Glucose Precursors Research Articles

Contribution of Several Amino Acids and Lactate to Gluconeogenesis in Hepatocytes Isolated from Rats Fed Various Diets

The contribution under various nutritional regimens of several amino acids and lactate to gluconeogenesis was estimated by measuring the glucose formation from 14C-labeled substrates. Isolated rat hepatocytes were incubated for 60 min in a Krebs-Ringer bicarbonate buffer pH 7.4 containing lactate, pyruvate and all the amino acids at concentrations similar to their physiological levels found in rat plasma, with one precursor labeled in each flask. In all conditions, lactate was the major glucose precursor, providing over 60% of the glucose formed. Glutamine and alanine were the major amino acid precursors of glucose, contributing 9.8% and 10.6% of the glucose formed, respectively, in hepatocytes isolated from starved rats. Serine, glycine and threonine also contributed to gluconeogenesis in the starved liver cells at 2.6, 2.1 and 3.8%, respectively, of the glucose formed. The rate of glucose formation from the isolated hepatocytes of the starved rats and those fed either high protein or high fat was higher than that from rats fed a nonpurified diet.

Energy metabolism in the digestive tract and liver of cattle: influence of physiological state and nutrition

Major functions of portal-drained viscera (PDV) and liver of cattle include absorption of digestion products and modification of the body's supply of intermediary metabolites. The disproportionately high metabolic rate of PDV and liver (7-13% of body tissues) is exemplified by their oxygen uptake (40-50% of whole body). Extensive metabolism of glucose, volatile fatty acids and amino acids by PDV modulates nutrient supply from the diet such that most responses to diet or physiological state are a function of level of diet intake. Similarly, blood flow through PDV is highly correlated with energy intake across a range of body weight, physiological state or diet composition. Most common dietary responses in metabolite uptake by PDV are changes in uptake of ammonia and volatile fatty acids, which emphasize the strong energy: nitrogen interrelationship in the rumen and subsequently the rest of the body. The liver (tissue in series with PDV) removes glucose precursors and ammonia from its blood supply as part of its functions in gluconeogenesis, ammonia detoxification and urea synthesis. The liver also alters amounts and proportions of amino acids supplied by PDV. Accountable percentages of metabolizable energy from net PDV supply include: organic acids, 41-59%; amino acids, 5-13%; and heat energy (from oxygen uptake), 11-22%.

Utilization of alanine for glucose formation in isolated rabbit kidney-cortex tubules

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis from low (up to 1 mM) 2-oxoglutarate concentrations and stimulates this process at higher (2.5–10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-[U- 14C]alanine to glucose from 8- to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low [U- 14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis.

Control of hepatic gluconeogenesis: Role of fatty acid oxidation

Octanoate has been found to activate the gluconeogenic pathway in perfused isolated rat liver. Whether a net increase in the production of glucose is observed is a function of the relative concentrations of the glucose precursor and the fatty acid. The kinetics of octanoate interaction with the gluconeogenic pathway are influenced by the rate changes induced by decreases in pyruvate concentration as a result of the increased NAD redox potential produced by the oxidation of fatty acid. Taking this into account, two distinct effects of octanoate were identified. The first is an increase in the V max even at the lowest (25 μ m) concentration of the fatty acid tested. The second is a progressive decrease in [pyruvate] 0.5 as a function of octanoate concentration. The latter occurs at low (<0.1 m m), presumably physiological, pyruvate concentrations, when its mitochondrial transport is limiting, indicating that this process must have been activated. The former is observable even at high (>0.5 m m), supraphysiological, concentrations of pyruvate, when its mitochondrial transport is not limiting, indicating that a distal step, presumably pyruvate carboxylation, is activated. The action of octanoate in increasing gluconeogenesis has been found not to be related to a decreased flux through pyruvate dehydrogenase, neither to changes in the NAD redox potential nor to its ability to increase energy production. Actually, the oxygen uptake induced by octanoate was largely accounted for by the production of ketone body and the latter process was found to be independent of variations in energy demand.

The finishing of zebu bulls on steam-hydrolysed sugar cane bagasse with different supplements

The fibrous residue remaining after juice extraction from the sugar cane stalk is commonly referred to as bagasse. It consists mainly of the ligno-cellulosic polymer matrix and small amounts of ash and water soluble solids which are mostly sucrose. In sugar industry mills, most of the bagasse is used as fuel, and although figures vary within each factory, energy efficient units can have 2 tonnes of surplus bagasse (dry basis) per 100 tonnes of fresh cane processed (Rolz et al). The bagasse can be supplemented with urea and legume protein in cattle diets. Research in Mauritius (Wong et al. 1974) showed that high pressure steam treatment (200°C) for 2-5 minutes raised rumen “in situ” digestibility from 30 to 60%. Subsequent work (Naidoo et al.1977) emphasised the critical role of supplements providing bypass nutrients (protein and glucose precursors) , in addition to urea-N and minerals for rumen microbes. In Colombia this technology is now being applied commercially for cattle fattening, using the foliage from the legume tree, Gliricidia sepium as the main source of bypass protein.

Splanchnic metabolism of amino acids in healthy subjects: Effect of 60 hours of fasting

Brief starvation is accompanied by decreased circulating levels of most amino acids, which has been attributed to an increased splanchnic uptake of amino acids, primarily alanine, for gluconeogenesis. However, quantitative data on splanchnic exchange of amino acids and gluconeogenic precursors is lacking. Consequently, arterial concentrations and splanchnic exchange of whole blood amino acids, ketone bodies, glucose, and gluconeogenic precursors were measured in 16 prolonged fasted (60 to 64 hours) and 15 overnight fasted (12 to 14 hours) healthy, nonobese subjects. After the 60-hour fast net splanchnic glucose production decreased by 41% to 0.31 ± 0.02 μmol/L ( P < .001), whereas the splanchnic uptake of gluconeogenic precursors increased and could account for the total glucose output. Net splanchnic uptake of taurine, threonine, serine, glycine, lysine, histidine, and arginine rose significantly in response to fasting ( P < .05 to .01) due to increased splanchnic fractional extraction. Although the splanchnic fractional extraction of alanine was augmented by 40% ( P < .001), net splanchnic uptake was not influenced by fasting. Total net splanchnic uptake of amino acids increased by 68%, from 231 ± 44 μmol/min in the postabsorptive state to 388 ± 63 μmol/min (mean ± SEM) ( P < .05) in the 60-hour fasted state. However, only one half of this rise was accounted for by gluconeogenic amino acids.

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [ 14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [ 14C]pyruvate and [ 14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the ‘cross-over’ plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD + ratio resulting in a decline of flux through lactate dehydrogenase.

Effects of thyroxine and growth hormone treatment of dairy cows on mammary uptake of glucose, oxygen and other milk fat precursors.

Four cows received thyroxine injections (T4; 20 mg/d) and three cows received growth hormone injections (GH, 44 mg/d) for 4 d during successive 16-d experimental periods. Milk fat, lactose output, mammary uptake of glucose, oxygen and milk fat precursors were determined with each treatment. Injection of T4 increased lactose yield by 25% and fat yield 42%. The injection of GH increased fat and lactose yields by 24%. Both GH and T4 increased mammary glucose uptake by 35% and 45%, respectively, while T4 administration was associated with an increase in plasma glucose concentration from 67 to 84%. Thyroxine, but not GH, increased the ratio of mammary glucose uptake to lactose output from 1.24 to 1.58. Blood plasma acetate concentration declined following GH and T4 treatment by 17%. Mammary acetate uptake increased in response to GH injection in two of three cows but did not change with T4 injection. The injection of GH had no effect on plasma propionate concentration or mammary uptake. Thyroxine reduced plasma propionate content and mammary uptake. Neither T4 nor GH changed plasma free fatty acid concentration or mammary uptake. Thyroxine had no effect on plasma triglyceride concentration or mammary uptake, whereas GH increased mammary triglyceride uptake to the end of the experimental period. Mammary oxygen uptake was increased by GH as milk production increased. Increased mammary oxygen uptake following T4 treatment was transient. Change in mammary metabolism with T4 treatment permitted increased milk output without change in mammary oxygen consumption. Such a change may involve increased mammary utilization of pre-formed long-chain fatty acid and increased metabolism of glucose via glycolysis.

Glycogen synthesis via the indirect gluconeogenic pathway in the periportal and via the direct glucose utilizing pathway in the perivenous zone of perfused rat liver.

The isolated liver from 24 h fasted rats was perfused in a non-recirculating manner in the ortho- and retrograde direction with erythrocyte-containing (20% v/v) media to provide adequate oxygenation of the liver. Glucose and/or gluconeogenic precursors were added as substrates. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the sole exogenous substrate glycogen was deposited in the perivenous area, with gluconeogenic precursors it was formed in the periportal zone during ortho- and retrograde flow. When glucose and gluconeogenic compounds were offered together, glycogen was deposited in both zones. The results corroborate the model of metabolic zonation predicting that periportal glycogen is synthesized indirectly from gluconeogenic precursors while perivenous glycogen is formed directly from glucose.

Effect of exercise on net hepatic uptake of lactate, pyruvate, alanine, and glycerol in sheep.

Gluconeogenesis increases during exercise, which is associated with elevated concentrations of lactate and glycerol in blood. This study was undertaken to determine if the exercise-induced increase in gluconeogenesis is due to increased hepatic extraction efficiency of glucose precursors. The net hepatic uptake and extraction ratios were determined for selected glucose precursors before and during exercise. The hepatic uptake of lactate and glycerol increased during exercise in fed and fasted animals, but extraction ratios of lactate and glycerol increased only in fed animals. Thus, the exercise-induced increase in gluconeogenesis is due to increased substrate supply and to hepatic extraction efficiency under certain circumstances, which is comparable to the situation in man.

Glycogen synthesis from pyruvate in the periportal and from glucose in the perivenous zone in perfused livers from fasted rats

The isolated liver of 24 h fasted rats was perfused in a non-recirculating manner in the orthograde or retrograde direction with media containing glucose and/or gluconeogenic precursors. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the only exogenous substrate glycogen was formed exclusively in the perivenous area during both orthograde and retrograde perfusion. With gluconeogenic precursors as the exogenous substrates glycogen was deposited in the periportal zone during orthograde perfusion and in the intermediate zone during retrograde perfusion. Supply of glucose and gluconeogenic substrates initiated glycogen synthesis only in the upstream region, i.e. in the periportal zone during orthograde and in the perivenous zone during retrograde perfusion. This localization of glycogen synthesis was probably due to an unavoidable, insufficient oxygen supply of the respective downstream area. In general, the results confirm the hypothesis that periportal and perivenous glycogen was synthesized from different substrates.

Hemidecortication selectively alters release of glutamate in perfusates collected from cerebral cortex of unrestrained rats.

The effect of hemidecortication on the in vivo release of amino acids was examined in different areas of the cerebral cortex of the freely-moving rat. After one side of the cortex was lesioned by aspiration, four guide tubes for push-pull perfusion were implanted chronically on the contralateral side so as to rest above the frontal, parietal, temporal and occipital areas of the cortex. After 10-14 days elapsed, each of these regions was perfused with an artificial cerebrospinal fluid (CSF) at a rate of 25.0 microliter/min. Two types of assays were undertaken to determine the release of either newly synthesized amino acids from [14C]glucose precursor or the actual endogenous content in samples of perfusate. The separation of the [14C]amino acids was performed by thin layer chromatography, whereas endogenous amino acids were separated by HPLC with electrochemical detection and quantitated in the range of 1.0-10.0 picomoles. When compared to the control group, samples collected in the hemidecorticate rat showed no significant differences in the new synthesis of glutamate, aspartate, glutamine, glycine, and GABA from the precursor. On the other hand, the analysis of the endogenous amino acid neurotransmitters revealed that the levels of glutamic acid and glutamine declined in samples obtained from the parietal and frontal cortex, respectively. These results implicate further the potential role of glutamic acid as a neurotransmitter of interhemispheric connections in the cerebral cortex.

Acute elevation of free fatty acid levels leads to hepatic insulin resistance in obese subjects

Raised levels of free fatty acids (FFA) compete with glucose for utilization by insulin-sensitive tissues, and, therefore, they may induce insulin resistance in the normal subject. The influence of experimental elevations in FFA levels on glucose metabolism in native insulin-resistant states is not known. We studied seven women with moderate obesity (63% above their ideal body weight) but normal glucose tolerance with the use of the insulin clamp technique with or without an infusion of Intralipid + heparin. Upon raising plasma insulin levels to ∼60 μU/mL while maintaining euglycemia, whole body glucose utilization ( 3H-3-glucose) rose similarly without (from 66 ± 7 to 113 ± 11 mg/min m 2, P < .02) or with (from 70 ± 7 to 137 ± 19 mg/min m 2, P < .02) concomitant lipid infusion. In contrast, endogenous glucose production was considerably (73%) suppressed (from 66 ± 7 to 15 ± 8 mg/min m 2, P < .001) during the clamp without lipid, but declined only marginally (from 70 ± 7 to 48 ± 7 mg/min m 2, NS) with lipid administration. The difference between the control and the lipid study was highly significant ( P < .02), and amounted to an average of 3.8 g of relative glucose overproduction during the second hour of the clamp. Blood levels of lactate rose by 34 ± 15% (.1 > P > .05) in the control study but only by 17 ± 10% (NS) during lipid infusion. Blood pyruvate concentrations fell in both sets of experiments (by ∼45% at the end of the study) with similar time courses. In contrast, blood alanine levels did not change from baseline during the control clamp (181 ± 18 v 185 ± 14 μmol/L) whereas they fell slightly (11 ± 3%) but consistently (175 ± 15 v 196 ± 14 μmol/L, P < .02) during lipid infusion. Furthermore, a positive correlation was found to exist in the pooled data between the decrease in blood alanine concentrations and the corresponding increase in endogenous glucose production ( r = .59, P < .02). We conclude that in the obese subject an increased supply of fatty fuels fails to inhibit insulin-mediated glucose disposal but induces hepatic insulin resistance. The greater availability of exogenous glucose precursors (glycerol) and the lower circulating levels of endogenous precursors (lactate and alanine) suggest that the observed increase in endogenous glucose production is sustained by hepatic gluconeogenesis.

Gluconeogenesis from acetone in diabetic rats.

Previous investigations have demonstrated that acetone is a true, if minor precursor of glucose in vivo. In diabetic rats 1.30% of the carbon atoms of circulating glucose arises from acetone, whereas 0.67% does in normal 3-day fasted animals. Calculated from these fractions and the turnover rate of glucose, 48 micrograms/kg. min acetone-carbon is converted to glucose-carbon in diabetic and 16 micrograms/kg. min in normal rats. In both groups of rats the labelling of plasma lactate was stronger than that of glucose. In view of these results we conclude that: the transfer of C-atoms from acetone to glucose increases in diabetes; acetone remains a minor source of glucose even in ketonemic diabetic rats.

The impact of sulfonylureas on hepatic glucose metabolism in type II diabetics.

Fasting hyperglycemia in subjects with NIDDM appears to be the final result of abnormalities in endogenous insulin secretion combined with diminished peripheral insulin action secondary to a combined receptor and postbinding defect in cellular insulin action and accelerated hepatic glucose production. Of these various abnormalities, the accelerated rate of hepatic glucose production appears to be of major importance in determining the overall severity of the hyperglycemia. The hyperglycemia, which is maintained by the accelerated rate of hepatic glucose release, appears to compensate for the decrease in insulin action at the level of the peripheral tissues, as well as the liver. Although this compensatory effect of hyperglycemia appears to match the decrease in insulin action in the peripheral tissues rather precisely, the compensation at the level of the liver does not totally restore normal homeostasis. These observations suggest that the liver and peripheral tissues are intimately linked via mechanisms that remain to be delineated. Recent observations of alterations in the activities of key rate-limiting enzymes in the pathway for oxidiative glucose metabolism suggest that an abundance of glucose precursors may be made in peripheral tissues, which then recycle to the liver to support and/or drive the accelerated rates of hepatic glucose production. Additional studies are clearly needed in these areas to further delineate these crucial issues. In a similar manner, the role of the liver in determining the success of various therapeutic modalities in these subjects appears to be of paramount importance. The data reviewed regarding the mechanisms of action of sulfonylurea agents indicate that the quality of glycemic control achieved in subjects treated with these agents is largely determined by the ability of the drug to lower the elevated rates of hepatic glucose production. This suggests that new compounds that are more effective in this regard will prove to be more efficacious.

Biosynthetic preparation of L-[13C]- and [15N]glutamate by Brevibacterium flavum

The biosynthesis of isotopically labeled L-glutamic acid by the microorganism Brevibacterium flavum was studied with a variety of carbon-13-enriched precursors. The purpose of this study was twofold: to develop techniques for the efficient preparation of labeled L-glutamate with a variety of useful labeling patterns which can be used for other metabolic studies, and to better understand the metabolic events leading to label scrambling in these strains. B. flavum, which is used commercially for the production of monosodium glutamate, has the capability of utilizing glucose or acetate as a sole carbon source, an important criterion from the standpoint of developing labeling strategies. Unfortunately, singly labeled glucose precursors lead to excessive isotopic dilution which reduces their usefulness. Studies with [3-13C]pyruvate indicate that this problem can in principle be overcome by using labeled three-carbon precursors; however, conditions could not be found which would lead to an acceptable yield of isotopically labeled L-glutamate. In contrast, [1-13C]- or [2-13C]acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, highly enriched L-glutamate. The preparation of L-[15N]glutamate from [15N]ammonium sulfate was carried out and is a very effective labeling strategy. Analysis of the isotopic distribution in labeled glutamate provides details about the metabolic pathways in these interesting organisms.

Glucose 6-phosphate plays a central role in the activation of glycogen synthase by glucose in hepatocytes

The state of activation of glycogen synthase enhanced by glucose, other sugars and gluconeogenic precursors shows a strong positive correlation with the intracellular concentrations of glucose 6-P when ATP concentrations remain constant. The concentrations of glucose 6-P achieved upon incubation of hepatocytes with glucose plus mannoheptulose, an inhibitor of glucokinase and hexokinase, were lower than those found when the incubation was carried out with glucose alone. Under these conditions, in keeping with the decrease in glucose 6-P, the activation of glycogen synthase by glucose was also impaired. On the other hand the inactivation of glycogen phosphorylase was not altered in the presence of mannoheptulose.

Identification of two forms of glycogen phosphorylase in Dictyostelium

It has been known for 20 years that during cellular differentiation of Dictyostelium discoideum, glycogen is degraded to provide the glucose precursors that are required for the synthesis of the end-products of development. Because this pathway provided a distinct developmentally regulated event, a number of laboratories have investigated the regulation of the first step in glycogen degradation, glycogen phosphorylase. Of particular interest was the possible regulation of this enzyme by cAMP. Cyclic AMP is know to act as a signal in this organism for both chemotaxis and cell differentiation. The phosphorylase activity was found to increase during development and, therefore, it has been used in many studies as a marker for late stage development. However, only one form of the phosphorylase was found, and therefore it was concluded that cAMP was not involved in regulation of this key step in the developmental pathway. Here we report the discovery of a second form of the enzyme. This form is completely dependent on AMP for activity and is found only in the undifferentiated stage. This second form contains several of the properties of the nonphosphorylated enzyme that occurs in systems that are regulated by cAMP. This result and the recent discovery of a cAMP-dependent protein kinase has rekindled the possibility that at least one of the effects of cAMP in this organism occurs via a cAMP-dependent cascade of phosphorylation; that is, the activation of glycogen phosphorylase and subsequent production of the precursors for the end-products of development.

Contrôle hormonal du métabolisme hépatique chez les ruminants

Insulin/glucagon control of hepatic metabolism, i.e. a endocrine-nervous system, is one of the general systems of integration in vertebrates. In this system, substrates coming from the digestive tract or from extrahepatic metabolism are important messenger molecules. Liver uptake of insulin and glucagon mainly accounts for high metabolic clearance rates of these hormones in both ruminants and non-ruminants. Glucagon infusion into ruminants results in an increase in the net hepatic uptake of glucose precursors and gluconeogenesis. Glucagon effects have also been demonstrated in isolated hepatocytes. Glucagon, through its effect on pyruvate carboxylase (EC 6.4.1.1.) may regulate gluconeogenesis. Insulin infusion induces hypoglycaemia. As a result, glucagon secretion increases and counterregulates insulin action. However, it has been shown that hepatic gluconeogenesis decreases during euglycaemic hyperinsulin clamp, mainly due to a decrease in the hepatic supply of glucose precursors following insulin action in extrahepatic tissues. Insulin fails to elicit any significant effect in vitro. Hepatocytes exhibit insulin and glucagon receptors. The apparent characteristics of hormone binding in vitro are similar in ruminants and non-ruminants, but the characteristics of postreceptor events are unknown in the former. Glucagon, which influences hepatic glucose synthesis, may be a major hormone in ruminants.

Role of insulin in regulating hepatic gluconeogenesis in sheep.

Ruminant animals, as a result of the fermentative nature of their digestion, ordinarily absorb little or no hexose sugar from the gut. Their glucose needs must be met by gluconeogenesis, even postprandially. The role of insulin in regulating hepatic gluconeogenesis in ruminants has not been assessed. In this study the effect of insulin on net hepatic removal of the major glucose precursors was determined. Insulin was infused with glucose matched to maintain euglycemia. The insulin concentrations attained in plasma were within the physiological range. Insulin at low concentrations reduced the hepatic removal of lactate, glutamine, and glycerol. At higher concentrations of insulin the hepatic extractions of pyruvate and alanine were also reduced. Thus, in sheep insulin at physiological concentrations may reduce hepatic glucose output by altering the uptake of glucose precursors.