Cytosolic Form Of Phosphoenolpyruvate Carboxykinase Research Articles

Effects of chronic dexamethasone administration on hyperglycemia and insulin release in goats

BackgroundDexamethasone (Dex), a synthetic glucocorticoid, is among the most commonly used drugs worldwide in animals and humans as an anti-inflammatory and immunosuppressive agent. GC has profound effects on plasma glucose level and other metabolic conditions. However, the effect of prolonged use of Dex on glucose metabolism in ruminants is still unclear.ResultsTen goats were randomly assigned to two groups: the control goats were injected with saline, and the Dex-treated goats were intramuscularly injected daily for 21 d with 0.2 mg/kg Dex. The results showed that plasma glucose and insulin concentrations were significantly increased after Dex administration (P < 0.05). Additionally, the content of hepatic glycogen was also markedly increased in Dex-treated goats (P < 0.01), while the content of glycogen in dorsal longissimus was unchanged by Dex (P > 0.05). The expression of several key genes, involved in blood glucose regulation, was detected by real-time PCR in the small intestine, skeletal muscle and liver. The expression of glucose transporter type 2 (GLUT2), sodium-glucose transporter 1 (SGLT1) and sodium-potassium ATPase (Na-K/ATPase) in the small intestine were generally increased by Dex, and GLUT2 mRNA expression was significantly up-regulated (P < 0.05). In liver, the expression of genes involved in gluconeogenesis including glucose-6-phosphatase catalytic subunit (G6PC), cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) and pyruvate carboxylase (PC), were significantly down-regulated by Dex. However, the protein expression levels of PCK1 & PCK2 were significantly increased by Dex, suggesting a post-transcriptional regulation. In dorsal longissimus, the mRNA expression of genes associated with gluconeogenesis and the insulin signaling pathway were generally up-regulated by Dex, but the mRNA expression of two markers of muscle atrophy, namely F-box protein 32 (FBXO32/Atrogin1) and muscle RING-finger protein 1 (MuRF1), was not altered by Dex.ConclusionsTaken together, these results indicate that chronic administration of a low dosage of Dex induces hyperglycemia mainly through gluconeogenesis activation in the goat liver.

Enhancement of porcine intramuscular fat content by overexpression of the cytosolic form of phosphoenolpyruvate carboxykinase in skeletal muscle

Intramuscular fat (IMF) content has been generally recognized as a desirable trait in pork meat because of its positive effect on eating quality. An effective approach to enhance IMF content in pork is the generation of transgenic pigs. In this study, we used somatic cell nuclear transfer (SCNT) to generate cloned pigs exhibiting ectopic expression of phosphoenolpyruvate carboxykinase (PEPCK-C) driven by an α-skeletal-actin gene promoter, which was specifically expressed in skeletal muscle. Using qRT-PCR and Western blot analysis, we demonstrated that PEPCK-C was functionally expressed and had a significant effect on total fatty acid content in the skeletal muscle of the transgenic pigs, while the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio showed no difference between transgenic and control pigs. Thus, genetically engineered PEPCK-Cmus pigs may be an effective solution for the production of IMF-enriched pork.

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn2+ ions for activity; Mg2+ ions reduce the Km for Mn2+ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (kcat=2.7s−1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.

Higher protein kinase C ζ in fatty rat liver and its effect on insulin actions in primary hepatocytes.

We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF) rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ), a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL) rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.

Impact of maternal overnutrition on gluconeogenic factors and methylation of the phosphoenolpyruvate carboxykinase promoter in the fetal and postnatal liver

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. 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. 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. 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.

Enhanced Energy Metabolism Contributes to the Extended Life Span of Calorie-restricted Caenorhabditis elegans

Caloric restriction (CR) markedly extends life span and improves the health of a broad number of species. Energy metabolism fundamentally contributes to the beneficial effects of CR, but the underlying mechanisms that are responsible for this effect remain enigmatic. A multidisciplinary approach that involves quantitative proteomics, immunochemistry, metabolic quantification, and life span analysis was used to determine how CR, which occurs in the Caenorhabditis elegans eat-2 mutants, modifies energy metabolism of the worm, and whether the observed modifications contribute to the CR-mediated physiological responses. A switch to fatty acid metabolism as an energy source and an enhanced rate of energy metabolism by eat-2 mutant nematodes were detected. Life span analyses validated the important role of these previously unknown alterations of energy metabolism in the CR-mediated longevity of nematodes. As observed in mice, the overexpression of the gene for the nematode analog of the cytosolic form of phosphoenolpyruvate carboxykinase caused a marked extension of the life span in C. elegans, presumably by enhancing energy metabolism via an altered rate of cataplerosis of tricarboxylic acid cycle anions. We conclude that an increase, not a decrease in fuel consumption, via an accelerated oxidation of fuels in the TCA cycle is involved in life span regulation; this mechanism may be conserved across phylogeny.

Effects of Enhanced Physical Activity on Hematopoietic Stem Cell Aging in PEPCK-Cmus Transgenic Mice

Abstract 1297Hematopoietic stem cells (HSCs) are self-renewing multipotent precursor cells that give rise to all blood cell lineages. As HSCs age, expansion of the pool of HSCs and abnormal production of blood cells occur, resulting in a loss of immune function and an increased incidence of myeloid leukemia. Exercise has been associated with an extended lifespan, yet the connection between HSC fitness and enhanced physical exercise is currently unclear. Transgenic mice that over-express the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) in their skeletal muscle (PEPCK-Cmus mice) have remarkable endurance, can run five to six kilometers at a speed of 20 meters per minute without stopping, and have a markedly longer lifespan. We thus determined the effects of enhanced physical activity on HSC aging by measuring the pool size and functional ability of HSCs in both aged PEPCK-Cmus transgenic and control mice. Cell surface immunostaining and flow cytometric analysis were performed to determine the total quantity of HSCs within the bone marrow cell population of both groups of animals. Preliminary studies showed that age-associated expansion of the HSC pool was absent in PEPCK-Cmus mice. However, the efficiency of HSCs to produce B lymphoid progenitors in PEPCK-Cmus mice was surprisingly 11-fold higher than that in control mice while the age-associated skewness of HSC differentiation toward the myeloid lineage was diminished in PEPCK-Cmus mice. Furthermore, reactive oxygen species (ROS), known to be associated with aging when at high levels, were lower in the HSCs of PEPCK-Cmus mice than was noted in the HSCs of control animals. Together, these preliminary data imply that the functional capacity of HSCs is maintained in old PEPCK-Cmus mice when compared to that of the HSCs in control animals of the same age. This suggests that enhanced physical activity may slow HSC aging, possibly attributed in part to decreased ROS levels. Disclosures:No relevant conflicts of interest to declare.

Metabolic Regulation by C1q/TNF-related Protein-13 (CTRP13): ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE AND SUPPRESSION OF FATTY ACID-INDUCED JNK SIGNALING

Members of the C1q/TNF family play important and diverse roles in the immune, endocrine, skeletal, vascular, and sensory systems. Here, we identify and characterize CTRP13, a new and extremely conserved member of the C1q/TNF family. CTRP13 is preferentially expressed by adipose tissue and the brain in mice and predominantly by adipose tissue in humans. Within mouse adipose tissue, CTRP13 is largely expressed by cells of the stromal vascular compartment. Due to sexually dimorphic expression patterns, female mice have higher transcript and circulating CTRP13 levels than males. CTRP13 transcript and circulating levels are elevated in obese male mice, suggesting a potential role in energy metabolism. The insulin-sensitizing drug rosiglitazone also increases the expression of CTRP13 in adipocytes, which correlates with the insulin-sensitizing action of CTRP13. In a heterologous expression system, CTRP13 is secreted as a disulfide-linked oligomeric protein. When co-expressed, CTRP13 forms heteromeric complexes with a closely related family member, CTRP10. This heteromeric association does not involve conserved N-terminal Cys residues. Functional studies using purified recombinant protein demonstrated that CTRP13 is an adipokine that promotes glucose uptake in adipocytes, myotubes, and hepatocytes via activation of the AMPK signaling pathway. CTRP13 also ameliorates lipid-induced insulin resistance in hepatocytes through suppression of the SAPK/JNK stress signaling that impairs the insulin signaling pathway. Further, CTRP13 reduces glucose output in hepatocytes by inhibiting the mRNA expression of gluconeogenic enzymes, glucose-6-phosphatase and the cytosolic form of phosphoenolpyruvate carboxykinase. These results provide the first functional characterization of CTRP13 and establish its importance in glucose homeostasis.

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

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

Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice

The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARgamma binding site in the promoter of Pck1 (PPARE(-/-)). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE(-/-) mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE(-/-) mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention.

Multi-scale characterization of the PEPCK-C mouse through 3D cryo-imaging.

We have developed, for the Case 3D Cryo-imaging system, a specialized, multiscale visualization scheme which provides color-rich volume rendering and multiplanar reformatting enabling one to visualize an entire mouse and zoom in to organ, tissue, and microscopic scales. With this system, we have anatomically characterized, in 3D, from whole animal to tissue level, a transgenic mouse and compared it with its control. The transgenic mouse overexpresses the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) in its skeletal muscle and is capable of greatly enhanced physical endurance and has a longer life-span and reproductive life as compared to control animals. We semiautomatically analyzed selected organs such as kidney, heart, adrenal gland, spleen, and ovaries and found comparatively enlarged heart, much less visceral, subcutaneous, and pericardial adipose tissue, and higher tibia-to-femur ratio in the transgenic animal. Microscopically, individual skeletal muscle fibers, fine mesenteric blood vessels, and intestinal villi, among others, were clearly seen.

Activation of SIRT1 by Resveratrol Represses Transcription of the Gene for the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) by Deacetylating Hepatic Nuclear Factor 4α

The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4alpha (-272/-252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (-248/-234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4alpha for binding to this site and also decreased HNF4alpha stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4alpha and AP-1, but not C/EBPbeta, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4alpha, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4alpha-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4alpha in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4alpha. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.

Glyceroneogenesis Is the Dominant Pathway for Triglyceride Glycerol Synthesis in Vivo in the Rat

Triglyceride synthesis in mammalian tissues requires glycerol 3-phosphate as the source of triglyceride glycerol. In this study the relative contribution of glyceroneogenesis and glycolysis to triglyceride glycerol synthesis was quantified in vivo in adipose tissue, skeletal muscle, and liver of the rat in response to a chow diet (controls), 48-h fast, and lipogenic (high sucrose) diet. The rate of glyceroneogenesis was quantified using the tritium ([(3)H(2)]O) labeling of body water, and the contribution of glucose, via glycolysis, was determined using a [U-(14)C]glucose tracer. In epididymal and mesenteric adipose tissue of control rats, glyceroneogenesis accounted for approximately 90% of triglyceride glycerol synthesis. Fasting for 48 h did not alter glyceroneogenesis in adipose tissue, whereas the contribution of glucose was negligible. In response to sucrose feeding, the synthesis of triglyceride glycerol via both glyceroneogenesis and glycolysis nearly doubled (versus controls); however, glyceroneogenesis remained quantitatively higher as compared with the contribution of glucose. Enhancement of triglyceride-fatty acid cycling by epinephrine infusion resulted in a higher rate of glyceroneogenesis in adipose tissue, as compared with controls, whereas the contribution of glucose via glycolysis was not measurable. Glyceroneogenesis provided the majority of triglyceride glycerol in the gastrocnemius and soleus. In the liver the fractional contribution of glyceroneogenesis remained constant (approximately 60%) under all conditions and was higher than that of glucose. Thus, glyceroneogenesis, in contrast to glucose, via glycolysis, is quantitatively the predominant source of triglyceride glycerol in adipose tissue, skeletal muscle, and liver of the rat during fasting and high sucrose feeding.

Overexpression of the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) in Skeletal Muscle Repatterns Energy Metabolism in the Mouse

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.

Identification and regulation of the promoter for PEPCK‐C in epithelial cells of mammary gland

The cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) has been proven to play an important role in both carbohydrate and lipid metabolism. PEPCK-C gene is expressed primarily in the liver, kidney, adipose tissue, and the mammary gland. Because the role of PEPCK-C in mammary gland is still unclear, we proposed to test the role of PEPCK-C gene in the mammary gland. The mammary gland is composed of two cell types, adipocytes and epithelial cells. We hypothesize that PEPCK-C is required for glyceroneogenesis. By the deletion analysis of PEPCK-C promoter, we found the region from −490bp to −260bp is required for the expression of PEPCK-C gene in NMuMG cells, a mammary epithelial cell line. We are investigating the regulation of PEPCK-C in the NMuMG cells.

Phosphoenolpyruvate carboxykinase and the critical role of cataplerosis in the control of hepatic metabolism

BackgroundThe metabolic function of PEPCK-C is not fully understood; deletion of the gene for the enzyme in mice provides an opportunity to fully assess its function.MethodsThe gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C) was deleted in mice by homologous recombination (PEPCK-C-/- mice) and the metabolic consequences assessed.ResultsPEPCK-C-/- mice became severely hypoglycemic by day two after birth and then died with profound hypoglycemia (12 mg/dl). The mice had milk in their stomachs at day two after birth and the administration of glucose raised the concentration of blood glucose in the mice but did not result in an increased survival. PEPCK-C-/- mice have two to three times the hepatic triglyceride content as control littermates on the second day after birth. These mice also had an elevation of lactate (2.5 times), β-hydroxybutyrate (3 times) and triglyceride (50%) in their blood, as compared to control animals. On day two after birth, alanine, glycine, glutamine, glutamate, aspartate and asparagine were elevated in the blood of the PEPCK-C-/- mice and the blood urea nitrogen concentration was increased by 2-fold. The rate of oxidation of [2-14C]-acetate, and [5-14C]-glutamate to 14CO2 by liver slices from PEPCK-C-/- mice at two days of age was greatly reduced, as was the rate of fatty acid synthesis from acetate and glucose. As predicted by the lack of PEPCK-C, the concentration of malate in the livers of the PEPCK-C-/- mice was 10 times that of controls.ConclusionWe conclude that PEPCK-C is required not only for gluconeogenesis and glyceroneogenesis but also for cataplerosis (i.e. the removal of citric acid cycle anions) and that the failure of this process in the livers of PEPCK-C-/- mice results in a marked reduction in citric acid cycle flux and the shunting of hepatic lipid into triglyceride, resulting in a fatty liver.

Glucocorticoids Regulate Transcription of the Gene for Phosphoenolpyruvate Carboxykinase in the Liver via an Extended Glucocorticoid Regulatory Unit

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%.

SREBP-1c and Sp1 Interact to Regulate Transcription of the Gene for Phosphoenolpyruvate Carboxykinase (GTP) in the Liver

The sterol regulatory element-binding protein-1c (SREBP-1c), as well as SREBP-1a and SREBP-2, inhibit transcription of the gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). There are two SREBP regulatory elements (SREs) in the PEPCK-C gene promoter (-322 to -313 and -590 to -581). The SRE at -590 overlaps an Sp1 site on the opposite strand of the DNA. These SREs bound SREBP-1a and SREBP-1c with low affinity but the addition of purified upstream stimulatory activity enhanced the binding of SREBP-1 to both of these sites. Mutating these SREs increased both unstimulated (5-fold) and protein kinase A-stimulated transcription (8-27-fold) from the PEPCK-C gene promoter; this was lost when both SREs were mutated. The SRE at -590 differs by a single base pair from the SRE in the low density lipoprotein (LDL) receptor gene (T in the PEPCK-C gene promoter at -582, compared with an A in the SRE of the gene for the LDL receptor promoter). Introduction of the LDL receptor SRE into the PEPCK-C gene promoter increased SREBP-1c binding and caused a 10-fold enhancement of basal transcription from the promoter, rather than an inhibition as observed with the SRE in the PEPCK-C gene promoter. The T/A change does not alter the binding of Sp1 to its site on the opposite strand of the DNA. Sp1 bound to the promoter independently of SREBP-1c but competed with SREBP-1c for binding. Sp1 does not bind to the SRE at -322. Chromatin immunoprecipitation analysis, using rat hepatocytes, demonstrated that SREBP-1 and Sp1 were associated in vivo with putative regulatory regions corresponding to the SREs in the PEPCK-C gene promoter. We propose that insulin represses transcription of the gene for PEPCK-C by inducing SREBP-1c production in the liver, which interferes with the stimulatory effect of Sp1 at -590 of the PEPCK-C gene promoter.

Glyceroneogenesis revisited

Glyceroneogenesis is the synthesis of 3-glycerol phosphate by an abbreviated version of gluconeogenesis. The research that led to the discovery of glyceroneogenesis in white adipose tissue is presented. This pathway is active during fasting in white and brown adipose tissue and in the liver as part of the triglyceride/fatty acid cycle. Glyceroneogenesis is critical for the extensive recycling of free fatty acid (FFA) back to triglyceride that occurs in mammals, including humans, after lipolysis, when up to 65% of the fatty acids are re-esterified back to triglyceride. The rate-limiting enzyme in this pathway is the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32) (PEPCK-C). Transcription of this gene is induced in adipose tissue and liver during fasting. Ablation of expression of the gene for PEPCK-C in white adipose tissue of mice results in lipodsytrophy, while overexpression of the gene for this enzyme in adipose tissue causes obesity. The critical role of glyceroneogenesis in diabetes was suggested by experiments in which the gene for PEPCK-C is induced in white adipose tissue by rosiglitazone, a drug used to control diabetes in humans. This was accompanied by a marked decrease in FFA release from adipose tissue due to an induction in glyceroneogenesis in the tissue. Since the chronic release of FFA by adipose tissue is a critical factor in the development Type 2 diabetes, it is likely that rosiglitazone acts in part by stimulating transcription of the gene for PEPCK-C, thereby increasing rate of glyceroneogenesis and lowering the rate of FFA release from adipose tissue.

The transcriptional regulation of phosphoenolpyruvate carboxykinase gene in the kidney requires the HNF-1 binding site of the gene

The transcription of the cytosolic form of phosphoenolpyruvate carboxykinase ( PEPCK-C) gene is differentially regulated in each of the several PEPCK-C-expressing tissues. In the kidney, it is regulated by glucocorticoids and acidosis. Previously, we reported that in LLC-PK1 and derived kidney cell lines, mutation of the hepatic nuclear factor 1 ( HNF-1) binding site in PEPCK-C gene promoter markedly reduced both the basal activity of the gene promoter and its response to acidic pH. Using the same kidney cell line, we now report that nuclear receptors robustly stimulate transcription from the PEPCK-C gene promoter. This stimulation is markedly reduced by mutation of the accessory factor 1 ( AF1) site in the glucocorticoid responsive unit ( GRU) residing within the glucocorticoid-responsive domain. The stimulation is likewise reduced by mutation of the HNF-1 site, residing outside the nuclear receptor-responsive domain of the PEPCK-C gene promoter. There is no binding similarity between HNF-1 and AF1 binding sites, as is evident from gel shift assays showing a lack of competition of either site for the binding of renal nuclear proteins to the other. We further assessed that the regulation of PEPCK-C gene transcription by acidosis is not mediated by nuclear receptors. This became evident from studies of transgenic mice harboring a rat PEPCK-C transgene driven by truncated 5′ flanking region of the gene, which contains the HNF-1 site but lacks the glucocorticoid responsive domain. The full transcriptional response of this transgene to acidosis establishes that the truncated 5′ flanking region (362 bp) of the PEPCK-C gene contains the information required for the acidosis-mediated regulation independent of the glucocorticoid domain. Taking together the previous and present results, it appears that acidosis and nuclear receptors regulate the renal transcription of the PEPCK-C gene via two independent domains in the 5′ flanking region of the gene. These two modulations, as well as the basal activity of the gene, require intact HNF-1 binding site in the gene promoter.