Liver glyconeogenesis: a pathway to cope with postprandial amino acid excess in high-protein fed rats?

This paper provides molecular evidence for a liver glyconeogenic pathway, that is, a concomitant activation of hepatic gluconeogenesis and glycogenesis, which could participate in the mechanisms that cope with amino acid excess in high-protein (HP) fed rats. This evidence is based on the concomitant upregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression, downregulation of glucose 6-phosphatase catalytic subunit (G6PC1) gene expression, an absence of glucose release from isolated hepatocytes and restored hepatic glycogen stores in the fed state in HP fed rats. These effects are mainly due to the ability of high physiological concentrations of portal blood amino acids to counteract glucagon-induced liver G6PC1 but not PEPCK gene expression. These results agree with the idea that the metabolic pathway involved in glycogen synthesis is dependent upon the pattern of nutrient availability. This nonoxidative glyconeogenic disposal pathway of gluconeogenic substrates copes with amino excess and participates in adjusting both amino acid and glucose homeostasis. In addition, the pattern of PEPCK and G6PC1 gene expression provides evidence that neither the kidney nor the small intestine participated in gluconeogenic glucose production under our experimental conditions. Moreover, the main glucose-6-phosphatase (G6Pase) isoform expressed in the small intestine is the ubiquitous isoform of G6Pase (G6PC3) rather than the G6PC1 isoform expressed in gluconeogenic organs.     

Effects of low protein intake on extra-hepatic gluconeogenic enzyme expression and peripheral glucose phosphorylation in rainbow trout (Oncorhynchus mykiss)

The objective of the study described here was to analyze in rainbow trout (Oncorhynchus mykiss) the effects of low protein intake on peripheral glucose phosphorylation capacities and gluconeogenic enzymes in kidney and intestine. Fish were food-deprived for 14 days or kept under a low and a high protein intake regime using a pair feeding protocol in order to maintain constant carbohydrate and lipid intakes. We analyzed the effect of protein restriction on (i) hepatic, renal and intestinal fructose-1.6-bisphophatase (FBPase) and glucose-6-phosphatase (G6Pase) enzymes at the molecular and enzymatic levels and (ii) glucose phosphorylation activities (hexokinases) in the liver, peri-visceral adipose tissue, red muscle and white muscle. Irrespective of the nutritional status, we observed the same levels of hexokinase activities in all the tissues studied. Renal G6Pase and FBPase gene expression and activities were not modified among the groups. In contrast, there was increased intestinal FBPase gene expression in fish under a low protein intake and higher G6Pase activities in both groups of fed fish. This result differs from what is observed in rats and suggest a role of intestine in the regulation of postprandial gluconeogenesis in fed trout. In conclusion, our data did not demonstrate any specific effect of low dietary protein intake to either gluconeogenic capacities or glucose phosphorylation capacities in rainbow trout.     

The role of hepatic, renal and intestinal gluconeogenic enzymes in glucose homeostasis of juvenile rainbow trout

Rainbow trout is unable to utilize high levels of dietary carbohydrates and experiences hyperglycemia after consumption of carbohydrate-rich meals. Carbohydrates stimulate hepatic glycolytic activity, but gene expression of the rate-limiting gluconeogenic enzymes glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) remains high. Although there is significant mRNA expression and activity of gluconeogenic enzymes in trout intestine and kidney, the regulation of these enzymes by diet is not known. We tested the hypothesis that dietary carbohydrate modulates intestinal and renal G6Pase, FBPase and PEPCK. Fish were either fasted or fed isocaloric carbohydrate-free (CF) or high carbohydrate (HC) diets for 14 days. As expected, fish fed HC exhibited postprandial hyperglycemia and enhanced levels of hepatic glucokinase mRNA and activity. Dietary carbohydrates had no significant effect on the expression and activity of PEPCK, FBPase and G6Pase in all three organs. In contrast, fasting enhanced the activity, but not the mRNA expression of both hepatic and intestinal PEPCK, as well as intestinal FBPase. Therefore, the activity of rate-limiting gluconeogenic enzymes in trout can be modified by fasting, but not by the carbohydrate content of the diet, potentially causing hyperglycemia when fed high levels of dietary carbohydrates. In this species consuming low carbohydrate diets at infrequent intervals in the wild, fasting-induced increases in hepatic and intestinal gluconeogenic enzyme activities may be a key adaptation to prevent perturbations in blood glucose during food deprivation. Presented in part at Experimental Biology, April 2006, San Francisco, CA [Kirchner S., Panserat S., Kaushik S. and Ferraris R. FASEB-IUPS-2006 A667.6].     

Transcriptional regulation of the glucose-6-phosphatase gene by cAMP/vasoactive intestinal peptide in the intestine. Role of HNF4alpha, CREM, HNF1alpha, and C/EBPalpha

Gluconeogenesis is induced in both the liver and intestine by increased cAMP levels. However, hepatic and intestinal glucose production can have opposite effects on glucose homeostasis. Glucose release into the portal vein by the intestine increases glucose uptake and reduces food intake. In contrast, glucose production by the liver contributes to hyperglycemia in type II diabetes. Glucose-6-phosphatase (Glc6Pase) is the key enzyme of gluconeogenesis in both the liver and intestine. Here we specify the cAMP/protein kinase A regulation of the Glc6Pase gene in the intestine compared with the liver. Similarly to the liver, the molecular mechanism of cAMP/protein kinase A regulation involves cAMP-response element-binding protein, HNF4alpha, CAAT/enhancer-binding protein, and HNF1. In contrast to the situation in the liver, we find that different isoforms of CAAT/enhancer-binding protein and HNF1 contribute to the specific regulation of the Glc6Pase gene in the intestine. Moreover, we show that cAMP-response element binding modulator specifically contributes to the regulation of the Glc6Pase gene in the intestine but not in the liver. These results allow us to identify intestine-specific regulators of the Glc6Pase gene and to improve the understanding of the differences in the regulation of gluconeogenesis in the intestine compared with the liver.     

Peroxisome proliferator-activated receptor {alpha} is responsible for the up-regulation of hepatic glucose-6-phosphatase gene expression in fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.     

O-GlcNAc modification of FoxO1 increases its transcriptional activity: A role in the glucotoxicity phenomenon?

O-GlcNAc glycosylations on serines or threonines are reversible post-translational modifications that control the localisation, the activity or the stability of cytosolic and nuclear proteins. These dynamic modifications are tightly dependent on the availability of glucose and on its flux through the hexosamine biosynthetic pathway. We recently showed that treatments that increase protein O-GlcNAc glycosylation (high-glucose concentrations, glucosamine) or inhibit their deglycosylation (PUGNAc), induced O-GlcNAc modification of FoxO1 in HEK293 cells. O-GlcNAc glycosylation of FoxO1 resulted in an increased of its activity towards a glucose 6-phosphatase promoter-luciferase reporter gene (G6Pase-luc). This effect appeared to be independent of FoxO1 sub-cellular re-localisation, since it was also observed with the constitutively nuclear FoxO1-AAA mutant. In liver-derived HepG2 cells, glucosamine and PUGNAc increased the expression of G6Pase mRNA, and synergistic effects were observed when both agents were present together. In addition, the expression of PGC1 alpha gene, which is known to be under the control of FoxO1, was also increased by glucosamine and PUGNAc. In HepG2 cells stably expressing the G6Pase-luc reporter gene, glucosamine and PUGNAc also increased the activity of the G6Pase promoter. The stimulation of the G6Pase reporter gene by these agents was abolished by two different FoxO1 siRNAs, thereby demonstrating the involvement of endogenous FoxO1 in the observed effects. Since G6Pase plays a key role in glucose production by the liver, increased in its expression through FoxO1 O-GlcNAc modification may be of considerable importance in the context of glucotoxicity associated with chronic hyperglycaemia. Moreover, since FoxO1 also plays important roles in several aspects of cell biology, including cell proliferation, survival and apoptosis, the regulation of FoxO1 activity by O-GlcNAc modification may have implications for other crucial biological processes.     

Developmental pattern of glucose-6-phosphatase activity in the small intestine of the mouse fetus.

The distribution of glucose-6-phosphatase (G6Pase) activity in the epithelium of the small intestine in mouse embryos (the last 4 days of gestation) was studied by electron microscope cytochemistry and by enzymatic assays. At 16 days, the lead phosphate deposited by the cytochemical reaction is localized on the rough endoplasmic reticulum (RER) and nuclear envelope of very few cells in the duodenum and jejunum. Positive cells are more frequently seen in the upper part of the developing villi. At 17 days of gestation, a tremendous burst in RER differentiation is noticed in all parts of the small intestine and concomitantly glycogen disappears. At 18 days of gestation all the principal cells of the intestinal mucosa show a well differentiated positive RER and the enzyme is also present in the smooth endoplasmic reticulum. Biochemically, G6Pase activity is detected in the proximal 2 thirds of the small intestine at 17 days of gestation and appears at 18 days in the last third. Afterwards the activity increases up until birth. These results suggest (1) that the endoplasmic reticulum differentiates very late in the intestinal mucosa of mouse embryos (2) that the differentiation with respect to G6Pase is asynchronous between the enterocytes, (3) that for a given cell all the cisternae of RER are involved in G6Pase synthesis at the same moment and (4) that the enterocytes of the duodenum differentiate sooner and faster that those of the jejunum and ileum.     

Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from gilthead sea bream (Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.