Search

Regulation of nutrient intake, utilization, and storage exhibits a circadian rhythmicity that allows organisms to anticipate and adequately respond to changes in the environment across day/night cycles. The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are important modulators of metabolism and metabolic health – for example, their catabolism yields carbon substrates for gluconeogenesis during periods of fasting. Krüppel-like factor 15 (KLF15) has recently emerged as a critical transcriptional regulator of BCAA metabolism, and the absence of this transcription factor contributes to severe pathologies such as Duchenne muscular dystrophy and heart failure. This review highlights KLF15’s role as a central regulator of BCAA metabolism during periods of fasting, throughout day/night cycles, and in experimental models of muscle disease. [ABSTRACT FROM AUTHOR]

Growing attention has been focused on the roles of the proximal tubules (PTs) of the kidney in glucose metabolism, including the mechanism of regulation of gluconeogenesis. In this study, we found that PT-specific insulin receptor substrate 1/2 double-knockout mice, established by using the newly generated sodium-glucose cotransporter 2 (SGLT2)-Cre transgenic mice, exhibited impaired insulin signaling and upregulated gluconeogenic gene expression and renal gluconeogenesis, resulting in systemic insulin resistance. In contrast, in streptozotocin-treated mice, although insulin action was impaired in the PTs, the gluconeogenic gene expression was unexpectedly downregulated in the renal cortex, which was restored by administration of an SGLT1/2 inhibitor. In the HK-2 cells, the gluconeogenic gene expression was suppressed by insulin, accompanied by phosphorylation and inactivation of forkhead box transcription factor 1 (FoxO1). In contrast, glucose deacetylated peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), a coactivator of FoxO1, via sirtuin 1, suppressing the gluconeogenic gene expression, which was reversed by inhibition of glucose reabsorption. These data suggest that both insulin signaling and glucose reabsorption suppress the gluconeogenic gene expression by inactivation of FoxO1 and PGC1α, respectively, providing insight into novel mechanisms underlying the regulation of gluconeogenesis in the PTs. [ABSTRACT FROM AUTHOR]

Sirtuin 7 (SIRT7), a member of the NAD+-dependent class III histone deacetylases, is involved in the regulation of various cellular processes and in resisting various stresses, such as hypoxia, low glucose levels, and DNA damage. Interestingly, SIRT7 is linked to the control of glycolysis, suggesting a role in glucose metabolism. Given the important roles of SIRT7, it is critical to clarify how SIRT7 activity is potentially regulated. It has been reported that some transcriptional and post-transcriptional regulatory mechanisms are involved. However, little is known how SIRT7 is regulated by the post-translational modifications. Here, we identified ubiquitin-specific peptidase 7 (USP7), adeubiquitinase, as a negative regulator of SIRT7. We showed that USP7 interacts with SIRT7 both in vitro and in vivo, and we further demonstrated that SIRT7 undergoes endogenous Lys-63-linked polyubiquitination, which is removed by USP7. Although the USP7-mediated deubiquitination of SIRT7 had no effect on its stability, the deubiquitination repressed its enzymatic activity. We also showed that USP7 coordinates with SIRT7 to regulate the expression of glucose-6-phosphatase catalytic subunit (G6PC), a gluconeogenic gene. USP7 depletion by RNA interference increased both G6PC expression and SIRT7 enzymatic activity. Moreover, SIRT7 targeted the G6PC promoter through the transcription factor ELK4 but not through forkhead box O1 (FoxO1). In summary, SIRT7 is a USP7 substrate and has a novel role as a regulator of gluconeogenesis. Our study may provide the basis for new clinical approaches to treat metabolic disorders related to glucose metabolism. [ABSTRACT FROM AUTHOR]

Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-β1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-β1/Smad3 signals suppressed endogenous glucose production. TGF-β1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-β1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-β1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance. [ABSTRACT FROM AUTHOR]

We aimed to quantify the rate of gluconeogenesis (GNG), non-essential amino-acid (NEAA) synthesis, and substrate partitioning to the Krebs cycle in embryonic (e) day e14 and e19 chicken embryos. An in ovo continuous tracer infusion approach was employed to test the hypotheses that GNG and NEAA synthesis in developing chicken embryo increases from e14 to e19. [13C6]Glucose or [13C3]glycerol was continuously infused (8 h) into the chorio-allantoic compartment of eggs on e14 and e19. Glucose entry rate, Cori cycling, and GNG were higher (P < 0.05) in e19 compared to e14 embryos, presumably to support higher glycogen deposition in liver and muscle. Whereas de novo synthesis of alanine, aspartate, and glutamate via glycolysis and the Krebs cycle was higher (P < 0.01) in e14 embryos, synthesis of these NEAA from glycerol was higher (P < 0.05) in e19 compared to e14 embryos. These patterns of glucose and glycerol utilization suggest a metabolic shift to conserve glucose for glycogen synthesis and an increased utilization of yolk glycerol (from triacylglyceride) after e14. Although the contribution of glycerol to GNG in e19 embryos was higher (P < 0.05) than that in e14 embryos, the contribution of glycerol to GNG (1.3 to 6.0%) was minor. Based on [13C6]glucose tracer kinetics, the activities of both pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) in the liver were higher (P < 0.05) in e19 embryos; whereas the higher (P < 0.01) relative activity of liver PC compared to PDH in e14 embryos suggests a greater anaplerotic flux into the Krebs cycle. In summary, the in ovo continuous tracer infusion approach allowed for a measurement of chicken embryo whole body and liver metabolism over a shorter window of development. This study provided quantitative estimates of the developmental shifts in substrate utilization, GNG, and NEAA synthesis by chicken embryos, as well as qualitative estimates of the activities of enzymes central to the Krebs cycle, glucose, and fatty acid metabolism. [ABSTRACT FROM AUTHOR]

Although the enzymatic activity of isocitrate dehydrogenase 1 (IDH1) was defined decades ago, its functions in vivo are not yet fully understood. Cytosolic IDH1 converts isocitrate to α-ketoglutarate (α-KG), a key metabolite regulating nitrogen homeostasis in catabolic pathways. It was thought that IDH1 might enhance lipid biosynthesis in liver or adipose tissue by generating NADPH, but we show here that lipid contents are relatively unchanged in both IDH1-null mouse liver and IDH1-deficient HepG2 cells generated using the CRISPR-Cas9 system. Instead, we found that IDH1 is critical for liver amino acid (AA) utilization. Body weights of IDH1-null mice fed a high-protein diet (HPD) were abnormally low. After prolonged fasting, IDH1-null mice exhibited decreased blood glucose but elevated blood alanine and glycine compared with wildtype (WT) controls. Similarly, in IDH1-deficient HepG2 cells, glucose consumption was increased, but alanine utilization and levels of intracellular α-KG and glutamate were reduced. In IDH1-deficient primary hepatocytes, gluconeogenesis as well as production of ammonia and urea were decreased. In IDH1-deficient whole livers, expression levels of genes involved in AA metabolism were reduced, whereas those involved in gluconeogenesis were up-regulated. Thus, IDH1 is critical for AA utilization in vivo and its deficiency attenuates gluconeogenesis primarily by impairing α-KG–dependent transamination of glucogenic AAs such as alanine. [ABSTRACT FROM AUTHOR]

Hepatitis B X-interacting protein (HBXIP) as an oncoprotein plays crucial roles in the development of cancer, involving glucose metabolism reprogramming. In this study, we are interested in whether the oncoprotein HBXIP is involved in the modulation of gluconeogenesis in liver cancer. Here, we showed that the expression level of phosphoenolpyruvate carboxykinase (PCK1), a key enzyme of gluconeogenesis, was lower in clinical hepatocellular carcinoma (HCC) tissues than that in normal tissues. Mechanistically, HBXIP inhibited the expression of PCK1 through down-regulating transcription factor FOXO1 in hepatoma cells, and up-regulated miR-135a targeting the 3'UTR of FOXO1 mRNA in the cells. In addition, HBXIP increased the phosphorylation levels of FOXO1 protein by activating PI3K/Akt pathway, leading to the export of FOXO1 from nucleus to cytoplasm. Strikingly, over-expression of PCK1 could abolish the HBXIP-promoted growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP is able to depress the gluconeogenesis through suppressing PCK1 to promote hepatocarcinogenesis, involving miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway. Our finding provides new insights into the mechanism by which oncoprotein HBXIP modulates glucose metabolism reprogramming in HCC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-13C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepaticTCAmetabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito. These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo. [ABSTRACT FROM AUTHOR]

Trehalose-6-P (T6P), an intermediate of trehalose biosynthesis, was identified as an important regulator of yeast sugar metabolism and signaling. tps1Λ mutants, deficient in T6P synthesis (TPS), are unable to grow on rapidly fermentable medium with uncontrolled influx in glycolysis, depletion of ATP and accumulation of sugar phosphates. However, the exact molecular mechanisms involved are not fully understood. We show that SNF1 deletion restores the tps1Λ growth defect on glucose, suggesting that lack of TPS hampers inactivation of SNF1 or SNF1-regulated processes. In addition to alternative, non-fermentable carbon metabolism, SNF1 controls two major processes: respiration and gluconeogenesis. The tps1Λ defect appears to be specifically associated with deficient inhibition of gluconeogenesis, indicating more downstream effects. Consistently, Snf1 dephosphorylation and inactivation on glucose medium are not affected, as confirmed with an in vivo Snf1 activity reporter. Detailed analysis shows that gluconeogenic Pck1 and Fbp1 expression, protein levels and activity are not repressed upon glucose addition to tps1Λ cells, suggesting a link between the metabolic defect and persistent gluconeogenesis. While SNF1 is essential for induction of gluconeogenesis, T6P/TPS is required for inactivation of gluconeogenesis in the presence of glucose, downstream and independent of SNF1 activity and the Cat8 and Sip4 transcription factors. [ABSTRACT FROM AUTHOR]

While in vitro studies have demonstrated that a glucocorticoid receptor (GR) splice isoform, β-isoform of human GR (hGRβ), acts as a dominant-negative inhibitor of the classic hGRβ and confers glucocorticoid resistance, the in vivo function of hGRβ is poorly understood. To this end, we created an adeno-associated virus (AAV) to express hGRβ in the mouse liver under the control of the hepatocyte-specific promoter. Genome-wide expression analysis of mouse livers showed that hGRβ significantly increased the expression of numerous genes, many of which are involved in endocrine system disorders and the inflammatory response. Physiologically, hGRβ antagonized GRβ's function and attenuated hepatic gluconeogenesis through downregulation of phosphoenolpyruvate carboxykinase (PEPCK) in wild-type (WT) mouse liver. Interestingly, however, hGRβ did not repress PEPCK in GR liver knockout (GRLKO) mice. In contrast, hGRβ regulates the expression of STAT1 in the livers of both WT and GRLKO mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that hGRβ binds to the intergenic glucocorticoid response element (GRE) of the STAT1 gene. Furthermore, treatment with RU486 inhibited the upregulation of STAT1 mediated by hGRβ. Finally, our array data demonstrate that hGRβ regulates unique components of liver gene expression in vivo by both GRβ-dependent and GRβ-independent mechanisms. [ABSTRACT FROM AUTHOR]

Cytoplasmic citrate serves as an important regulator of gluconeogenesis and carbon source for de novo lipogenesis in the liver. For this reason, the sodium-coupled citrate transporter (NaCT), a plasma membrane transporter that governs hepatic influx of plasma citrate in human, is being explored as a potential therapeutic target for metabolic disorders. As cytoplasmic citrate also originates from intracellular mitochondria, the relative contribution of these two pathways represents critical information necessary to underwrite confidence in this target. In this work, hepatic influx of plasma citrate was quantified via pharmacokinetic modeling of published clinical data. The influx was then compared to independent literature estimates of intracellular citrate flux in human liver. The results indicate that, under normal conditions, <10% of hepatic citrate originates from plasma. Similar estimates were determined experimentally in mice and rats. This suggests that NaCT inhibition will have a limited impact on hepatic citrate concentrations across species. [ABSTRACT FROM AUTHOR]

Biotin is a water-soluble B-complex vitamin and is well-known as a co-factor for 5 indispensable carboxylases. Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of carboxylases and other proteins, whereas biotinidase catalyzes the release of biotin from biotinylated peptides. Previous studies have reported that nutritional biotin deficiency and genetic defects in either HLCS or biotinidase induces cutaneous inflammation and immunological disorders. Since biotin-dependent carboxylases involve various cellular metabolic pathways including gluconeogenesis, fatty acid synthesis, and the metabolism of branched-chain amino acids and odd-chain fatty acids, metabolic abnormalities may play important roles in immunological and inflammatory disorders caused by biotin deficiency. Transcriptional factors, including NF-κB and Sp1/3, are also affected by the status of biotin, indicating that biotin regulates immunological and inflammatory functions independently of biotin-dependent carboxylases. An in-vivo analysis with a murine model revealed the therapeutic effects of biotin supplementation on metal allergies. The novel roles of biotinylated proteins and their related enzymes have recently been reported. Non-carboxylase biotinylated proteins induce chemokine production. HLCS is a nuclear protein involved in epigenetic and chromatin regulation. In this review, comprehensive knowledge on the regulation of immunological and inflammatory functions by biotin and its potential as a therapeutic agent is discussed. [ABSTRACT FROM AUTHOR]

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein- protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K+. We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose- 1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the coexpression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Physiology & Pharmacology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In metabolically versatile bacteria, carbon catabolite repression ( CCR) facilitates the preferential assimilation of the most efficient carbon sources, improving growth rates and fitness. In P seudomonas putida, the Crc and Hfq proteins and the CrcZ and CrcY small RNAs, which are believed to antagonize Crc/ Hfq, are key players in CCR. Unlike that seen in other bacterial species, succinate and glucose elicit weak CCR in this bacterium. In the present work, metabolic, transcriptomic and constraint-based metabolic flux analyses were combined to clarify whether P . putida prefers succinate or glucose, and to identify the role of the Crc protein in the metabolism of these compounds. When provided simultaneously, succinate was consumed faster than glucose, although both compounds were metabolized. CrcZ and CrcY levels were lower when both substrates were present than when only one was provided, suggesting a role for Crc in coordinating metabolism of these compounds. Flux distribution analysis suggested that, when both substrates are present, Crc works to organize a metabolism in which carbon compounds flow in opposite directions: from glucose to pyruvate, and from succinate to pyruvate. Thus, our results support that Crc not only favours the assimilation of preferred compounds, but balances carbon fluxes, optimizing metabolism and growth. [ABSTRACT FROM AUTHOR]

To maintain euglycemia in healthy organisms, hepatic glucose production is increased during fasting and decreased during the postprandial period. This whole process is supported by insulin levels. These responses are associated with the insulin signaling pathway and the reduction in the activity of key gluconeogenic enzymes, resulting in a decrease of hepatic glucose production. On the other hand, defects in the liver insulin signaling pathway might promote inadequate suppression of gluconeogenesis, leading to hyperglycemia during fasting and after meals. The hepatocyte nuclear factor 4, the transcription cofactor PGC1-α, and the transcription factor Foxo1 have fundamental roles in regulating gluconeogenesis. The loss of insulin action is associated with the production of pro-inflammatory biomolecules in obesity conditions. Among the molecular mechanisms involved, we emphasize in this review the participation of TRB3 protein (a mammalian homolog of Drosophila tribbles), which is able to inhibit Akt activity and, thereby, maintain Foxo1 activity in the nucleus of hepatocytes, inducing hyperglycemia. In contrast, physical exercise has been shown as an important tool to reduce insulin resistance in the liver by reducing the inflammatory process, including the inhibition of TRB3 and, therefore, suppressing gluconeogenesis. The understanding of these new mechanisms by which physical exercise regulates glucose homeostasis has critical importance for the understanding and prevention of diabetes. [ABSTRACT FROM AUTHOR]

Production of xylitol and ethanol, as well as activities of the key enzymes of D-xylose consumption, were studied in Pachysolen tannophilus mutants with altered growth on D-xylose, xylitol, ethanol, or D-glucose as sole carbon sources. Suppressed activity of xylose reductase with preferential affinity for NADPH and of xylitol dehydrogenase to 4.40 and 4.80 μmol mg min, respectively, resulted in accumulation of xylitol (0.25 g per 1 g D-xylose consumed). The highest levels of NADH/NADPH-xylose reductase and xylitol dehydrogenase (6.00-6.80 and 6.80-8.40 μmol mg min, respectively) were found in the strains producing 0.24-0.26 g ethanol per 1 g D-xylose. Application of Pa. tannophilus mutants for analysis of the regulation of D-xylose catabolism in yeasts is discussed. [ABSTRACT FROM AUTHOR]

Background & Aims It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38α in gluconeogenesis during fasting by applying a loss-of-function genetic approach. Methods We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38α in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38α (p38α-AF) and the constitutively active mitogen-activated protein kinase 6, respectively. Adenoviral dominant-negative form of AMP-activated protein kinase α (DN-AMPKα) was employed to test our proposed model. Results Mice lacking hepatic p38α exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38α did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKα phosphorylation. Adenoviral DN-AMPKα could abolish the effect of p38α-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor β-activated kinase 1 decreased the AMPKα phosphorylation induced by p38α-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKα phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38α-AF treatment ameliorated hyperglycemia in diabetic mice. Conclusions Our study provides evidence that hepatic p38α functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38α could be a drug target for hyperglycemia. [ABSTRACT FROM AUTHOR]

CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bis-phosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs. [ABSTRACT FROM AUTHOR]

Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of micro- RNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinaseAsignaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat dietinduced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1(FOXO1) transcriptional activity. Finally, liver-specific miR- 214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. [ABSTRACT FROM AUTHOR]

We undertook this study to determine whether an acute glucose stimulus during first feeding period and dietary carbohydrates can modify the gluconeogenesis pathway in the Siberian sturgeon ( Acipenser baerii ). The sturgeon larvae were fed a high glucose diet (57% glucose, 21.5% protein) or a carbohydrate-free diet (67.7% protein) from first feeding to yolk absorption (8–12 days post-hatching, dph). At 13 dph, each group of fish was assigned to two treatments. One group was fed a high-carbohydrate diet (35% dextrin, 39.3% protein), and the other group was fed a carbohydrate-free diet until the 20th wk. The results indicated that the high carbohydrate intake inhibited mRNA expression and activity of gluconeogenic enzymes at 13, 30 dph and the 20th wk. The early high glucose stimulus depressed final body weight of sturgeon at 13, 30 dph and the 20th wk. This stimulus also inhibited mRNA expressions of fructose-1,6-bisphosphatase at 30 dph and increased mRNA expressions of phosphoenolpyruvate carboxykinase cytosolic forms and glucose-6-phosphatase (G6Pase), while it inhibited activities of G6Pase at 20th wk when the fish were fed a carbohydrate-free diet. Results suggested that the Siberian sturgeon could regulate gluconeogenesis pathway response to dietary carbohydrates content at both transcriptional and post-translational levels. High glucose low protein intake during first feeding disturbs gluconeogenesis regulation in later life. However, the Siberian sturgeon might have a capacity to adapt to later nutritional conditions by reversing the changes in the gluconeogenesis pathway which induced by early stimulation at the transcriptional level during the 20 weeks feeding. [ABSTRACT FROM AUTHOR]

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells. [ABSTRACT FROM AUTHOR]

Our aim was to investigate the potential role of TOR (target of rapamycin) signaling pathway in the regulation of hepatic glucose metabolism in rainbow trout. Fasted fish were first treated with a single intraperitoneal injection of rapamycin or vehicle and then submitted to a second intraperitoneal administration of glucose 4 h later. Our results revealed that intraperitoneal administration of glucose induced hyperglycemia for both vehicle and rapamycin treatments, which peaked at 2 h. Plasma glucose level in vehicle-treated fish was significantly higher than in rapamycin-treated fish at 8 and 17 h, whereas it remained at the basal level in rapamycin-treated fish. Glucose administration significantly enhanced the phosphorylation of Akt and ribosomal protein S6 kinase (S6K1) in vehicle-treated fish, while rapamycin completely abolished the activation of S6K1 in rapamycin-treated fish, without inhibiting the phosphorylation of Akt on Thr-308 or Ser-473. Despite the lack of significant variation in phosphoenolpyruvate carboxykinase mRNA abundance, mRNA abundance for glucokinase (GK), glucose 6-phosphatase (G6Pase) I and II, and fructose 1,6-bisphosphatase (FBPase) was reduced by rapamycin 17 h after glucose administration. The inhibition effect of rapamycin on GK and FBPase was further substantiated at the activity level. The suppression of GK gene expression and activity by rapamycin provided the first in vivo evidence in fish that glucose regulates hepatic GK gene expression and activity through a TORC1-dependent manner. Unlike in mammals, we observed that acute rapamycin treatment improved glucose tolerance through the inhibition of hepatic gluconeogenesis in rainbow trout. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) is hallmarked by insulin resistance, impaired insulin secretion, and increased hepatic glucose production. The worldwide increasing prevalence of T2D calls for efforts to understand its pathogenesis in order to improve disease prevention and management. Recent genome-wide association studies have revealed strong associations between the CDKN2A/B locus and T2D risk. The CDKN2A/B locus contains genes encoding cell cycle inhibitors, including p16Ink4a, which have not yet been implicated in the control of hepatic glucose homeostasis. Here, we show that p16Ink4a deficiency enhances fasting-induced hepatic glucose production in vivo by increasing the expression of key gluconeogenic genes. p16Ink4a downregulation leads to an activation of PKA-CREB-PGC1α signaling through increased phosphorylation of PKA regulatory subunits. Taken together, these results provide evidence that p16Ink4a controls fasting glucose homeostasis and could as such be involved in T2D development. [ABSTRACT FROM AUTHOR]

Background/Objectives:Glycerol represents an important metabolite for the control of lipid accumulation and hepatic gluconeogenesis. We investigated whether hepatic expression and functionality of aquaporin-9 (AQP9), a channel mediating glycerol influx into hepatocytes, is impaired in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in the context of insulin resistance.Subjects/Methods:Liver biopsies were obtained from 66 morbid obese patients undergoing bariatric surgery (66% women, mean body mass index (BMI) 46.1±1.0 kg m−2) with available liver echography and pathology analysis of the biopsies in this cross-sectional study. Subjects were classified according to normoglycemia (NG), impaired glucose tolerance (IGT) or type 2 diabetes (T2D). Hepatic expression of AQP9 was analyzed by real-time PCR, western blotting and immunohistochemistry, while glycerol permeability (Pgly) was measured by stopped-flow light scattering.Results:AQP9 was the most abundantly (P<0.0001) expressed aquaglyceroporin in human liver (AQP9>>>AQP3>AQP7>AQP10). Obese patients with T2D showed increased plasma glycerol as well as lower Pgly and hepatic AQP9 expression. The prevalence of NAFLD and NASH in T2D patients was 100 and 65%, respectively. Interestingly, AQP9 expression was decreased in patients with NAFLD and NASH as compared with those without hepatosteatosis, in direct relation to the degree of steatosis and lobular inflammation, being further reduced in insulin-resistant individuals. The association of AQP9 with insulin sensitivity was independent of BMI and age. Consistent with these data, fasting insulin and C-reactive protein contributed independently to 33.1% of the hepatic AQP9 mRNA expression variance after controlling for the effects of age and BMI.Conclusions:AQP9 downregulation together with the subsequent reduction in hepatic glycerol permeability in insulin-resistant states emerges as a compensatory mechanism whereby the liver counteracts further triacylglycerol accumulation within its parenchyma as well as reduces hepatic gluconeogenesis in patients with NAFLD. [ABSTRACT FROM AUTHOR]

Highlights: [•] RPS showed different metabolic alterations in normal and cancer mice. [•] RPS inhibited gluconeogenesis, alanine and glycine metabolism in cancer mice. [•] RPS inhibited glucose and valine to transform ketones in the normal mice. [Copyright &y& Elsevier]

3-Iodothyronamine (T1AM) is an endogenous relative of thyroid hormone with profound metabolic effects. In different experimental models, T1AM increased blood glucose, and it is not clear whether this effect is entirely accounted by changes in insulin and/or glucagone secretion. Thus, in the present work, we investigated the uptake of T1AM by hepatocytes, which was compared with the uptake of thyroid hormones, and the effects of T1AM on hepatic glucose and ketone body production. Two different experimental models were used: HepG2 cells and perfused rat liver. Thyronines and thyronamines (T0AMs) were significantly taken up by hepatocytes. In HepG2 cells exposed to 1 mM T1AM, at the steady state, the cellular concentration of T1AM exceeded the medium concentration by six- to eightfold. Similar accumulation occurred with 3,5,30-triiodothyronine and thyroxine. Liver experiments confirmed significant T1AM uptake. T1AM was partly catabolized and the major catabolites were 3-iodothyroacetic acid (TA1) (in HepG2 cells) and T0AM (in liver). In both preparations, infusion with 1μMT1AM produced a significant increase in glucose production, if adequate gluconeogenetic substrates were provided. This effect was dampened at higher concentration (10 mM) or in the presence of the amine oxidase inhibitor iproniazid, while TA1 was ineffective, suggesting that T1AM may have a direct gluconeogenetic effect. Ketone body release was significantly increased in liver, while variable results were obtained in HepG2 cells incubated with gluconeogenetic substrates. These findings are consistent with the stimulation of fatty acid catabolism, and a shift of pyruvate toward gluconeogenesis. Notably, these effects are independent from hormonal changes and might have physiological and pathophysiological importance. [ABSTRACT FROM AUTHOR]

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-erb 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. [ABSTRACT FROM AUTHOR]

We showed recently that apoA-IV improves glucose homeosta- sis by enhancing pancreatic insulin secretion in the presence of elevated levels of glucose. Therefore, examined whether apolipo- proteinA-IV(apoA-IV) also regulates glucosemetabolismthrough the suppression of hepatic gluconeogenesis.The ability of apoA-IV to lower gluconeogenic gene expression and glucose production/ was measured in apoA-IV and wild-type mice and primary mouse hepatocytes. The transcriptional regulation of Glc-6-Pase and phosphoenolpyruvate carboxykinase (PEPCK) by apoA-IV-/- was determined by luciferase activity assay. Using bacterial two-hybrid library screening,NR1D1was identified as a putative apoA-IV-binding protein. The colocalization and interaction between apoA-IV and NR1D1 were confirmed by immunofluorescence, in situ proximity ligation assay, and coimmunoprecipitation. Enhanced recruitment of NR1D1 and activity by apoA-IV to Glc- 6-Pase promoter was verified with ChIP and a luciferase assay. Down-regulation of apoA-IV on gluconeogenic genes is mediated through NR1D1, as illustrated in cells with NR1D1 knockdown by siRNA. We found that apoA-IV suppresses the expression of PEPCK and Glc-6-Pase in hepatocytes; decreases hepatic glucose production; binds and activates nuclear receptorNR1D1 and stimulates NR1D1 expression; in cells lacking NR1D1, fails to inhibit PEPCK and Glc-6-Pase gene expression; and stimulates higher hepatic glucose production and higher gluconeogenic gene expression in apoA-IV-/- mice. We conclude that apoA-IV inhibits hepatic gluconeogenesis by decreasing Glc-6-Pase and PEPCK gene expression through NR1D1. This novel regulatory pathway connects an influx of energy as fat from the gut (and subsequent apoA-IV secretion) with inhibition of hepatic glucose production. [ABSTRACT FROM AUTHOR]

Highlights: [•] Brain μ-opioid receptors are involved in the reward system and influence food intake. [•] Periportal μ-opioid receptors control intestinal gluconeogenesis via a reflex arc. [•] Protein-digests induce gut gluconeogenesis and satiety by activating this reflex arc. [Copyright &y& Elsevier]

Various hydrocarbons have been released into the environment as a result of industrialization. An effective way of removing these materials without further environmental contamination is microbial bioremediation. Mycobacterium gilvum PYR-GCK, a bacteria isolated from a PAH polluted estuary, was studied using comparative shotgun proteomics to gain insight on its molecular activity while using pyrene and glucose as sole carbon and energy sources. Based on annotated genomic information, a confirmation analysis was first performed to confirm its pyrene degradation activity, using gas chromatography-mass spectrometry technology. One dimensional gel electrophoresis and liquid chromatography-mass spectrometry technologies employed in the proteomics analysis revealed the expression of pyrene degrading gene products along with upregulated expression of proteins functioning in the glyoxylate and shikimate pathways, in the pyrene-induced cells. The study also revealed the pathway of pyrene degraded intermediates, via partial gluconeogenesis, into the pentose phosphate pathway to produce precursors for nucleotides and amino acids biosynthesis. [ABSTRACT FROM AUTHOR]

Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element-binding protein (CREB) and the dephos- phorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat-containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small- molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment. [ABSTRACT FROM AUTHOR]

Background & Aims: The expression levels of microRNA-29 (miR-29) family members (miR-29a, miR-29b, miR-29c, here denoted collectively as miR-29a-c) are increased in livers of Goto-Kakizaki diabetic rats and db/db diabetic mice. However, the functional consequences of miR-29a-c upregulation in diabetic livers are not explored. The objective of this study was to evaluate the roles of miR-29a-c in the regulation of hepatic glucose production and blood glucose levels using different mouse models. Methods: db/m, db/db diabetic and diet-induced obese (DIO) mice were injected with adenovirus expressing miR-29a-c through the tail vein. Blood glucose levels were measured and glucose-tolerance tests and pyruvate-tolerance tests were performed. To explore the molecular mechanism by which miR-29a-c regulate hepatic glucose metabolism, gain or loss of miR-29a-c function studies were performed in primary mouse hepatocytes and the direct effectors of miR-29-mediated effects on glucose metabolism were identified. Results: Adenovirus-mediated overexpression of miR-29a-c in the livers of db/m, db/db, and DIO mice decreased fasting blood glucose levels and improved glucose tolerance. Overexpression of miR-29a-c in primary hepatocytes and mouse livers decreased the protein levels of PGC-1α and G6Pase, the direct targets of miR-29a-c, thereby reducing cellular, and hepatic glucose production. In contrast, loss of miR-29a-c function in primary hepatocytes increased the protein levels of PGC-1α and G6Pase and increased cellular glucose production. Finally, enforced expression of PGC-1α increased miR-29a-c expression levels in primary hepatocytes, thus forming a negative feedback regulation loop. Conclusions: miR-29a-c can regulate hepatic glucose production and glucose tolerance in mice. [ABSTRACT FROM AUTHOR]

The regulation of carbon metabolism in the diatom Phaeodactylum tricornutum at the cell, metabolite, and gene expression levels in exponential fed-batch cultures is reported. Transcriptional profiles and cell chemistry sampled simultaneously at all time points provide a comprehensive data set on carbon incorporation, fate, and regulation. An increase in Nile Red fluorescence (a proxy for cellular neutral lipids) was observed throughout the light period, and water-soluble glucans increased rapidly in the light period. A near-linear decline in both glucans and lipids was observed during the dark period, and transcription profile data indicated that this decline was associated with the onset of mitosis. More than 4,500 transcripts that were differentially regulated during the light/dark cycle are identified, many of which were associated with carbohydrate and lipid metabolism. Genes not previously described in algae and their regulation in response to light were integrated in this analysis together with proposed roles in metabolic processes. Some very fast light-responding genes in, for example, fatty acid biosynthesis were identified and allocated to biosynthetic processes. Transcripts and cell chemistry data reflect the link between light energy availability and light energy-consuming metabolic processes. Our data confirm the spatial localization of processes in carbon metabolism to either plastids or mitochondria or to glycolysis/gluconeogenesis, which are localized to the cytosol, chloroplast, and mitochondria. Localization and diel expression pattern may be of help to determine the roles of different isoenzymes and the mining of genes involved in light responses and circadian rhythms. [ABSTRACT FROM AUTHOR]

Few studies have investigated liver gluconeogenesis in cancer and there is no agreement as to whether the activity of this pathway is increased or decreased in this disease. The aim of this study was to evaluate gluconeogenesis from alanine, pyruvate and glycerol, and related metabolic parameters in perfused liver from Walker-256 tumour-bearing rats on days 5 ( WK5 group), 8 ( WK8 group) and 12 ( WK12 group) of tumour development. There was reduction ( P < 0.05) of liver glucose production from alanine and pyruvate in WK5, WK8 and WK12 groups, which was accompanied by a decrease ( P < 0.05) in oxygen consumption. Moreover, there was higher ( P < 0.05) pyruvate and lactate production from alanine in the WK5 group and a marked reduction ( P < 0.05) of pyruvate and urea production from alanine in the WK12 group. In addition, liver glucose production and oxygen consumption from glycerol were not reduced in WK5, WK8 and WK12 groups. Thus the, the results show inhibition of hepatic gluconeogenesis from alanine and pyruvate, but not from glycerol, on days 5, 8 and 12 of Walker-256 tumour development, which can be attributed to the metabolic step in which the substrate enters the gluconeogenic pathway. [ABSTRACT FROM AUTHOR]

Abstract: This study describes the metabolic capacities of the African cichlid Pseudocrenilabrus multicolor victoriae from four sites in Uganda, East Africa. Fish were captured during the dry season, from two aquatic systems in different regions (Lake Nabugabo and Mpanga River). Within the Lake Nabugabo region, individuals were sampled from Lake Kayanja (normoxic) and Lwamunda Swamp (hypoxic); within the Mpanga River system, individuals were sampled from Bunoga and Kahunge (characterized by seasonal variation in dissolved oxygen (D.O.)). Enzyme activity levels of pyruvate kinase, lactate dehydrogenase, citrate synthase, and cytochrome C oxidase were measured in four tissues: white skeletal muscle, heart, brain, and liver. Two additional enzymes were measured in the liver, malate dehydrogenase and fructose 1,6-bisphosphatase. Regional differences between enzyme activities in most tissues were evident; however, little variation was observed between two sites within a region despite differences in D.O. In general, P. multicolor from the Mpanga River system displayed greater anaerobic enzyme activity in white skeletal muscle, lower gluconeogenic enzyme activity in the liver, and an overall higher enzyme activity in the heart and brain tissues than fish from the Nabugabo region. The latter may reflect a long-term adaptation to low-oxygen conditions at the metapopulation level in the Nabugabo region. [Copyright &y& Elsevier]

The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water (2H2O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with 2H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific 2H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal 2H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment (2H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/ z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/ z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used. [ABSTRACT FROM AUTHOR]

Abstract: The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca2+-influx via voltage-gated Ca2+-channels, elevation of [Ca2+]i and initiation of Ca2+-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca2+-channel present in α- and β-cells and the modulation of hormone secretion by Ca2+-dependent and -independent processes. We also consider how subtle changes in Ca2+-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes. [Copyright &y& Elsevier]

Abstract: NADPH oxidase, catalysing superoxide radical (O2 −) formation, is considered as a main source of reactive oxygen species in kidneys and its increased activity is supposed to be involved in the development of diabetic nephropathy. The aim of this study was to investigate the effect of NADPH oxidase inhibitor, apocynin, on renal gluconeogenesis, which is an important source of endogenous glucose under diabetic conditions. The following observations were made during the experiments performed on isolated renal proximal tubules of control and alloxan diabetic rabbits: (1) apocynin (200μM) inhibited the rate of glucose synthesis by 45–80%, depending on the substrate applied; (2) the rate of glucose production was also significantly diminished in the presence of TEMPOL (5mM), a superoxide radical scavenger, suggesting that the decrease in O2 − formation might be involved in apocynin-evoked gluconeogenesis inhibition; (3) the activities of phosphoenolpyruvate carboxykinase (PEPCK) and/or aldolase were lowered in the presence of either apocynin or TEMPOL, as concluded from the intracellular levels of gluconeogenic intermediates. The data from in vivo experiments indicated that apocynin treatment (2g/l of drinking water): (1) significantly (by about 30%) attenuated serum glucose concentration in diabetic rabbits and did not affect glycaemia in control animals; (2) normalized diabetes-stimulated rate of glucose synthesis and slightly inhibited gluconeogenesis in control rabbits; (3) normalized diabetes-increased activity of mitochondrial PEPCK and lowered cytosolic PEPCK activity by about 20% below the value for untreated control animals; (4) slightly decreased the activity of mitochondrial PEPCK and did not change the activity of cytosolic one in control rabbits. Thus, it is concluded that: (1) the inhibition of NADPH oxidase might contribute to lowered rate of renal gluconeogenesis, probably due to decreasing PEPCK activity; (2) inhibition of renal gluconeogenesis is involved in apocynin hypoglycaemic action in vivo; (3) apocynin might be beneficial for diabetes treatment. [Copyright &y& Elsevier]

Inhibitors of 11β-HSD1 are in clinical trials for the treatment of type 2 diabetes. These compounds act by decreasing the cortisol generated in liver and adipose tissue, and therefore reducing tissue-specific gluconeogenesis and fatty acid metabolism. However, there is concern that reduction in tissue-regenerated cortisol might decrease feedback to the hypothalamic–pituitary–adrenal (HPA) axis, resulting in upregulation of cortisol from the adrenal gland. This review considers evidence from 11β-HSD1 knockout and transgenic mice, inhibitor studies and results from clinical trials evaluating HPA axis biomarkers. It is clear that analysis of the HPA axis is not sufficiently detailed, and there is a need to understand the subtle changes in the axis associated with pulsatility, diurnal rhythm and stress. [ABSTRACT FROM AUTHOR]

To shed some light on gluconeogenesis in mammalian retina, we have focused on fructose-1,6-bisphosphatase (FBPase), a regulatory enzyme of the process. The abundance of the enzyme within the layers of the rat retina suggests that, in mammals in contrast to amphibia, gluconeogenesis is not restricted to one specific cell of the retina. We propose that FBPase, in addition to its gluconeogenic role, participates in the protection of the retina against reactive oxygen species. Additionally, the nuclear localization of FBPase and of its binding partner, aldolase, in the retinal cells expressing the proliferation marker Ki-67 indicates that these two gluconeogenic enzymes are involved in non-enzymatic nuclear processes. [ABSTRACT FROM AUTHOR]

Abstract: Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of gluconeogenesis. In the yeast Saccharomyces cerevisiae, it is only expressed when cells are grown in medium with nonfermentable carbon sources. Addition of glucose to cells leads to inactivation of FBPase and degradation via the ubiquitin–proteasome system. Polyubiquitination of FBPase is carried out by the Gid complex, a multi-subunit ubiquitin ligase. Using tandem affinity purification and subsequent mass spectrometry we identified the Hsp70 chaperone Ssa1 as a novel interaction partner of FBPase. Studies with the temperature-sensitive mutant ssa1-45ts showed that Ssa1 is essential for polyubiquitination of FBPase by the Gid complex. Moreover, we show that degradation of an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is also affected in ssa1-45ts cells demonstrating that Ssa1 plays a general role in elimination of gluconeogenic enzymes. [Copyright &y& Elsevier]

Metformin has become a mainstay in the modest therapeutic armamentarium for the treatment of the insulin resistance of type 2 diabetes mellitus. Although metformin functions primarily by reducing hepatic glucose output, the molecular mechanism mediating this effect had remained elusive until recently. Metformin impairs ATP production, activating the conserved sensor of nutritional stress AMP-activated protein kinase (AMPK), thus providing a plausible and generally accepted model for suppression of gluconeogenic gene expression and glucose output. In this issue of the JCI, Foretz et al. refute this hypothesis by showing that AMPK is dispensable for the effects of metformin on hepatic glucose output in primary hepatocytes; rather, their data suggest that the antidiabetic effects of metformin in the liver are mediated directly by reducing energy charge. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Insulin-mediated suppression of hepatic glucose production (HGP) is associated with sensitive intracellular signaling and molecular inhibition of gluconeogenic (GNG) enzyme mRNA expression. We determined, for the first time, the time course and relevance (to metabolic flux) of these molecular events during physiological hyperinsulinemia in vivo in a large animal model. RESEARCH DESIGN AND METHODS--24 h fasted dogs were infused with somatostatin, while insulin (basal or 8x basal) and glucagon (basal) were replaced intraportally. Euglycemia was maintained and glucose metabolism was assessed using tracer, 2H2O, and arterio-venous difference techniques. Studies were terminated at different time points to evaluate insulin signaling and enzyme regulation in the liver. RESULTS--Hyperinsulinemia reduced HGP due to a rapid transition from net glycogen breakdown to synthesis, which was associated with an increase in glycogen synthase and a decrease in glycogen phosphorylase activity. Thirty minutes of hyperinsulinemia resulted in an increase in phospho-FOXO1, a decrease in GNG enzyme mRNA expression, an increase in F2,6P2, a decrease in fat oxidation, and a transient decrease in net GNG flux. Net GNG flux was restored to basal by 4 h, despite a substantial reduction in PEPCK protein, as gluconeogenically-derived carbon was redirected from lactate efflux to glycogen deposition. CONCLUSIONS--In response to acute physiologic hyperinsulinemia, 1) HGP is suppressed primarily through modulation of glycogen metabolism; 2) a transient reduction in net GNG flux occurs and is explained by increased glycolysis resulting from increased F2,6P2 and decreased fat oxidation; and 3) net GNG flux is not ultimately inhibited by the rise in insulin, despite eventual reduction in PEPCK protein, supporting the concept that PEPCK has poor control strength over the gluconeogenic pathway in vivo. Diabetes 59:1302-1311, 2010 [ABSTRACT FROM AUTHOR]

Summary: Endoplasmic reticulum (ER)-bound transcription factor families are shown to be involved in the control of various metabolic pathways. Here, we report a critical function of ER-bound transcription factor, CREBH, in the regulation of hepatic gluconeogenesis. Expression of CREBH is markedly induced by fasting or in the insulin-resistant state in rodents in a dexamethasone- and PGC-1α-dependent manner, which results in the accumulation of active nuclear form of CREBH (CREBH-N). Overexpression of constitutively active CREBH activates transcription of PEPCK-C or G6Pase by binding to its enhancer site that is distinct from the well-characterized CREB/CRTC2 regulatory sequences in vivo. Of interest, knockdown of CREBH in liver significantly reduces blood glucose levels without altering expression of genes involved in the ER stress signaling cascades in mice. These data suggest a crucial role for CREBH in the regulation of hepatic glucose metabolism in mammals. [Copyright &y& Elsevier]

The kidney plays a major role in glucose homeostasis because of its role in gluconeogenesis and the glomerular filtration and reabsorption of glucose in the proximal convoluted tubules. Approximately 180 g of glucose is filtered daily in the glomeruli of a normal healthy adult. Typically, all of this glucose is reabsorbed with <1% being excreted in the urine. The transport of glucose from the tubule into the tubular epithelial cells is accomplished by sodiumglucose co-transporters (SGLTs). SGLTs encompass a family of membrane proteins that are responsible for the transport of glucose, amino acids, vitamins, ions and osmolytes across the brush-border membrane of proximal renal tubules as well as the intestinal epithelium. SGLT2 is a high-capacity, low-affinity transporter expressed chiefly in the kidney. It accounts for approximately 90% of glucose reabsorption in the kidney and has thus become the focus of a great deal of interest in the field of diabetes mellitus. SGLT2 inhibitors block the reabsorption of filtered glucose leading to glucosuria. This mechanismof action holds potential promise for patients with type 2 diabetes mellitus (T2DM) in terms of improvements in glycaemic control. In addition, the glucosuria associated with SGLT2 inhibition is associated with caloric loss, thus providing a potential benefit of weight loss. Dapagliflozin is the SGLT2 inhibitor with the most clinical data available to date, with other SGLT2 inhibitors currently in the developmental pipeline. Dapagliflozin has demonstrated sustained, dose-dependent glucosuria over 24 hours with once-daily dosing in clinical trials. Although long-term safety data are lacking, studies to date have generally found dapagliflozin to be safe and well tolerated. Concerns related to SGLT2 inhibition include the fact that by their very nature they cause glucose elevation in the urine that can theoretically lead to urinary tract and genital infections, electrolyte imbalances and increased urinary frequency. Although studies to date have been promising in terms of these and other concerns, longer-term studies evaluating the usual safety and efficacy outcomes will need to be conducted. Similarly, head-to-head comparator trials are needed to determine the role of SGLT2 inhibitors in relation to the many other therapeutic options available for the treatment of T2DM. If significant reductions in haemoglobin A1c are associated with SGLT2 inhibitor therapy, and these agents are determined to be safe and well tolerated in the long term, they could become a major breakthrough in the T2DM treatment armamentarium. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response is a physiological process enabling cells to cope with altered protein synthesis demands. However, under conditions of obesity, prolonged activation of the UPR has been shown to have deteriorating effects on different metabolic pathways. Here we identify Bax inhibitor-1 (BI-1), an evolutionary conserved ER-membrane protein, as a novel modulator of the obesity-associated alteration of the UPR. BI-1 partially inhibits the UPR by interacting with IRE1n and inhibiting IRE1n endonuclease activity as seen on the splicing of the transcription factor Xbp-1. Because we observed a down-regulation of BI-1 expression in liver and muscle of genetically obese ob/ob and db/db mice as well as in mice with diet-induced obesity in vivo, we investigated the effect of restoring BI-1 expression on metabolic processes in these mice. Importantly, BI-1 overexpression by adenoviral gene transfer dramatically improved glucose metabolism in both standard diet-fed mice as well as in mice with diet-induced obesity and, critically, reversed hyperglycemia in db/db mice. This improvement in whole body glucose metabolism and insulin sensitivity was due to dramatically reduced gluconeogenesis as shown by reduction of glucose-6phosphatase and phosphoenolpyruvate carboxykinase expression. Taken together, these results identify BI-1 as a critical regulator of ER stress responses in the development of obesity associated insulin resistance and provide proof of concept evidence that gene transfer-mediated elevations in hepatic BI1 may represent a promising approach for the treatment of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Summary: Dynamic regulation of insulin signaling and metabolic gene expression is critical to nutrient homeostasis; dysregulation of these pathways is widely implicated in insulin resistance and other disease states. Though the metabolic effects of insulin are well established, the components linking insulin signal transduction to a metabolic response are not as well understood. Here, we show that Cdc2-like kinase 2 (Clk2) is an insulin-regulated suppressor of hepatic gluconeogenesis and glucose output. Clk2 protein levels and kinase activity are induced as part of the hepatic refeeding response by the insulin/Akt pathway. Clk2 directly phosphorylates the SR domain on PGC-1α, resulting in repression of gluconeogenic gene expression and hepatic glucose output. In addition, Clk2 is downregulated in db/db mice, and reintroduction of Clk2 largely corrects glycemia. Thus, we have identified a role for and regulation of the Clk2 kinase as a component of hepatic insulin signaling and glucose metabolism. [Copyright &y& Elsevier]

Abstract: Coordinated recovery of hepatic glucose metabolism is prerequisite for normal liver regeneration. To examine roles of hypoxia inducible factor-1α (HIF-1α) for hepatic glucose homeostasis during the reparative process, we inactivated the gene in hepatocytes in vivo. Following partial hepatectomy (PH), recovery of residual liver weight was initially retarded in the mutant mice by down-regulation of hepatocyte proliferation, but occurred comparably between the mutant and control mice at 72h after PH. At this time point, the mutant mice showed lowered blood glucose levels with enhanced accumulation of glycogen in the liver. The mutant mice exhibited impairment of hepatic gluconeogenesis as assessed by alanine tolerance test. This appeared to result from reduced expression of PGK-1 and PEPCK since 3-PG, PEP and malate were accumulated to greater extents in the regenerated liver. In conclusion, these findings provide evidence for roles of HIF-1α in the regulation of gluconeogenesis under liver regeneration. [Copyright &y& Elsevier]