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1.
J Nutr Biochem ; 19(5): 305-12, 2008 May.
Article in English | MEDLINE | ID: mdl-17683927

ABSTRACT

The Koletsky (SHROB) strain of rats is spontaneously hypertensive and displays insulin resistance, hyperglucagonemia and hypertriglyceridemia but is normoglycemic under fasting conditions. The aim of this study was to unravel the pattern of expression of genes encoding key regulatory enzymes involved in carbohydrate metabolism in the liver and kidney that may be impacted in this strain. We found that SHROB animals have decreased beta-adrenergic receptor density and, consequently, blunted increases in cAMP levels in response to beta-adrenergic agonists. They also have lower levels of hepatic as well as renal phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) mRNA and protein than their lean littermates. Expression of the genes for glycogen phosphorylase and glycogen synthase was also decreased. Hepatocytes from the SHROB animals exhibited glycogen depletion of only 50% compared to 86% by hepatocytes from lean littermates when challenged with either glucagon or forskolin to stimulate adenylyl cyclase. The expression of C/EBPalpha and C/EBPbeta, two key transcription factors that are essential for the coordinated expression of genes involved in glucose homeostasis, was depressed in livers of the SHROB rats, as were levels of HNF-4alpha, PPARalpha and PGC-1alpha. We conclude that overproduction of glucose is prevented in the SHROB rats by decreased expression of the genes for glycogen phosphorylase and the gluconeogenic enzymes PEPCK and G6Pase, which may prevent progression to diabetes in this model.


Subject(s)
Carbohydrate Metabolism , Enzymes/metabolism , Gene Expression Regulation , Liver/metabolism , Transcription Factors/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Carbohydrate Metabolism/genetics , Cells, Cultured , Cyclic AMP/metabolism , Enzymes/genetics , Female , Gene Expression Regulation/drug effects , Gluconeogenesis/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Hormones/pharmacology , Kidney/enzymology , Liver/drug effects , Liver/enzymology , Mice , Phosphodiesterase Inhibitors/pharmacology , Rats , Rats, Inbred SHR , Receptors, Adrenergic, beta/metabolism , Transcription Factors/genetics
2.
J Biol Chem ; 282(45): 32844-55, 2007 Nov 09.
Article in English | MEDLINE | ID: mdl-17716967

ABSTRACT

Transgenic mice, containing a chimeric gene in which the cDNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) was linked to the alpha-skeletal actin gene promoter, express PEPCK-C in skeletal muscle (1-3 units/g). Breeding two founder lines together produced mice with an activity of PEPCK-C of 9 units/g of muscle (PEPCK-C(mus) mice). These mice were seven times more active in their cages than controls. On a mouse treadmill, PEPCK-C(mus) mice ran up to 6 km at a speed of 20 m/min, whereas controls stopped at 0.2 km. PEPCK-C(mus) mice had an enhanced exercise capacity, with a VO(2max) of 156 +/- 8.0 ml/kg/min, a maximal respiratory exchange ratio of 0.91 +/- 0.03, and a blood lactate concentration of 3.7 +/- 1.0 mm after running for 32 min at a 25 degrees grade; the values for control animals were 112 +/- 21 ml/kg/min, 0.99 +/- 0.08, and 8.1 +/- 5.0 mm respectively. The PEPCK-C(mus) mice ate 60% more than controls but had half the body weight and 10% the body fat as determined by magnetic resonance imaging. In addition, the number of mitochondria and the content of triglyceride in the skeletal muscle of PEPCK-C(mus) mice were greatly increased as compared with controls. PEPCK-C(mus) mice had an extended life span relative to control animals; mice up to an age of 2.5 years ran twice as fast as 6-12-month-old control animals. We conclude that overexpression of PEPCK-C repatterns energy metabolism and leads to greater longevity.


Subject(s)
Cytosol/enzymology , Energy Metabolism , Gene Expression Regulation, Enzymologic , Muscle, Skeletal/enzymology , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Aging/physiology , Animals , Behavior, Animal , Female , Male , Mice , Mice, Transgenic , Organ Specificity , Pedigree , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Physical Conditioning, Animal , Respiration
3.
J Nutr ; 137(3): 554-9, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17311939

ABSTRACT

Fatty acids and glucose are strong modulators of the expression of glucose-6-phosphatase (Glc-6-Pase), an enzyme that plays a key role in glucose homeostasis. PUFA inhibit, whereas SFA and monounsaturated fatty acids induce the expression of the Glc-6-Pase gene. Palmitate and oleate are the most abundant fatty acid species in circulation during food deprivation in mammals. Although dietary fats have been shown to modulate the expression of genes involved in both lipid and carbohydrate metabolism in liver, little is known regarding the molecular mechanism of transcriptional response of the Glc-6-Pase gene to long-chain fatty acids. Using H4IIE hepatoma cells and hepatocytes from adult rats, we investigated the mechanism of the induction of this gene by palmitate and oleate. Both of these fatty acids stimulated Glc-6-Pase gene transcription but did not affect the stability of its mRNA. In transient transfection assays, transcription from the Glc-6-Pase gene promoter was markedly enhanced by both palmitate and oleate but not by arachidonate. Chromatin immunoprecipitation analysis was used to show that palmitate induced the recruitment of an array of transcription factors viz hepatic nuclear factor(NF)-4alpha, CAAT/enhancer binding proteinbeta, PPARalpha, chicken ovalbumin upstream promoter transcription factor (COUP-TF), cAMP regulatory element binding protein, and NF-kappaB to this gene promoter. Although it is presently unclear how these various transcription factors interact at this promoter, the data are consistent with the view that multiple regulatory elements in the Glc-6-Pase gene promoter are responsible for the modulation of gene transcription by fatty acids.


Subject(s)
Glucose-6-Phosphatase/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Palmitic Acid/pharmacology , Promoter Regions, Genetic , Transcription Factors/metabolism , Animals , Base Sequence , Cell Line, Tumor , DNA/genetics , DNA/metabolism , Gene Expression/drug effects , In Vitro Techniques , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Models, Biological , Oleic Acid/pharmacology , Promoter Regions, Genetic/genetics , Protein Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats
4.
FEBS Lett ; 580(15): 3746-52, 2006 Jun 26.
Article in English | MEDLINE | ID: mdl-16777101

ABSTRACT

G6P translocase (G6PT) is thought to play a crucial role in transducing intracellular signaling events in brain tumor-derived cancer cells. In this report, we investigated the contribution of G6PT to the control of U-87 brain tumor-derived glioma cell survival using small interfering RNA (siRNA)-mediated suppression of G6PT. Three siRNA constructs were generated and found to suppress up to 91% G6PT gene expression. Flow cytometry analysis of propidium iodide/annexin-V-stained cells indicated that silencing the G6PT gene induced necrosis and late apoptosis. The anticancer agent curcumin, also inhibited G6PT gene expression by more than 90% and triggered U-87 glioma cells death. Overexpression of recombinant G6PT rescued the cells from curcumin-induced cell death. Targeting G6PT expression may provide a new mechanistic rationale for the action of chemopreventive drugs and lead to the development of new anti-cancer strategies.


Subject(s)
Antineoplastic Agents/pharmacology , Antiporters/genetics , Curcumin/pharmacology , Gene Silencing , Glioma/enzymology , Glioma/pathology , Microsomes/enzymology , Monosaccharide Transport Proteins/genetics , Antiporters/deficiency , Antiporters/metabolism , Cell Death/drug effects , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Glioma/genetics , Humans , Monosaccharide Transport Proteins/deficiency , Monosaccharide Transport Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
5.
J Biol Chem ; 280(40): 33873-84, 2005 Oct 07.
Article in English | MEDLINE | ID: mdl-16100117

ABSTRACT

The hepatic transcriptional regulation by glucocorticoids of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) gene is coordinated by interactions of specific transcription factors at the glucocorticoid regulatory unit (GRU). We propose an extended GRU that consists of four accessory sites, two proximal AF1 and AF2 sites and their distal counterpart dAF1 (-993) and a new site, dAF2 (-1365); together, these four sites form a palindrome. Sequencing and gel shift binding assays of hepatic nuclear proteins interacting with these sites indicated similarity of dAF1 and dAF2 sites to the GRU proximal AF1 and AF2 sites. Chromatin immunoprecipitation assays demonstrated that glucocorticoids enhanced the binding of FOXO1 and peroxisome proliferator-activated receptor-alpha to AF2 and dAF2 sites and not to dAF1 site but enhanced the binding of hepatic nuclear transcription factor-4alpha only to the dAF1 site. Insulin inhibited the binding of these factors to their respective sites but intensified the binding of phosphorylated FOXO1. Transient transfections in HepG2 human hepatoma cells showed that glucocorticoid receptor interacts with several non-steroid nuclear receptors, yielding a synergistic response of the PEPCK-C gene promoter to glucocorticoids. The synergistic stimulation by glucocorticoid receptor together with peroxisome proliferator-activated receptor-alpha or hepatic nuclear transcription factor-4alpha requires all four accessory sites, i.e. a mutation of each of these markedly affects the synergistic response. Mice with a targeted mutation of the dAF1 site confirmed this requirement. This mutation inhibited the full response of hepatic PEPCK-C gene to diabetes by reducing PEPCK-C mRNA level by 3.5-fold and the level of circulating glucose by 25%.


Subject(s)
Gene Expression Regulation , Glucocorticoids/pharmacology , Phosphoenolpyruvate Carboxykinase (GTP)/genetics , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Receptors, Glucocorticoid/physiology , Animals , Carcinoma, Hepatocellular/pathology , Chromatin/chemistry , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Hypoglycemic Agents/pharmacology , Immunoprecipitation , Insulin/pharmacology , Liver/enzymology , Liver Neoplasms/pathology , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , PPAR alpha , Promoter Regions, Genetic , Transcription, Genetic , Transfection , Tumor Cells, Cultured
6.
J Biol Chem ; 278(42): 40694-701, 2003 Oct 17.
Article in English | MEDLINE | ID: mdl-12915406

ABSTRACT

Mechanisms underlying dietary nutrient regulation of glucose-6-phosphatase (Glc-6-Pase) gene expression are not well understood. Here we investigated the effects of short-chain fatty acids on the expression of this gene in primary cultures of rat hepatocytes and H4IIE hepatoma cells. Propionate, butyrate, valerate, and caproate induced severalfold increases in the expression of Glc-6-Pase mRNA. In reporter gene assays, propionate, valerate, caproate, and also octanoate increased Glc-6-Pase promoter activity by 6-16-fold. Butyrate, by itself, had little or no effect on promoter activity, but it induced a robust increase (45-fold) in promoter activity in cells co-transfected with a plasmid expressing the transcription factor HNF-4alpha (alpha isoforms of hepatic nuclear factor 4). HNF-4alpha also enhanced promoter activity induced by other short-chain fatty acids. A dominant negative form of HNF-4alpha abrogated the fatty acid-induced promoter activity, a finding that accentuates a role for HNF-4alpha in the transcription process studied here. In cells transfected with HNF-4alpha, short-chain fatty acids and trichostatin A, an inhibitor of histone deacetylase, synergistically enhanced promoter activity, suggesting that hyperacetylation of histones is an important component of the transactivation of the Glc-6-Pase gene promoter by HNF-4alpha. Region-751/-466 of this promoter contains seven putative HNF-4alpha-binding motifs. Binding of HNF-4alpha to this region was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, indicating that HNF-4alpha is recruited to the Glc-6-Pase gene promoter during short-chain fatty acid-induced transcription from this promoter.


Subject(s)
DNA-Binding Proteins , Gene Expression Regulation, Enzymologic , Glucose-6-Phosphatase/biosynthesis , Glucose-6-Phosphatase/genetics , Hepatocytes/enzymology , Phosphoproteins/physiology , Transcription Factors/physiology , Amino Acid Motifs , Animals , Blotting, Northern , Blotting, Western , Cell Nucleus/metabolism , Cells, Cultured , Chromatin/metabolism , Fatty Acids, Volatile/metabolism , Genes, Dominant , Genes, Reporter , Hepatocyte Nuclear Factor 4 , Histone Deacetylases/metabolism , Histones/metabolism , Hydroxamic Acids/pharmacology , Models, Genetic , Plasmids/metabolism , Precipitin Tests , Promoter Regions, Genetic , Protein Isoforms , RNA, Messenger/metabolism , Rats , Time Factors , Transcription, Genetic , Transcriptional Activation , Transfection
7.
Am J Physiol Endocrinol Metab ; 283(5): E946-57, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12376321

ABSTRACT

The purpose of this study was to compare the assessment of gluconeogenesis (GNG) in the overnight- and prolonged-fasted states and during chronic hypercortisolemia using the arteriovenous difference and [14C]phosphoenolpyruvate-liver biopsy techniques as well as a combination of the two. Two weeks before a study, catheters and flow probes were implanted in the hepatic and portal veins and femoral artery of dogs. Animals were studied after an 18-h fast (n = 8), a 42- or 66-h fast (n = 7), and an 18-h fast plus a continuous infusion of cortisol (3.0 microg. kg(-1). min(-1)) for 72 h (n = 7). Each experiment consisted of an 80-min tracer ([3-(3)H]glucose and [U-(14)C]alanine) and dye equilibration period (-80 to 0 min) and a 45-min sampling period. In the cortisol-treated group, plasma cortisol increased fivefold. In the overnight-fasted group, total GNG flux rate (GNG(flux)), conversion of glucose 6-phosphate to glucose (GNG(G-6-P-->Glc)), glucose cycling, and maximal GNG flux rate (GNG(max)) were 0.95 +/- 0.14, 0.65 +/- 0.06, 0.62 +/- 0.06, and 0.70 +/- 0.09 mg. kg(-1). min(-1), respectively. In the prolonged-fasted group, they were 1.50 +/- 0.18, 1.18 +/- 0.13, 0.40 +/- 0.07, and 1.28 +/- 0.10 mg. kg(-1). min(-1), whereas in the cortisol-treated group they were 1.64 +/- 0.33, 0.99 +/- 0.29, 1.32 +/- 0.24, and 0.91 +/- 0.13 mg. kg(-1). min(-1). These results demonstrate that GNG(G-6-P-->Glc) and GNG(max) were almost identical. However, these rates were 15-38% lower than GNG(flux) generated by a combination of the two methods. This difference was most apparent in the steroid-treated group, where the combination of the two methods (GNG(flux)) detected a significant increase in gluconeogenic flux.


Subject(s)
Fasting/physiology , Gluconeogenesis/physiology , Hydrocortisone/pharmacology , Liver/metabolism , Physiology/methods , Amino Acids/metabolism , Animals , Biopsy , Blood Glucose/metabolism , Carbon Radioisotopes , Dogs , Female , Gluconeogenesis/drug effects , Glucose-6-Phosphate/metabolism , Glycerol/blood , Hydrocortisone/blood , Lactic Acid/blood , Liver/cytology , Liver/drug effects , Male , Phosphoenolpyruvate/pharmacokinetics
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