Sex-specific regionality of liver metabolism during starvation; with special reference to the heterogeneity of the lobular periphery

Staining procedures for glucose-6-phosphatase and 3-hydroxybutyrate dehydrogenase activity and for glycogen were used to investigate adaptive changes in the regionality of hepatic gluconeogenesis and ketogenesis in fasting male and female rats. A reciprocal distribution of gluconeogenic and ketogenic capacities was found in both sexes, but male and female animals were different with respect to: a) the time necessary for full induction of glucose-6-phosphatase activity (24 h in females, 48 h in males); b) the overall activity of 3-hydroxybutyrate dehydrogenase; and c) glycogen content. The activity of the latter enzyme and the glycogen content did increase with time of starvation, but at all times, were higher in males, than in females. Results, thus, indicate that the extent to which ketone bodies replace glucose as major fuel for the brain is larger in males than in females. This may explain the delayed induction of glucose-6-phosphatase activity and the higher glycogen content in the male during starvation. Distributions of enzyme activities and of glycogen, furthermore, revealed the heterogeneity of the lobular periphery, i.e. functional differences among sinusoids dependent upon whether they originate from the portal tract or the vascular septum, and thus confirm the lobular concept proposed by Matsumoto et al. (1979).     

Activity of gluconeogenetic enzymes of rat kidney cortex during acute hypoxia]

The activities of gluconeogenic enzymes of the rat kidney cortex was studied after exposure to lowered atmospheric pressure (200 mm Hg) for 3 hours. The hypoxic stress was found to cause an increase in the activities of phosphoenolpyruvate carboxykinase and alanine aminotransferase, but failed to affect significantly the activities of fructose-1,6-diphosphatase, glucose-6-phosphatase, and aspartate aminotranspherase. The ratio of glucose-6-phosphatase/hexokinase activities was increased under these conditions.     

Amiloride activation of hepatic microsomal glucose-6-phosphatase; activation of T1?

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) in vitro by amiloride has been investigated in both intact and fully disrupted microsomes. The major effect of amiloride is a 4.5-fold reduction in the Km of glucose-6-phosphatase activity in intact diabetic rat liver microsomes. Amiloride also decreased the Km of glucose-6-phosphatase activity in intact liver microsomes isolated from starved rats 2.5-fold. Kinetic calculations, direct enzyme assays and direct transport assays all demonstrated that the site of amiloride action was T1, the hepatic microsomal glucose 6-phosphate transport protein. This is, to our knowledge, the first report of an activation of any of the proteins of the multimeric hepatic microsomal glucose-6-phosphatase complex.     

Effect of normal and waxy maize starch on growth, food utilization and hepatic glucose metabolism in European sea bass (Dicentrarchus labrax) juveniles

We determined the effect of dietary starch on growth performance and feed utilization in European sea bass juveniles. Data on the dietary regulation of key hepatic enzymes of the glycolytic, gluconeogenic, lipogenic and amino acid metabolic pathways (hexokinase, HK; glucokinase, GK; pyruvate kinase, PK; fructose-1,6-bisphosphatase, FBPase; glucose-6-phosphatase, G6Pase; glucose-6-phosphate dehydrogenase, G6PD; alanine aminotransferase, ALAT; aspartate aminotransferase, ASAT and glutamate dehydrogenase, GDH) were also measured. Five isonitrogenous (48% crude protein) and isolipidic (14% crude lipids) diets were formulated to contain 10% normal starch (diet NS10), 10% waxy starch (diet WS10), 20% normal starch (diet NS20), 20% waxy starch (diet WS20) or no starch (control diet). Another diet was formulated with no carbohydrate, and contained 68% crude protein and 14% crude lipids (diet HP). Each experimental diet was fed to triplicate groups of 30 fish (initial weight: 23.3 g) on an equivalent feeding scheme for 12 weeks. The best growth performance and feed efficiency were achieved with fish fed the HP diet. Neither the level nor the nature of starch had measurable effects on growth performance of sea bass juveniles. Digestibility of starch was higher with waxy starch and decreased with increasing levels of starch in the diet. Whole-body composition and plasma metabolites, mainly glycemia, were not affected by the level and nature of the dietary starch. Data on enzyme activities suggest that dietary carbohydrates significantly improve protein utilization associated with increased glycolytic enzyme activities (GK and PK), as well as decreased gluconeogenic (FBPase) and amino acid catabolic (GDH) enzyme activities. The nature of dietary carbohydrates tested had little influence on performance criteria.     

Hormonal and lipogenic and gluconeogenic enzymatic responses in LA/N-corpulent rats

Genetically obese normotensive rats, LA/N-corpulent (cp), were fed ad libitum diets containing either 54% sucrose or cooked corn starch for 12 weeks. Twenty-four rats were used for the study; half were corpulent (cp/cp) and half were lean (cp/+ or +/+). Fasting levels of plasma insulin, glucose, corticosterone, glucagon and growth hormone, and activities of liver and epididymal fat pad glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), and liver and kidney glucose-6-phosphatase (G6Pase), fructose 1,6-diphosphatase (FDPase), and phosphoenolpyruvate carboxykinase (PEPCK) were measured. A significant phenotype effect was observed in insulin, corticosterone, growth hormone, and liver G6PD, ME, FDPase, and kidney PEPCK, G6Pase, FDPase, and epididymal fat pad G6PD and ME (corpulent greater than lean), and glucagon (lean greater than corpulent). Diet effect (sucrose greater than starch) was significant for plasma glucose, liver ME, and kidney G6Pase. Although not significant at the P less than 0.05 level, insulin, corticosterone, liver G6PD and FDPase and kidney FDPase tended to be higher in sucrose-fed rats. This study suggests that the corpulent rat is more lipogenic and gluconeogenic than the lean, and that the hormones responsible are effective in keeping both the lipogenic and gluconeogenic enzyme activity elevated.     

Sex-specific regionality of liver metabolism during starvation; with special reference to the heterogeneity of the lobular periphery

Summary Staining procedures for glucose-6-phosphatase and 3-hydroxybutyrate dehydrogenase activity and for glycogen were used to investigate adaptive changes in the regionality of hepatic gluconeogenesis and ketogenesis in fasting male and female rats. A reciprocal distribution of gluconeogenic and ketogenic capacities was found in both sexes, but male and female animals were different with respect to: a) the time necessary for full induction of glucose-6-phosphatase activity (24 h in females, 48 h in males); b) the overall activity of 3-hydroxybutyrate dehydrogenase; and c) glycogen content. The activity of the latter enzyme and the glycogen content did increase with time of starvation, but at all times, were higher in males than in females. Results, thus, indicate that the extent to which ketone bodies replace glucose as major fucl for the brain is larger in males than in females. This may explain the delayed induction of glucose-6-phosphatase activity and the higher glycogen content in the male during starvation. Distributions of enzyme activities and of glycogen, furthermore, revealed the heterogeneity of the lobular periphery, i.e. functional differences among sinusoids dependent upon whether they originate from the portal tract or the vascular septum, and thus confirm the lobular concept proposed by Matsumoto et al. (1979).Parts of this study were carried out in the Anatomisches Institut, Universität Freiburg, Federal Republic of GermanySupported by grants from the Deutsche Forschungsgemeinschaft, the Bochringer-Ingelheim Fonds and Grant AM 32654 from the National Institutes of Health     

Effect of tumor necrosis factor on enzymes of gluconeogenesis in the rat.

The effect of human recombinant tumor necrosis factor (TNF)-alpha on enzymes of gluconeogenesis in the rat was investigated by determining the activity of glucose 6-phosphatase, fructose 1,6-diphosphatase (FDP), and phosphoenolpyruvate carboxykinase in the liver and kidney of fed and fasted rats. The activity of transaldolase in the pentose phosphate pathway was also measured. Starvation of rats for 24 hr resulted in a 1.6- to 3.1-fold increase in liver and kidney glucose 6-phosphatase and phosphoenolpyruvate carboxykinase (P less than or equal to 0.05), a decrease in liver and kidney FDP (P less than 0.002), and an increase in liver and kidney transaldolase (P = 0.0001). Injection of 50 and 100 micrograms/kg/day of TNF for 5 days resulted in a significant (P less than or equal to 0.03) decrease in kidney FDP only. Injection of 100 micrograms/kg/day of TNF for 5 days with a 24-hr fast on Day 5 resulted in a significant (P = 0.04) increase in liver transaldolase, and a significant decrease in kidney FDP and phosphoenolpyruvate carboxykinase. Comparison of the enzyme activities of rats injected with 100 micrograms/kg/day of TNF for 5 days with those of their pair-fed control partners revealed additionally a significant decrease in glucose 6-phosphatase in the liver (P less than 0.001). It is concluded that TNF administration in the rat has different effects on the enzymes of gluconeogenesis in the liver and kidney, and these effects differ from those seen in starved or tumor-bearing rats.     

The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Biochemical compartmentation of fish tissues. Distribution of gluconeogenic enzymes in the brain

1. Specific glucose-6-phosphatase and fructose-1,6-diphosphatase activity were found to be biochemically compartmentalized in four parts of the brain in nine nutritionally important fishes. 2. Glucose-6-phosphatase and fructose-1,6-diphosphatase activity were highest in the cerebrum and lowest in the cerebellum. 3. Piscivorous fishes had the highest gluconeogenic enzyme content, followed by catfishes and major carps. 4. After the liver and muscles, the various parts of the brain play an important role in carbohydrate metabolism. 5. A direct relationship between the stage of evolution and elevation of gluconeogenic enzyme levels was observed. 6. It is evident from the results and the discussion that evolution modifies the biochemical organization of fishes in general and of their brain in particular.     

Regulation of carbohydrate metabolism by indole-3-carbinol and its metabolite 3,3′-diindolylmethane in high-fat diet-induced C57BL/6J mice

Indole glucosinolates, present in cruciferous vegetables have been investigated for their putative pharmacological properties. The current study was designed to analyse whether the treatment of the indole glucosinolates—indole-3-carbinol (I3C) and its metabolite 3,3′-diindolylmethane (DIM) could alter the carbohydrate metabolism in high-fat diet (HFD)-induced C57BL/6J mice. The plasma glucose, insulin, haemoglobin (Hb), glycosylated haemoglobin (HbA1c), glycogen and the activities of glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were analysed in liver and kidney of the treated and HFD mice. Histopathological examination of liver and pancreases were also carried out. The HFD mice show increased glucose, insulin and HbA1c and decreased Hb and glycogen levels. The elevated activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase and subsequent decline in the activity of glucokinase and glucose-6-phosphate dehydrogenase were seen in HFD mice. Among treatment groups, the mice administered with I3C and DIM, DIM shows decreased glucose, insulin and HbA1c and increased Hb and glycogen content in liver when compared to I3C, which was comparable with the standard drug metformin. The similar result was also obtained in case of carbohydrate metabolism enzymes; treatment with DIM positively regulates carbohydrate metabolic enzymes by inducing the activity of glucokinase and glucose-6-phosphate dehydrogenase and suppressing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase when compared to I3C, which were also supported by our histopathological observations.     

A putative second messenger of insulin action regulates hepatic microsomal glucose-6-phosphatase

Physiological concentrations of insulin suppressed rat liver microsomal glucose-6-phosphatase activity in vitro. To attest a hypothesis that a putative second messenger of insulin action (insulin mediator) mediated this process, we isolated the low molecular factor from insulin-treated plasma membranes of rat liver, which was acid- and heat-stable substance of a peptide nature. The insulin mediator which was proved to activate the mitochondria pyruvate dehydrogenase suppressed microsomal glucose-6-phosphatase. The insulin mediator was linked to suppression of the gluconeogenic enzyme through the control of non-specific phosphohydroxylase.     

On the mechanism of a radiation-induced change in enzymic differentiation during development

In experiments on glucose-6-phosphatase and tyrosine aminotransferase it was shown that radiation induces changes in enzymic differentiation in perinatal rat liver. A study was made of the probable reasons for the observed changes. It was shown that the macromolecular system of the protein enzyme synthesis was not damaged by the radiation doses used. The observed decrease in glucose-6-phosphatase activity during late embryogenesis, after pre-irradiation at early organogenesis, is eliminated by administration of exogenous thyroxine. A radiation-induced rise in the tyrosine aminotransferase activity during the perinatal period correlated with the cyclic AMP system status. It is proposed that modification of enzymic differentiation after irradiation results from the change in the amount of inductors.     

The flavonoid silibinin decreases glucose-6-phosphate hydrolysis in perfused rat hepatocytes by an inhibitory effect on glucose-6-phosphatase.

BACKGROUND/AIMS: The flavonoid silibinin has been reported to be beneficial in several hepatic disorders. Recent evidence also suggests that silibinin could be beneficial in the treatment of type 2 diabetes, owing to its anti-hyperglycemic properties. However, the mechanism(s) underlying these metabolic effects remains unknown. METHODS: The effects of silibinin on liver gluconeogenesis were studied by titrating hepatocytes from starved rats with sub-saturating concentrations of various exogenous substrates in a perifusion system. Hepatocytes from fed rats were also used to investigate glycogenolysis from endogenous glycogen. The effect of silibinin on glucose-6-phosphatase kinetics was determined in intact and permeabilized rat liver microsomes. RESULTS: Silibinin induced a dose-dependent inhibition of gluconeogenesis associated with a potent decrease in glucose-6-phosphate hydrolysis. This effect was demonstrated whatever the gluconeogenic substrates used, i.e. dihydroxyacetone, lactate/pyruvate, glycerol and fructose. In addition, silibinin decreased the glucagon-induced stimulation of both gluconeogenesis and glycogenolysis, this being associated with a reduction of glucose-6-phosphate hydrolysis. Silibinin inhibits glucose-6-phosphatase in rat liver microsomes in a concentration-dependent manner that could explain the decrease in glucose-6-phosphate hydrolysis seen in intact cells. CONCLUSION: The inhibitory effect of silibinin on both hepatic glucose-6-phosphatase and gluconeogenesis suggests that its use may be interesting in treatment of type 2 diabetes.     

Effect of dibutyryl cyclic AMP on glucose-6-phosphatase activity in human fetal liver explants

Glucose-6-phosphatase (EC 3.1.3.9) activity in human fetal liver remains constant at 8–28 nmoles/min per mg protein from the 8th week of gestation to at least week 28 and this value is approximately 25–35% of that found in the adult. This enzyme activity was well maintained for 2–3 days in organ culture of fetal liver explants. Incubation with dibutyryl cyclic AMP (0.1 mM) and theophylline (0.5 mM) increased glucose-6-phosphatase activity 4–8-fold within 24 h. Theophylline alone was ineffective, but markedly potentiated the effects of dibutyryl cyclic AMP. This increase in enzyme activity was completely abolished by simultaneous incubation with cycloheximide or actinomycin D. Insulin clearly decreased glucose-6-phosphatase activity in control tissues after 24 h incubation and tended to diminish the elevated glucose-6-phosphatase activity which resulted from pre-incubation with dibutyryl cyclic AMP.The smallest specimen obtained (36 mm crown-rump length = 6 weeks gestation) was capable of elevating glucose-6-phosphatase activity more than 3-fold in response to dibutyryl cyclic AMP incubation, suggesting that the human fetal liver has the competence to respond to hormonal agents at a very early stage of development.     

Relationship between microsomal membrane permeability and the inhibition of hepatic glucose-6-phosphatase by pyridoxal phosphate.

Arion et al; (Arion, W. J., Wallin, B. K., Lange A. J., and Ballas, L. M. (1975) Mol. Cell. Biochem. 6, 75-83) propsed a model for glucose-6-phosphatase in which the substrate was transported across the microsomal membrane by a carrier before hydrolysis on the cisternal side. Evidence to support this model has been obtained by studying the inhibition of the enzyme by pyridoxal-P. Pyridoxal-P was a linear noncompetitive inhibitor of glucose-6-phosphatase (EC 3.1.3.9) in freshly isolated ("intact") microsomes from rat liver. Pyridoxol-P was a much less effective inhibitor and no inhibition was observed with pyridoxamine-P. When microsomes were subjected to nitrogen cavitation, treatment with solium deoxycholate, or glutaraldehyde fixation, the Km of glucose-6-phosphatase for glucose-6 P decreased from approximately 6 mM to approximately 2.5 mM; the corresponding change in the Vmax ranged from-10% to +40%. The same procedures decreased the inhibition of glucose-6-phosphatase by pyridoxal-P several-fold. No inhibition by pyridoxal-P was observed in a preparation of glucose-6-phosphatase purified approximately 20 fold (on the basis of Vmax) from micoromes. A nondialyzable inhibitor was apparently formed when intact microsomes were reacted with pyridoxal-P and NaBH4; this inhibition was also reversed by procedures which changed the kinetic properties of glucose-6-phosphatase.     

Biochemical compartmentation of gluconeogenic enzymes in fish tissues

Compartmentation of liver, kidney muscle and gill tissues in relation to glucose-6-phosphatase and fructose 1,6-diphosphatase was examined in the fishes Labeo rohita, Clarias batrachus and Channa punctatus. The anterior region of the right and left lobes of the liver contained the maximum of fructose 1,6-diphosphatase and glucose-6-phosphatase, while the minimum was in the right and left lobes of gill tissue. Herbivore fish had the highest gluconeogenic enzyme content followed by carnivore and piscivore species. The observed enzymatic variations in the three fish species were discussed.     

Development of gluconeogenic enzymes in fetal sheep liver and kidney

In the sheep, the system of enzymes necessary for conversion of nonhexose substrates to glucose becomes active during late fetal life. Glucose-6-phosphatase and fructose-1,6-diphosphatase, two of the four key gluconeogenic enzymes, appear in significant amounts between 100 and 120 days gestation. Phosphoenolpyruvate carboxykinase activity is comparable to mature animals as early as 45 days gestation. Two aminotransferases, necessary to allow amino acid access to the gluconeogenic pathway, likewise have substantial activity as early as 45 days gestation. Hence, the surge of glucose-6-phosphatase and fructose-1,6-diphosphatase at 100-120 days gestation makes possible the endogenous production of new glucose by fetal sheep at a time when the amount of glucose transferred from the maternal circulation is less than the total aerobic substrate utilized by the fetus. Both renal cortex and liver have similar developmental patterns for the gluconeogenic enzymes, although renal cortex generally shows greater activity than liver. This observation holds true for tissue from both fetal and mature animals.