Co-ordinated regulation of muscle glycolysis and hepatic glucose output in exercise by catecholamines acting via alpha-receptors.

Recent findings indicate that glucose uptake by contracting hindlimb #Acta Physiol. Scand. (1982) 116, 215-222 #and heart #Biochem. Biophys. Res. Commun. (1982) 108, 124-131 # of the rat is stimulated by epinephrine acting through alpha-adrenergic mechanisms. Since in exercise hepatic glucose output may be increased markedly by activation of alpha-adrenergic receptors and matched by the increase in muscle glucose uptake (maintaining blood glucose levels relatively constant), it is now proposed that a general coordination of glucose metabolism may operate via alpha-adrenergic receptor mechanisms. The basis for this proposal is discussed.

Plasma half-lives of native and modified insulin-like growth factor-I in lambs.

The clearance of labelled insulin-like growth factor-I (IGF-I) has been measured in lambs following acid gel-permeation chromatography and immunoprecipitation of plasma samples. The half-lives obtained in three experiments were between 5 and 7h. Chromatography at neutral pH on a Fractogel HW55(S) column demonstrated that all the radioactivity associated with undegraded peptide in plasma was bound to a carrier protein. Similar studies with IGF-I that had been reduced by prior dithiothreitol treatment showed that two-thirds of the initial radioactivity in plasma decayed with a much shorter half-life and represented material that did not bind to carrier proteins in plasma. The remaining radioactivity was both associated with a binding protein and exhibited the characteristically long half-life of the native growth factor. Analysis of plasma samples using reversed-phase chromatography demonstrated that the radioactive component with a long half-life was IGF-I while that with a short half-life had been reoxidized to an incorrect form of the growth factor. When reoxidation of reduced IGF-I was blocked by S-carboxymethylation before injection of the radioactive peptide into lambs, it remained unbound in plasma and had a 0.8-0.9 h half-life. We suggest that reduced IGF-I only associates with the binding protein upon oxidation and correct folding and that this association is necessary in order for IGF-I to have a relatively long half-life.

Obesity and the regulation of phosphofructokinase in heart: an apparent insensitivity to adrenergic activation in mature-age genetically obese rats.

The activity ratio of phosphofructokinase in perfused rat heart and its activation by epinephrine was examined in non-obese, fat-fed obese, and genetically obese rats. For non-obese colony rats there was an age-dependent increase in the activity ratio of phosphofructokinase from 0.2 at 40 days to 0.4 at mature age (greater than 200 days). Epinephrine (10 microM) treatment of the heart for 5 min increased the ratio at all ages but the proportional increase diminished with age. For mature-age lean Zucker rats carrying the genetic determinant for obesity the results were similar to those obtained for comparable non-obese colony rats. For fat-fed obese rats the activity ratio of phosphofructokinase at 200 days of age was 0.2 and was increased to 0.6 by epinephrine treatment. For mature-age obese Zucker rats the activity ratio was 0.2 and no significant response to epinephrine occurred. The activity ratio of glycogen phosphorylase and its response to epinephrine (beta-adrenergic receptor mediated) in heart was unaffected by age, diet or the gene for obesity. The present findings indicate a specific defect in the adrenergic regulatory mechanism for phosphofructokinase in genetically obese rats.

Amino acid uptake and output by the sheep hind limb.

Arteriovenous differences across the hind limb of fed sheep show release of alanine, glutamine, and tyrosine and uptake of serine, glutamate, and possibly lysine. In starved animals there is a net output of most amino acids, although the amount of alanine released, 26 nmol/ml blood, is much lower than reported for human muscle and is less than the cumulative release of valine, leucine, and isoleucine. Since it has been argued that the carbon of alanine is derived from glucose and the nitrogen from the deamination of branched chain amino acids, we suggest that either nutrient availability is limiting alanine output or else sheep muscle has an impaired ability to degrade the branched chain amino acids.

Utilization of oxidizable substrates by the sheep hind limb: effects of starvation and exercise.

Measurements of substrate uptake by the sheep hind limb show a pattern similar to human and other monogastric animals. Thus free fatty acids (FFA) are the principal substrates at rest and during exercise while beta-hydroxybutyrate and acetoacetate are major nutrients in starved animals. The hind limb has arteriovenous differences for glucose and lactate which indicate that glucose supplies about 27% of the fuel of respiration during exercise, but the hind limb in resting, fed, and starved animals returns essentially all of the glucose carbon to the blood in the form of lactate. This finding is consistent with a conservation of glucose in aminals which obtain very little dietary glucose. Although some acetate is extracted from the blood in fed sheep, the utilization of this nutrient can account for only 2% or less of the oxygen uptake in the hind limb of starved or exercising animals. Thus, while acetate is the major product of the sheep rumen it is not used directly as a major energy source. We propose that most of the actate is converted to FFA which can be stored as triglyceride or oxidized in muscle.

An effect of extracellular redox state on the glucagon-stimulated glucose release by rat hepatocytes and perfused liver.

The effect of changes in the extracellular redox-state on glucagon-stimulated glucose release by intact isolated rat hepatocytes and the perfused liver was examined. For hepatocytes from the fed rat an increase in pyruvate, ammonium ion or oxygen concentration or a decrease in the lactate/pyruvate or sorbitol/fructose ratios decreased the ability of 1 microM-glucagon to stimulate glucose release without significantly altering the control rate. These changes coincided with a decrease in the lactate/pyruvate ratio of the cell suspension. A decrease in the lactate/pyruvate ratio also decreased the ability of 1 microM-glucagon to stimulate glycogen breakdown measured by loss of contained radioactivity. For the isolated perfused rat liver (fed rat) maximal effects of glucagon as a stimulant of glucose release occurred when lactate instead of pyruvate was present in the perfusion medium. It is concluded that the efficacy of glucagon as a stimulant of glucose release by isolated hepatocytes and the perfused liver depends upon the cytoplasmic redox-state represented by the intracellular lactate/pyruvate ratio.

Interference by ATPase in the assay of rat heart phosphofructokinase.

A high molecular-weight protein was found in heart extracts which, in the assay for phosphofructokinase, artificially activated the enzyme. The protein could be removed by gel-exclusion chromatography or high-speed centrifugation. The mechanism of activation appeared to be due to the hydrolysis of ATP to ADP, AMP and inorganic phosphate which was inhibited by Mn2+. Phosphofructokinase is thus activated by the production of activators and by the lowered inhibitory concentration of ATP. Since the adrenergic/Ca2+-activated form of the enzyme is the more sensitive to activators, the difference between the two forms of phosphofructokinase is amplified in the presence of the ATPase and diminished upon its removal or its inhibition by Mn2+. The Mn2+-sensitive ATPase appears to play no part in the adrenergic/Ca2+-mediated control of cardiac phosphofructokinase or the interconverting reactions.

Effects of fructose concentration on carbohydrate metabolism, heat production and substrate cycling in isolated rat hepatocytes.

1. Hepatocytes from starved rats were incubated with 5mm-glucose, labelled uniformly with (14)C and specifically with (3)H at positions 1, 2, 3 or 6, and with fructose at concentrations of 2.5, 7.5 or 25mm. 2. In the absence of other substrates only 1% of the radioactivity initially present in [U-(14)C]glucose appeared in the metabolic products, CO(2), lactate, pyruvate, amino acids and glycogen. 3. Fructose at 2.5mm caused a 30% increase in the glucose concentration and a 4-fold increase in the apparent oxidation of [U-(14)C]-glucose. 4. The formation of (3)H(2)O from [1-(3)H]-, [2-(3)H]-, [3-(3)H]- or [6-(3)H]-glucose was 2.4, 4.3, 2.15 or 1.6% respectively in the control incubations and 4.1, 10.4, 7.7 or 5.1% with 2.5mm-fructose. 5. Fructose at 7.5 and 25mm decreased the (3)H(2)O yields to less than the control values, but had no apparent effect on the amount of [U-(14)C]glucose metabolized. 6. In the incubations with 5mm-glucose and 25mm-fructose there were significant decreases in heat production, O(2) consumption and in the ratio of O(2) uptake to heat output. 7. Fructose at 2.5mm caused a 64% increase in heat output, but only a 43% increase in O(2) uptake. 8. The radioisotopic and calorimetric data demonstrate that physiological concentrations of fructose greatly increase metabolism in hepatocytes from starved rats. These data also indicate increased cycling at glucose/glucose 6-phosphate and at fructose 6-phosphate/fructose 1,6-bisphosphate in the presence of 2.5mm-fructose, although the rates of cycling were actually decreased relative to the amount of glucose catabolized. 9. At concentrations of 2.5, 7.5 and 25mm, fructose depressed hepatocyte ATP concentrations by 20, 65 and 80% respectively. Although fructose at 7.5 and 25mm increased glucose and lactate release, O(2) consumption, production of heat and formation of(3)H(2)O from [1-(3)H]-, [2-(3)H]-, [3-(3)H]- or [6-(3)H]-glucose were lowered to values equal to, or less than, controls. These effects probably reflect a severe derangement of hepatic metabolism due to excess phosphorylation of fructose when present at high concentrations.

Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate.

1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogeneous precursors, 10 mM-propionate and 10mM-galactose, were linear for 1 h and were both stimulated by 1 muM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37 degrees, 22 degrees and * degrees C. Even under the best of the three conditions of storage that were tested (i.e. at 22 degrees C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceeded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 muM) and 2mM-butyrate accelerated the rate of glucose formation of liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 muM-glucagon. For lactate+pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1muM-glucagon and for both lactate+pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-cell gluconeogenesis by different mechanisms.

Glucose and acetate metabolism in sheep at rest and during exercise.

Total entry rate of blood glucose and the rate of irreversible loss of blood acetate and its oxidation have been examined in sheep at rest and while walking on a horizontal treadmill at 5 km/h for 2 h. Sheep were given their daily ration of 1000 g chaff in 24 eaual portions at hourly intervals and received multiple intravenous injections of [2-3H]glucose and intravenous infusions of [1-14C]acetate and NaH14CO3. At rest the total entry rate of blood glucose was 0-44 +/- 0-03 mmol/min (values given as mean +/- s.e.m. for four sheep), the glucose pool was 23 +/- 1 mmol and the rate of irreversible loss of blood acetate was 2-3 +/- 0-1 mmol/min. During exercise, the total entry rate of blood glucose was 0-84 +/- 0-04 mmol/min, the glucose pool was 27 +/- 2 mmol and the rate of irreversible loss of blood acetate was 2-6 +/- 0-1 mmol/min. Gluconeogenesis apparently increased markedly in response to exercise as indicated by the incorporation of 14C from blood bicarbonate into blood glucose. Despite the substantial increase in the rate of irreversible loss of blood bicarbonate (from 11-6 +/- 1 to 20-2 +/- 2 mmol C/min), and hence energy expenditure with exercise, only a slight change was recorded in the proportion of the irreversible loss rate of acetate that was oxidized.

Effects of carbohydrate availability on lipogenesis in sheep.

1. Lipogenesis in sheep liver and adipose tissue was investigated by incorporation studies in vitro with radioactive glucose and acetate and by assays of key enzymes. 2. Carbohydrate availability to sheep was increased by feeding on a diet containing 70% soluble carbohydrate, by infusing glucose into the abomasum or by direct intravenous infusion of glucose. 3. Under these conditions lipogenesis from glucose and acetate was increased from very low values in lìver and adipose tissue, especially in those animals where rumen fermentation was by-passed by glucose infusion. 4. Large increases in the activities of ATP citrate lyase (EC 4.1.3.8) and NADP-malate dehydrogenase (EC 1.1.1.40) occurred in both tissues when lipogenesis was increased. 5. No adaptations were found in the activities of pyruvate carboxylase (EC 6.4.1.1) in adipose tissue, glucokinase (EC 2.7.1.2) in liver or 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in liver. It is proposed that the absence of these enzymes is not related to glucose availability. 6. The effect of glucose on liver lipogenesis was to increase conversion of acetate into lipid. 7. This effect also occurred in adipose tissue, but in this tissue glucose also became a quantitatively important precursor of triglyceride fatty acid.

Catecholamines and Ca2+ mediate an increase in activity and reactive thiols of rat heart phosphofructokinase.

Catecholamines and Ca2+ mediate an increase in reactive-thiol status of phosphofructokinase, associated with an increase in activation of the enzyme. A correlation was observed between the number of reactive thiols and activation (r = 0.878; P less than 0.001) for 14 different treatments, with approx. 1 group per 85,000-Mr subunit becoming available over the range from the lowest to the highest state of activation.

Insensitivity of cardiac phosphofructokinase to adrenergic activation in Zucker rats. A post-receptor defect.

The apparent insensitivity of phosphofructokinase to activation by adrenaline in hearts from genetically obese Zucker (fa/fa) rats [Patten, Filsell & Clark (1982) Metab. Clin. Exp. 31, 1137-1141] was examined in detail. Perfusion of hearts from obese (fa/fa) animals with medium containing 1nM-100 microM-adrenaline for 4 min did not significantly activate phosphofructokinase, but fully activated glycogen phosphorylase. Activation of phosphofructokinase occurred in hearts from lean (Fa/?) animals, with half-maximal activation at 10nM-adrenaline. Binding characteristics of cardiac alpha 1-adrenergic receptors were comparable between lean and obese animals. Properties of phosphofructokinase from obese and lean hearts were similar. High extracellular Ca2+ concentration, which activated phosphofructokinase in hearts from lean animals and phosphorylase in all hearts, failed to activate phosphofructokinase in hearts from obese rats. The data indicate that the failure of adrenaline to activate phosphofructokinase in hearts from obese (fa/fa) rats results from a post-receptor defect in the as-yet unidentified Ca2+-dependent phosphofructokinase-activating reactions.

Is orally-derived epidermal growth factor beneficial following premature birth or intestinal resection?

Epidermal growth factor (EGF), a major growth factor in human milk, may stimulate growth and development of crucial infant tissues, particularly in conditions where the infant's growth is compromised. Here, we provide evidence of a role for EGF in two such conditions, premature birth and intestinal resection. Compared with women delivering at term, mothers of premature infants produce milk containing higher concentrations of EGF, an effect that is probably maintained throughout lactation. The increased EGF in milk could not be accounted for by a decrease in the volume of milk production or by events surrounding birth, but may instead represent a maternal compensatory mechanism to accelerate growth in pre-term infants. A role for EGF in stimulating adaptive intestinal regrowth following gut resection was identified using weanling rats. Following removal of 50% of the small intestine of weanling rats, inclusion of EGF (20 micrograms/100 g per day) in the diet (S-26 infant formula) significantly accelerated intestinal growth, as determined by wet weight, protein and sucrase activity per unit length. These studies support the concept that administration of EGF could prove beneficial to infants in which crucial, EGF-responsive tissues are poorly developed, e.g. following premature birth or intestinal resection.

Production and utilization of acetate in mammals.

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.

No major thermogenic role for (Na+ + K+)-dependent adenosine triphosphatase apparent in hepatocytes from hyperthyroid rats.

(Na+ + K+)-dependent ATPase activity, heat production and oxygen consumption were increased by 59%, 62% and 75% respectively in hepatocytes from tri-iodothyronine-treated rats. Ouabain at concentrations of 1 and 10 mM decreased oxygen uptake by 2--8% in hepatocytes from euthyroid rats and by 5--15% in hepatocytes from hyperthyroid animals. Heat output was decreased by 4--9% with the glycoside in isolated liver parenchymal cells from the control animals and by 11% in the cells from the tri-iodothyronine-treated animals. These results do not support the hypothesis that hepatic (Na+ + K+)-ATPase plays a major role in increased heat production in hepatocytes from hyperthyroid rats.

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.

The application of microcalorimetry to the assessment of metabolic efficiency in isolated rat hepatocytes.

1. Heat output by suspensions of isolated rat hepatocytes was determined by using a modified batch-type microcalorimeter. 2. The ratio of O(2) uptake (determined polarographically) to heat output was used to assess the metabolic efficiency of isolated hepatocytes. 3. Cells from starved or fed rats incubated in either bicarbonate-buffered physiological saline containing gelatin, or bicarbonate-buffered physiological saline containing amino acids, serum albumin and glucose showed no significant difference with respect to the ratio of O(2) uptake to heat output. 4. For liver cells from 24h-starved rats, the addition of 10mm-dihydroxyacetone and 2.5mm-fructose significantly decreased the ratio of O(2) uptake to heat output from 1.94+/-0.05 in the controls to 1.52+/-0.04 and 1.54+/-0.01mumol/J respectively. 5. Glucagon (1mum), which slightly increased both O(2) uptake and heat output, did not significantly alter the ratio. 6. The addition of extracellular 10mm-NH(4)Cl and urease to provide an energetically wasteful cycle by ensuring hydrolysis of newly synthesized urea, lowered the ratio of O(2) uptake to heat output from 1.81+/-0.08 to 1.47+/-0.06mumol/J, indicating a reduced metabolic efficiency. 7. Metabolic efficiency in rats of different dietary regimen, age and genetically based obesity was also assessed. No differences in the ratio of O(2) uptake to heat output were found between liver cell suspensions prepared from rats maintained on colony diet and high-fat diet or sucrose-rich diet nor between animals ranging from 38 to 179 days of age. Comparison of the ratio of liver cell O(2) uptake to heat output between homozygote Zucker fa/fa obese rats and their lean littermates showed no significant difference. 8. It is concluded that the ratio of O(2) uptake to heat output for isolated hepatocytes is relatively constant unless perturbed by conditions that markedly enhance substrate cycling.