDH was induced by response to the amount of surplus amino acids from dietary protein taken beyond the body's requirement. The increase in SDH activity was accompanied by an increase in the level of SDH mRNA. Since the half-life of this mRNA did not change significantly, the induction was mainly controlled at the level of transcription. In addition, the induction seems not to be related to gluconeogenesis, since the mRNA levels of tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), other gluconeogenic enzymes, were not changed under these experimental conditions.

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme for gluconeogenesis. To investigate underlying mechanisms of corticosteroid (CS) action in regulating glucose, temporal patterns of hepatic PEPCK gene expression, enzyme activity, and cAMP content were examined in adrenalectomized rats receiving acute and chronic methylprednisolone (MPL) treatments. After single MPL intravenous doses, PEPCK mRNA showed a fast increase, reaching a maximum at around 0.75 h, which was followed by an immediate decline to below baseline after 4 h, an apparent acute tolerance/rebound phenomenon. However, PEPCK enzyme showed continuous hyperactivity for over 72 h. This may be the result of generation of cAMP, an important inducer of PEPCK activity, which peaked at around 6 h. During 7-day subcutaneous infusion of MPL, PEPCK mRNA showed profiles consistent with single-dose results, whereas PEPCK activity increased to a comparable maximum followed by a slow decline. However, the extent of cAMP induction was markedly higher during infusion, which could be attributed to amplification of cAMP synthesis and/or a stabilizing effect of MPL on cAMP degradation. A pharmacokinetic/pharmacodynamic model was developed based on receptor/gene mechanisms of CS action. It successfully described the dual effects of MPL on regulating PEPCK message and the post-transcriptional control by cAMP. Our results exemplify the importance of the extent and duration of steroid exposure in mediating pharmacological effects. The model provides quantitation of multiple controlling factors regulating PEPCK and presents insights into its function in glucose metabolism.

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.

Fibrates and thiazolidinediones are used clinically to treat hypertriglyceridemia and hyperglycemia, respectively. Fibrates bind to the peroxisome proliferator-activated receptor (PPAR)-alpha, and thiazolidinediones are ligands of PPAR-gamma. These intracellular receptors form heterodimers with retinoid X receptor to modulate gene transcription. To elucidate the target genes regulated by these compounds, we treated Zucker diabetic fatty rats (ZDF) for 15 days with a PPAR-alpha-specific compound, fenofibrate, a PPAR-gamma-specific ligand, rosiglitazone, and a PPAR-alpha/-gamma coagonist, GW2331, and measured the levels of several messenger RNAs (mRNAs) in liver by real-time polymerase chain reaction. All 3 compounds decreased serum glucose and triglyceride levels. Fenofibrate and GW2331 induced expression of acyl-coenzyme A (CoA) oxidase and enoyl-CoA hydratase and reduced apolipoprotein C-III and phosphoenolpyruvate carboxykinase mRNAs. Rosiglitazone modestly increased apolipoprotein C-III mRNA and had no effect on expression of the other 2 genes in the liver but increased the expression of glucose transporter 4 and phosphoenolpyruvate carboxykinase in adipose tissue. We identified a novel target in liver, mitogen-activated phosphokinase phosphatase 1, whose down-regulation by PPAR-alpha agonists may improve insulin sensitivity in that tissue by prolonging insulin responses. The results of these studies suggest that activation of PPAR-alpha as well as PPAR-gamma in therapy for type 2 diabetes will enhance glucose and triglyceride control by combining actions in hepatic and peripheral tissues., (Copyright 2001 by W.B. Saunders Company)

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.

The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4-14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.

The objective of this study was to profile phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) mRNA expression in the liver of dairy cattle during the peripartum transition and determine changes in abundance of these mRNA in response to protein fed during the prepartum period. Thirty-eight multiparous Holstein cows were fed diets containing either 12% crude protein (CP) and 26% rumen undegradable protein (RUP), 16% CP and 26% RUP, 16% CP and 33% RUP, or 16% CP and 40% RUP on a dry-matter basis beginning 28 d before expected calving. After calving, all cows were fed a common diet through 56 d in milk (DIM). Northern analysis of RNA from liver biopsy samples obtained on days -28, -14, +1, +28, and +56 relative to calving indicated that PC and PEPCK mRNA expression were responsive to onset of lactation but not to prepartum protein or RUP concentration. Abundance of PEPCK mRNA was similar at -28, -14, and +1 DIM but was elevated by +28 and +56 DIM relative to precalving levels. Liver PC mRNA abundance was elevated on +1 DIM, remained elevated through 28 DIM, and declined to precalving levels by 56 DIM. The activity of PC enzyme was correlated (r2 = 0.89) with PC mRNA abundance. The data demonstrate increased abundance of PC mRNA during the early transition period followed by increased abundance of PEPCK mRNA during the postpartum period and suggest increased potential metabolism of lactate, pyruvate, and amino acids that contribute to the liver pyruvate pool.

In vitro, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. 11beta-HSD-1 is highly expressed in liver, where the reaction direction is 11beta-reduction, thus potentially increasing intrahepatic active glucocorticoid levels. Inhibition of 11beta-HSD-1 increases insulin sensitivity in humans in vivo suggesting that hepatic 11beta-HSD-1 plays a role in the maintenance or control of key glucocorticoid-regulated metabolic functions. We have selectively repressed hepatic 11beta-HSD-1 in rats by oestradiol administration for 42 days. This nearly completely repressed hepatic 11beta-HSD-1 mRNA expression and enzyme activity and reduced expression of hepatic glucocorticoid-inducible genes including phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting step in gluconeogenesis. Similar effects were seen after 3 weeks of oestradiol treatment. To examine whether this was due to any direct effect of oestradiol upon PEPCK, the experiment was repeated in adrenalectomised rats+/-glucocorticoid replacement. In adrenalectomised rats, oestradiol did not attenuate hepatic PEPCK, whilst glucocorticoid replacement restored this action. Oestradiol did not alter hepatic metabolism of corticosterone by pathways other than 11beta-HSD-1. These data suggest 11beta-HSD-1 plays an important role in maintaining expression of key glucocorticoid-regulated hepatic transcripts. Enzyme inhibition may provide a useful therapeutic target for manipulating glucose homeostasis.