Compartmentation of glucose 6-phosphate in hepatocytes.

Rat hepatocytes were incubated with 14C-labelled hexoses, and the specific radioactivities of glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate were determined. (1) When suspensions of freshly isolated hepatocytes were incubated with [14C]glucose, the specific radioactivities of glucose 1-phosphate and fructose 6-phosphate were severalfold higher than that of glucose 6-phosphate. The ratios of the specific radioactivities decreased with time of incubation. These relationships were also found when incubations were carried out with primary cultures of rat hepatocytes or with crude homogenates of hepatocytes, but not with isolated nuclei. (2) When cells were incubated with [14C]fructose, the ratios of the specific radioactivities were higher than with [14C]glucose, and also decreased with time. (3) Paired incubations were carried out with a mixture of galactose and fructose, with one or other sugar being labelled with 14C. The specific radioactivity of glucose released into the medium was greater than that of glucose 6-phosphate when fructose was labelled, but not when galactose was labelled. Furthermore, glucose 6-phosphate and glucose in the medium differed with regard to the distribution of 14C between C-1 and C-6. These results are interpreted as evidence that glucose 6-phosphate in hepatocytes does not exist as a homogeneous pool, but that subcompartments exist which are associated with glucose phosphorylation, gluconeogenesis and glycogenolysis.     

Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

Glycogen synthesis in the fungus Neurospora crassa.

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [14C]glucose from ADP-[14C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose : glycogen glucosyltransferase as compared with those of the already known UDP glucose : glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-14C]glucose or ADP-[14C]glucose released all the radioactivity as maltose after alpha or beta amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[14C]glucose was the substrate but the stimulation was much greater with ADP-[14C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent &on the presence of a 'primer' of the alpha 1 leads to 4 glucan type.     

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.     

Metabolism of glucose in hyper- and hypo-thyroid rats in vivo. Glucose-turnover values and futile-cycle activities obtained with 14C- and 3H-labelled glucose.

1. A trace amount of glucose labelled with 14C uniformly and with 3H at position 2, 3 or 6 was injected intravenously into starved rats to measure the turnover rate of blood glucose. 2. Reliable estimates were made based on the semilogarithmic plot of specific radioactivity of the glucose contained in whole blood samples taken from the tail vein. 3. Glucose turned over more rapidly in hyperthyroid and more slowly in hypothyroid than in euthyroid rats. The percentage contribution of glucose recycling (determined from the difference in replacement rates between [U-14C]glucose and [6-3H]glucose) to the glucose utilization increased on induction of hyperthyroidism. 4. Futile cycles between glucose and glucose 6-phosphate (determined from the difference between replacement rates of [2-3H]glucose and [6-3H]glucose) were activated and inactivated by induction of hyperthyroid and hypothyroid states respectively. 5. The hepatic content of glycogen was much lower in hyper- and hypo-thyroid than in euthyroid rats. The enhanced glucose production in hyperthyroid rats resulted from not only activationof hepatic gluconeogenesis but also diversion of the final product of gluconeogenesis from liver glycogen to blood glucose. In hypothyroidism, the inhibition of gluconeogensis led to suppression of both glucose production and glycogenesis in the liver.     

Evidence for extracellular enzymic activity of the isolated perfused rat heart

1. The dissimilation of a number of externally added hexose phosphates and 5′-nucleotides by the perfused rat heart is described, and non-specific esterase and 5′-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of ¹⁴CO₂ from [U-¹⁴C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-¹⁴C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-¹⁴C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.     

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.     

Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells.     

Glycogen synthesis in the fungus Neurospora crassa

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [¹⁴C]glucose from ADP-[¹⁴C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose: glycogen glucosyltransferase as compared with those of the already known UDP glucose: glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-[¹⁴C]glucose or ADP-[¹⁴C]glucose released all the radioactivity as maltose after α or β amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[¹⁴C]glucose was the substrate but the stimulation was much greater with ADP-[¹⁴C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent on the presence of a ‘primer’ of the α 1 → 4 glucan type.     

Difference in glucose sensitivity of liver glycolysis and glycogen synthesis. Relationship between lactate production and fructose 2,6-bisphosphate concentration.

Incubation of isolated rat hepatocytes from fasted rats with 0-6 mM-glucose caused an increase in [fructose 2,6-bisphosphate] (0.2 to about 5 nmol/g) without net lactate production. A release of 3H2O from [3-3H]glucose was, however, detectable, indicating that phosphofructokinase was active and that cycling occurred between fructose 6-phosphate and fructose 1,6-bisphosphate. A relationship between [fructose 2,6-bisphosphate] and lactate production was observed when hepatocytes were incubated with [glucose] greater than 6 mM. Incubation with glucose caused a dose-dependent increase in [hexose 6-phosphates]. The maximal capacity of liver cytosolic proteins to bind fructose 2,6-bisphosphate was 15 nmol/g, with affinity constants of 5 X 10(6) and 0.5 X 10(6) M-1. One can calculate that, at 5 microM, more than 90% of fructose 2,6-bisphosphate is bound to cytosolic proteins. In livers of non-anaesthetized fasted mice, the activation of glycogen synthase was more sensitive to glucose injection than was the increase in [fructose 2,6-bisphosphate], whereas the opposite situation was observed in livers of fed mice. Glucose injection caused no change in the activity of liver phosphofructokinase-2 and decreased the [hexose 6-phosphates] in livers of fed mice.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

Glycogen metabolism in the liver of the neonatal gsd/gsd and control (GSD/GSD) rat.

1. The metabolism of hepatic glycogen, labelled with [6-3H]glucose at day 19.5 of gestation and with 14C from [U-14C]galactose at delivery, was followed for 10 h in food-deprived gsd/gsd and control (GSD/GSD) neonatal rats. 2. In the affected pups glycogen was maintained at 12% (w/w) and there was no loss of incorporated radioactivity. 3. The 3H and 14C in glycogen from the controls were both decreased by 80%, but 14C was removed at 0--5 h and [6-3H]glucose at 5--10 h. 4. Blood glucose concentrations in the unaffected neonatal rats fell from 5.3 mM at 20 min to 1.7 mM after 10 h. In the gsd/gsd pups blood glucose concentration was decreased from 2 mM at birth to 0.3 mM at 2.5 h: it was maintained at 0.8 mM between 5 and 10 h. 5. In neonatal rats that had been dead for 10 h, hepatic glycogen was decreased by 34% in the controls and by 22% in the gsd/gsd pups. These results demonstrate that liver from the affected rats contains glycogenolytic activity, but that it is not expressed in living tissue.     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis.

Toluene-treated cells of Streptococcus bovis JB1 phosphorylated cellobiose, glucose, maltose, and sucrose by the phosphoenolpyruvate-dependent phosphotransferase system. Glucose phosphorylation was constitutive, while all three disaccharide systems were inducible. Competition experiments indicated that separate phosphotransferase systems (enzymes II) existed for glucose, maltose, and sucrose. [14C]maltose transport was inhibited by excess (10 mM) glucose and to a lesser extent by sucrose (90 and 46%, respectively). [14C]glucose and [14C]sucrose transports were not inhibited by an excess of maltose. Since [14C]maltose phosphorylation in triethanolamine buffer was increased 160-fold as the concentration of Pi was increased from 0 to 100 mM, a maltose phosphorylase (Km for Pi, 9.5 mM) was present, and this activity was inducible. Maltose was also hydrolyzed by an inducible maltase. Glucose 1-phosphate arising from the maltose phosphorylase was metabolized by a constitutive phosphoglucomutase that was specific for alpha-glucose 1-phosphate (Km, 0.8 mM). Only sucrose-grown cells possessed sucrose hydrolase activity (Km, 3.1 mM), and this activity was much lower than the sucrose phosphotransferase system and sucrose-phosphate hydrolase activities.     

Mechanism and contribution of the pentose phosphate cycle to glucose metabolism in epididymal fat tissue

1. Glucose 6-phosphate, fructose 6-phosphate and altroheptulose 7-phosphate are the major products formed non-oxidatively from ribose 5-phosphate by rat epididymal fat pad enzymes. 2. Arabinose 5-phosphate was detected among the reaction products and significant activity of the new enzyme of the L-type pentose pathway, D-glycero D-ido octulose 1,8-bisphosphate: D-altroheptulose 7-phosphotransferase was found. 3. The glucose moieties of glucose 1-phosphate, glucose 6-phosphate and glucose 1,6-bisphosphate were degraded and showed that epididymal fat pad enzymes relocate 14C from [2-14C]glucose into C-1, C-2, and C-3 of each hexose-phosphate. 4. The 14C-distribution patterns in the hexose-phosphates revealed that these intermediates were not in isotopic equilibrium and the rate of the transaldolase exchange reaction was relatively small. 5. The 14C-distribution data suggest that glucose 1-phosphate, rather than glucose 6-phosphate, is the first intermediate in the path of glycogen synthesis from glucose in this tissue. 6. The data provide the first proof of the mechanism of the pentose pathway in adipose tissue.     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

The nature of the pentose pathway in liver

[2-14C]Glucose, [3,4-14C]glucose, [5-14C]glucose, [4,5,6-14C]glucose, and [1-14C]ribose were perfused through livers of rats. The rats were fed or fasted and refed. In one experiment the liver perfused was regenerating and in another phenazine methosulfate was in the perfusate. Perfusion was for 30 or 90 min. Glucose from each perfusate and liver glucose-6-P and glycogen were isolated, purified, and degraded. The distributions of 14C in the carbons of the glucoses from the glycogens are similar to the distributions from the glucose 6-phosphates. The distributions of 14C are in accord with metabolism of glucose by the classical pentose pathway and not by the L-type pathway that has been proposed to function in liver.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

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.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.