Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae.
(9/1508)
External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms. (+info)
Effect of a selective rise in hepatic artery insulin on hepatic glucose production in the conscious dog.
(10/1508)
In the present study we compared the hepatic effects of a selective increase in hepatic sinusoidal insulin brought about by insulin infusion into the hepatic artery with those resulting from insulin infusion into the portal vein. A pancreatic clamp was used to control the endocrine pancreas in conscious overnight-fasted dogs. In the control period, insulin was infused via peripheral vein and the portal vein. After the 40-min basal period, there was a 180-min test period during which the peripheral insulin infusion was stopped and an additional 1.2 pmol. kg-1. min-1 of insulin was infused into the hepatic artery (HART, n = 5) or the portal vein (PORT, n = 5, data published previously). In the HART group, the calculated hepatic sinusoidal insulin level increased from 99 +/- 20 (basal) to 165 +/- 21 pmol/l (last 30 min). The calculated hepatic artery insulin concentration rose from 50 +/- 8 (basal) to 289 +/- 19 pmol/l (last 30 min). However, the overall arterial (50 +/- 8 pmol/l) and portal vein insulin levels (118 +/- 24 pmol/l) did not change over the course of the experiment. In the PORT group, the calculated hepatic sinusoidal insulin level increased from 94 +/- 30 (basal) to 156 +/- 33 pmol/l (last 30 min). The portal insulin rose from 108 +/- 42 (basal) to 192 +/- 42 pmol/l (last 30 min), whereas the overall arterial insulin (54 +/- 6 pmol/l) was unaltered during the study. In both groups hepatic sinusoidal glucagon levels remained unchanged, and euglycemia was maintained by peripheral glucose infusion. In the HART group, net hepatic glucose output (NHGO) was suppressed from 9.6 +/- 2.1 micromol. kg-1. min-1 (basal) to 4.6 +/- 1.0 micromol. kg-1. min-1 (15 min) and eventually fell to 3.5 +/- 0.8 micromol. kg-1. min-1 (last 30 min, P < 0.05). In the PORT group, NHGO dropped quickly (P < 0.05) from 10.0 +/- 0.9 (basal) to 7.8 +/- 1.6 (15 min) and eventually reached 3.1 +/- 1.1 micromol. kg-1. min-1 (last 30 min). Thus NHGO decreases in response to a selective increase in hepatic sinusoidal insulin, regardless of whether it comes about because of hyperinsulinemia in the hepatic artery or portal vein. (+info)
Postnatal profiles of glycogenolysis and gluconeogenesis are modified in rat pups by maternal dietary glucose restriction.
(11/1508)
Because glucose is an important metabolic fuel during perinatal development, the effect of restriction of maternal dietary glucose on the developmental profile of neonatal glucoregulatory pathways was investigated. Pregnant rats were fed isoenergetic diets (0, 12, 24 or 60% glucose) and offspring were killed at seven postpartum time periods: 0-2, 4-6, 12-16 and 24 h, and 3, 6 and 15 d. Failure of the most restricted pups (0%) to survive 24 h was explained by persistent hypoglycemia resulting from the following: 1) insufficient tissue glycogen reserves at birth; 2) lower liver glycogen mobilization; 3) delayed phosphorylase a induction; and 4) low phosphoenolpyruvate carboxykinase (PEPCK) gene expression, all of which occurred despite the lower insulin:glucagon ratio. Differences in liver glycogen stores, which had been exhausted in all dietary groups by 16 h, could not account for the high d 1 pup mortality in the moderately restricted (12 and 24% glucose) groups. However, a certain metabolic distress was suggested because these moderately restricted neonates had significantly higher liver PEPCK gene expression at 12-16 h but significantly lower plasma glucose at 24 h. The high d 3 mortality, confirmed by analysis of deviance, was not supported by significant differences in any of the measured glucoregulatory indices. We conclude that dietary glucose during pregnancy is required for neonatal survival; its restriction not only lowers tissue glycogen reserves, but can disrupt the normal gene expression of liver PEPCK and the neonatal profile of phosphorylase a activity. Importantly, these observations show that the development of neonatal glucoregulatory mechanisms is modified by the availability of maternal dietary glucose. (+info)
Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.
(12/1508)
Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo. (+info)
Molecular and functional characterization of the Rhodopseudomonas palustris no. 7 phosphoenolpyruvate carboxykinase gene.
(13/1508)
The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of the pckA initiation codon and an upstream sequence similar to sigma70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, the pckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK. (+info)
The transcription factor CCAAT/enhancer-binding protein beta regulates gluconeogenesis and phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes.
(14/1508)
CCAAT/enhancer-binding protein (C/EBP) beta and C/EBPalpha are members of the c/ebp gene family and are highly expressed in mammalian liver and adipose tissue. C/EBPalpha is essential for adipogenesis and neonatal gluconeogenesis, as shown by the C/EBPalpha knockout mouse. C/EBPbeta binds to several sequences of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter with high affinity, and C/EBPbeta protein is increased 200% in the livers of streptozotocin-diabetic mice, concurrent with increased PEPCK mRNA. To elucidate the role of C/EBPbeta in the control of gluconeogenesis during diabetes, we studied the levels of plasma metabolites and hormones related to energy metabolism during diabetes in adult mice heterozygous and homozygous for a null mutation of the gene for C/EBPbeta. We also examined the expression of PEPCK and glucose 6-phosphatase mRNAs and regulation of blood glucose, including the contribution of gluconeogenesis to blood glucose in c/ebpbeta-/- mice. C/EBPbeta was not essential to basal PEPCK mRNA levels. However, C/EBPbeta deletion affected streptozotocin-diabetic response by: (a) delaying hyperglycemia, (b) preventing the increase of plasma free fatty acids, (c) limiting the full induction of PEPCK and glucose 6-phosphatase genes, and (d) preventing the increase in gluconeogenesis rate. Gel supershifts of transcription factor C/EBPalpha, bound to CRE, P3I, and AF-2 sites of the PEPCK promoter, was not increased in diabetic c/ebpbeta-/- mouse liver nuclei, suggesting that C/EBPalpha does not substitute for C/EBPbeta in the diabetic response of liver gene transcription. These results link C/EBPbeta to the metabolic and gene regulatory responses to diabetes and implicate C/EBPbeta as an essential factor underlying glucocorticoid-dependent activation of PEPCK gene transcription in the intact animal. (+info)
Structure, function and regulation of pyruvate carboxylase.
(15/1508)
Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145-->Ala, Arg451-->Cys, Ala610-->Thr and Met743-->Thr. (+info)
Role of the human kidney in glucose counterregulation.
(16/1508)
Animal experiments indicate that the kidney may play an important role in glucose counterregulation. Because the human kidney normally takes up and releases glucose, and since patients with end-stage renal disease are prone to hypoglycemia, we examined whether the kidney is also involved in human glucose counterregulation. Accordingly, we compared renal glucose release (RGR) and uptake (RGU) during 4-h hyperinsulinemic-hypoglycemic (approximately 3.2 mmol/l, n = 9) and -euglycemic (approximately 5 mmol/l, n = 10) control clamp experiments in normal postabsorptive subjects. A combination of renal balance and isotopic ([3H]glucose, [14C]glutamine) techniques was used, which permitted hepatic glucose release (HGR) and glutamine gluconeogenesis to be calculated as the difference between systemic (overall) and renal values. In both experiments, infusion of insulin increased plasma insulin comparably (approximately 210 pmol/l). In euglycemic control experiments, RGR and HGR decreased more than 50% (both P<0.001) and RGU increased approximately 35% (P = 0.02). In hypoglycemic experiments, both HGR (P = 0.034) and RGR (P<0.001) increased to a comparable extent (1.69+/-0.47 and 1.67+/-0.15 pmol x kg-(-1) x min(-1), respectively, P = 0.96) above rates observed in control experiments; hepatic and renal glutamine gluconeogenesis increased by 0.19+/-0.06 (P<0.008) and 0.30+/-0.07 pmol x kg(-1) x min(-1) (P< 0.001), respectively. RGU decreased by 65% compared with control experiments (P<0.001), so that renal glucose balance changed from a net uptake of 80+/-19 micromol/min to a net release of 130+/-9 micromol/min, P< 0.001. These observations provide evidence that the kidney may play an important role in human glucose counterregulation. (+info)