Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

Effects in vitro of alloxan on the glucose metabolism of mouse pancreatic B-cells

To facilitate detailed studies of the B-cytotoxic action of alloxan we developed a model using isolated pancreatic islets of normal mice. An essential feature of this model is the low temperature employed during exposure to alloxan, which minimizes the degradation of the drug. The islets were incubated with alloxan for 30min at 4 degrees C and subsequently various aspects of their metabolism were studied. The O(2) consumption was measured by the Cartesian-diver technique. Islets exposed to 2mm-alloxan and control islets had the same endogenous respiration, whereas the O(2) uptake of the alloxan-treated islets was inhibited and that of the control islets stimulated when they were incubated with 28mm-glucose as an exogenous substrate. The islet glucose oxidation was estimated by measurement of the formation of (14)CO(2) from [U-(14)C]glucose at 37 degrees C. Compared with the controls, alloxan-treated islets showed a decrease in the glucose-oxidation rate in a dose-dependent manner. Pretreatment of the islets with 28mm-glucose for 30min at 37 degrees C completely protected against this effect, whereas preincubations at glucose concentrations below 16.7mm failed to exert any protective effect. The glucose utilization was estimated as the formation of (3)H(2)O from [5-(3)H]glucose. Alloxan (2mm) failed to affect islet glucoseutilization rate in the presence of either 2.8 or 28mm-glucose. In contrast, islets exposed to 5 or 10mm-alloxan exhibited lowered glucose utilization. It is concluded that in vitro alloxan has an acute inhibitory effect on the islet glucose metabolism, and that this action can be prevented by previous exposure to a high glucose concentration. The results are consistent with the idea that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell.     

The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro

1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-(14)C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-(14)C]glucose, except that incorporation into [(14)C]lactate was decreased, and significant amounts of [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-(14)C]glucose 6-phosphate into all metabolites except [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-(14)C]glucose other than to increase markedly the incorporation into, and amount of, [(14)C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12mumol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-P(i) or 26mm-HCO(3) (-) was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; comparison with phosphomannoisomerase

The isotopic discrimination, diastereotopic specificity and intramolecular hydrogen transfer characterizing the reaction catalyzed by phosphomannoisomerase are examined. During the monodirectional conversion of D-[2-3H]mannose 6-phosphate to D-fructose 6-phosphate and D-fructose 1,6-bisphosphate, the reaction velocity is one order of magnitude lower than with D-[U-14C]mannose 6-phosphate and little tritium (less than 6%) is transferred intramolecularly. Inorganic phosphate decreases the reaction velocity but favours the intramolecular transfer of tritium. Likewise, when D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose is exposed solely to phosphomannoisomerase, the generation of tritiated metabolites is virtually restricted to 3H2O and occurs at a much lower rate than the production of D-[U-14C]mannose 6-phosphate from D-[U-14C]fructose 6-phosphate. However, no 3H2O is formed when D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose is exposed to phosphomannoisomerase, indicating that the diastereotopic specificity of the latter enzyme represents a mirror image of that of phosphoglucoisomerase. Advantage is taken of such a contrasting enzymic behaviour to assess the back-and-forth flow through the reaction catalyzed by phosphomannoisomerase in intact cells exposed to D-[1-3H]glucose, D-[5-3H]glucose or D-[6-3H]glucose. Relative to the rate of glycolysis, this back-and-forth flow amounted to approx. 4% in human erythrocytes and rat parotid cells, 9% in tumoral cells of the RINm5F line and 47% in rat pancreatic islets.     

Nutritional and hormonal control of glucose and fructose utilization by lung

Whereas glucose is a major substrate for pulmonary lipid synthesis, fructose has also been suggested as a potential substrate. In vivo pulmonary fatty acid synthesis is depressed in hormonally deprived conditions, such as diabetes, and this can be modified by fructose feeding, but not by glucose feeding. In this study the glucose and fructose utilizations were compared in normal, diabetic and fasting states using isolated perfused rat lungs. When (U-14C)- or (5-3H)-glucose was used as substrate, glucose utilization by lung was reduced by 50% in both the fasting and diabetic animals compared to the normal controls. Using (U-14C)-glucose as substrate, the incorporation of (14C)-label in various metabolites of glucose was significantly depressed. For example, this reduction was 50% in lactate, pyruvate and CO2, 15% in ethanol-insoluble fraction, 65% in neutral lipids, 75% in phospholipids, 80% in fatty acid moiety, 40% in deacylated fraction and 10% in the polysaccharide fractions. Refeeding the fasted animals or insulin treatment to the diabetic animals restored these depressed (14C)-recoveries to the normal levels. Fructose utilization was less than 10% of glucose utilization, but remained unaffected by fasting and diabetic states. In addition, pulmonary hexokinase enzyme activity was lowered significantly in fasting and diabetic animals, whereas fructokinase enzyme activity was not altered. Despite the low rate of fructose utilization, these results suggest that fructose may serve as an alternative substrate for pulmonary phospholipid synthesis when glucose utilization is significantly depressed.     

Comparison between D-[3-3H]- and D-[5-3H]glucose and fructose utilization in pancreatic islets from control and hereditarily diabetic rats

The metabolism of D-glucose and/or D-fructose was investigated in pancreatic islets from control rats and hereditarily diabetic GK rats. In the case of both D-glucose and D-fructose metabolism, a preferential alteration of oxidative events was observed in islets from GK rats. The generation of 3HOH from D-[5-3H]glucose (or D-[5-3H]fructose) exceeded that from D-[3-3H]glucose (or D-[3-3H]fructose) in both control and GK rats. This difference, which is possibly attributable to a partial escape from glycolysis of tritiated dihydroxyacetone phosphate, was accentuated whenever the rate of glycolysis was decreased, e.g., in the absence of extracellular Ca(2+) or presence of exogenous D-glyceraldehyde. D-Mannoheptulose, which inhibited D-glucose metabolism, exerted only limited effects upon D-fructose metabolism. In the presence of both hexoses, the paired ratio between D-[U-14C]fructose oxidation and D-[3-3H]fructose or D-[5-3H]fructose utilization was considerably increased, this being probably attributable, in part at least, to a preferential stimulation by the aldohexose of mitochondrial oxidative events. Moreover, this coincided with the fact that D-mannoheptulose now severely inhibited the catabolism of D-[5-3H]fructose and D-[U-14C]fructose. The latter situation is consistent with both the knowledge that D-glucose augments D-fructose phosphorylation by glucokinase and the findings that D-mannoheptulose, which fails to affect D-fructose phosphorylation by fructokinase, inhibits the phosphorylation of D-fructose by glucokinase.     

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.     

Palmitate inhibits liver glycolysis. Involvement of fructose 2,6-bisphosphate in the glucose/fatty acid cycle.

In hepatocytes from overnight-fasted rats incubated with glucose, palmitate decreased the production of lactate, the detritiation of [2-3H]- and [3-3H]-glucose, and the concentration of fructose 2,6-bisphosphate. Similarly, perfusion of hearts from fed rats with beta-hydroxybutyrate resulted in an inhibition of the detritiation of [3-3H]glucose and a fall in fructose 2,6-bisphosphate concentration. This fall could result from an increase in citrate (hepatocytes and heart) and sn-glycerol 3-bisphosphate concentration. It is suggested that a fall in fructose 2,6-bisphosphate concentration participates in the inhibition of glycolysis by fatty acids and ketone bodies.     

Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with (14)C and specifically with (3)H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24mumol/ml of blood the rate of utilization was 2.8mumol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of (3)H(2)O from specifically (3)H-labelled glucoses increased in the order [6-(3)H]<[3-(3)H]<[2-(3)H]. Production of (3)H(2)O from [2-(3)H]glucose was significantly greater than that from [3-(3)H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased (3)H(2)O formation from all [(3)H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-(14)C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose-glucose 6-phosphate and at fructose 6-phosphate-fructose 1,6-bisphosphate.     

The pentose cycle and insulin release in mouse pancreatic islets

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.     

Calditol tetraether lipids of the archaebacterium Sulfolobus solfataricus. Biosynthetic studies.

Lipids from the archaebacterium Sulfolobus solfataricus are based on 72-membered macrocyclic tetraethers made up from two C40 diol units differently cyclized and either two glycerol moieties or one glycerol moiety and a unique branched-chain nonitol named calditol (glycerodialkylnonitol tetraethers, GDNTs). To elucidate the biosynthesis of calditol and related tetraethers, labelled precursors, [U-14C,1(3)-3H]glycerol, [U-14C,2-3H]glycerol, D-[1-14C,6-3H]glucose, D-[6-14C,1-3H]glucose, D-[1-14C,2-3H]glucose, D-[1-14C,6-3H]fructose and D-[1-14C]galactose, were fed to S. solfataricus. Without regard to stereochemistry or phosphorylation, incorporation experiments provided evidence that the biosynthesis of calditol occurs via an aldolic condensation between dihydroxyacetone and fructose, through a 2-oxo derivative of calditol as an intermediate. The latter is in turn reduced and then alkylated to yield the GDNTs. The biogenetic origins of both glycerol and C40 isoprenoid moieties of GDNTs are also discussed.     

Effect of dihydroxyacetone and pyruvate on plasma glucose concentration and turnover in noninsulin-dependent diabetes mellitus

Consumption of dihydroxyacetone and pyruvate (DHP) increases muscle extraction of glucose in normal men. To test the hypothesis that these three-carbon compounds would improve glycemic control in diabetes, we evaluated the effect of DHP on plasma glucose concentration, turnover, recycling, and tolerance in 7 women with noninsulin-dependent diabetes. The subjects consumed a 1,500-calorie diet (55% carbohydrate, 30% fat, 15% protein), randomly containing 13% of the calories as DHP (1/1) or Polycose (placebo; PL), as a drink three times daily for 7 days. On the 8th day, primed continuous infusions of [6-3H]-glucose and [U-14C]-glucose were begun at 05.00 h, and at 09.00 h a 3-hour glucose tolerance test (75 g glucola) was performed. Two weeks later the subjects repeated the study with the other diet. The fasting plasma glucose level decreased by 14% with DHP (DHP = 8.0 +/- 0.9 mmol/l; PL = 9.3 +/- 1.0 mmol/l, p less than 0.05) which accounted for lower postoral glucose glycemia (DHP = 13.1 +/- 0.8 mmol/l, PL = 14.7 +/- 0.8 mmol/l, p less than 0.05). [6-3H]-glucose turnover (DHP = 1.50 +/- 0.19 mg.kg-1.min-1, PL = 1.77 +/- 0.21 mg.kg-1.min-1, p less than 0.05) and glucose recycling, the difference in [6-3H]-glucose and [U-14C]-glucose turnover rates, decreased with DHP (DHP = 0.25 +/- 0.07 mg.kg-1.min-1, PL = 0.54 +/- 0.10 mg.kg-1.min-1, p less than 0.05). Fasting and postoral glucose, plasma insulin, glucagon, and C peptide levels were unaffected by DHP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction and suppression of the key enzymes of glycolysis and gluconeogenesis in isolated perfused rat liver in response to glucose, fructose and lactate

1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.     

Pathways of glycogen synthesis from glucose during the glycogenic response to insulin in cultured foetal hepatocytes.

The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.     

Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.     

Hexose metabolism in pancreatic islets stimulation by D-glucose of [2-3H]glycerol detritiation

1. In pancreatic islets, a rise in glucose concentration is known to increase the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization. The opposite situation was found to prevail in parotid cells. 2. In rat pancreatic islets, D-glucose caused a concentration-related stimulation of 3H2O production from [2-3H]glycerol, but failed to affect 3H2O production from [1(3)-3H]glycerol or 14CO2 production from [U-14C]glycerol. At the low concentration used in most of these experiments (i.e. 1.0 mM), glycerol failed to affect D-[U-14C]glucose oxidation. 3. These findings suggest that the preferential stimulation by D-glucose of mitochondrial oxidative events in pancreatic islets represents an unusual situation in secretory cells and involves an accelerated circulation in the glycerol phosphate shuttle.     

Fiber type-specific determinants of Vmax for insulin-stimulated muscle glucose uptake in vivo

The aim of this study was to determine barriers limiting muscle glucose uptake (MGU) during increased glucose flux created by raising blood glucose in the presence of fixed insulin. The determinants of the maximal velocity (V(max)) of MGU in muscles of different fiber types were defined. Conscious rats were studied during a 4 mU x kg(-1) x min(-1) insulin clamp with plasma glucose at 2.5, 5.5, and 8.5 mM. [U-(14)C]mannitol and 3-O-methyl-[(3)H]glucose ([(3)H]MG) were infused to steady-state levels (t = -180 to 0 min). These isotope infusions were continued from 0 to 40 min with the addition of a 2-deoxy-[(3)H]glucose ([(3)H]DG) infusion. Muscles were excised at t = 40 min. Glucose metabolic index (R(g)) was calculated from muscle-phosphorylated [(3)H]DG. [U-(14)C]mannitol was used to determine extracellular (EC) H(2)O. Glucose at the outer ([G](om)) and inner ([G](im)) sarcolemmal surfaces was determined by the ratio of [(3)H]MG in intracellular to EC H(2)O and muscle glucose. R(g) was comparable at the two higher glucose concentrations, suggesting that rates of uptake near V(max) were reached. In summary, by defining the relationship of arterial glucose to [G](om) and [G](im) in the presence of fixed hyperinsulinemia, it is concluded that 1) V(max) for MGU is limited by extracellular and intracellular barriers in type I fibers, as the sarcolemma is freely permeable to glucose; 2) V(max) is limited in muscles with predominantly type IIb fibers by extracellular resistance and transport resistance; and 3) limits to R(g) are determined by resistance at multiple steps and are better defined by distributed control rather than by a single rate-limiting step.     

Carbohydrate metabolism in fructose-fed and food-restricted running rats

In chronically catheterized rats hepatic glycogen was increased by fructose (approximately 10 g/kg) gavage (FF rats) or lowered by overnight food restriction (FR rats). [3-3H]- and [U-14C]glucose were infused before, during, and after treadmill running. During exercise the increase in glucose production (Ra) was always directly related to work intensity and faster than the increase in glucose disappearance, resulting in increased plasma glucose levels. At identical work-loads the increase in Ra and plasma glucose as well as liver glycogen breakdown were higher in FF and control (C) rats than in FR rats. Breakdown of muscle glycogen was less in FF than in C rats. Incorporation of [14C]glucose in glycogen at rest and mobilization of label during exercise partly explained that 14C estimates of carbohydrate metabolism disagreed with chemical measurements. In some muscles glycogen depletion was not accompanied by loss of 14C and 3H, indicating futile cycling of glucose. In FR rats a postexercise increase in liver glycogen was seen with 14C/3H similar to that of plasma glucose, indicating direct synthesis from glucose. In conclusion, in exercising rats the increase in glucose production is subjected to feedforward regulation and depends on the liver glycogen concentration. Endogenous glucose may be incorporated in glycogen in working muscle and may be used directly for liver glycogen synthesis rather than after conversion to trioses. Fructose ingestion may diminish muscular glycogen breakdown. The [14C]glucose infusion technique for determination of muscular glycogenolysis is of doubtful value in rats.     

Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.     

Decreased islet sensitivity to insulin in hamsters with dietary-induced insulin resistance

In the present study, we evaluated the autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from hamsters with insulin resistance (IR) induced by administration of a sucrose-rich diet (SRD) during 5 weeks. We used an approach of two metabolic pathways (glucose oxidation and utilization) based on the measurement of 14CO2 and 3H2O production from D-[U-14C]-glucose and D-[5-(3)H]-glucose, respectively, in isolated islets incubated with 3.3 and 16.7 mM glucose alone, or with 5 or 15 mU/ml insulin, anti-insulin guinea-pig serum (1:500), 25 microM nifedipine, or 150 nM wortmannin. Insulin release was measured by radioimmunoassay in islets incubated with 3.3 or 16.7 mM glucose, with or without 75, 150, and 300 nM wortmannin. Results showed that the stimulatory effect of insulin upon 14CO2 and 3H2O production in control islets was not observed in SRD islets. Addition of anti-insulin serum, nifedipine or wortmannin to the medium with 16.7 mM glucose decreased 14CO2 and 3H2O production in control but not in SRD islets. Whereas wortmannin did not decrease insulin release induced by 16.7 mM glucose in SRD hamsters, it did in controls. We can conclude that the autocrine stimulatory effect of insulin upon glucose metabolism observed in normal islets is attenuated or even absent in islets from IR animals. Such decreased islet sensitivity to insulin did not prevent the compensatory secretion of insulin from maintaining glucose homeostasis, suggesting that, at least in this model, the islets can put forward alternative mechanisms to overcome such defect.