Lipogenesis and gluconeogenesis in the liver of irradiated rats

The incorporation of 14C from [U-14C] glucose and 3H from 3H2O into the total lipids fatty acids and glycogen of the liver incorporation of 3H from 3H2O into blood glucose was studied in rats totally irradiated in a dose of 14.4 Gy. It is shown that in the liver of irradiated rats glucose is accumulated in considerable amounts as glycogen but it is slightly used as a source of carbon for lipid synthesis. The study of 3H incorporation shows that irradiation stimulates glucogenesis, glyconeogenesis and lipogenesis in the liver.     

Studies on the existence of a pathway in liver and muscle for the conversion of glucose into glycogen without glucose 6-phosphate as an intermediate

Mixtures of (14)C-labelled glucose plus pyruvate were incubated either with rat diaphragm or slices of rat liver. Incorporation of glucose carbon into glycogen was compared with its incorporation into glucose 6-phosphate relative to the incorporation of pyruvate carbon into these metabolic products. There was no preferential incorporation of glucose carbon relative to pyruvate carbon into glycogen compared with glucose 6-phosphate in the liver slices, but there was in diaphragm. On the assumption that glucose 6-phosphate is a necessary intermediate in the conversion of pyruvate carbon into glycogen, this is evidence for the existence in muscle, but not in liver, of more than one pool of glucose 6-phosphate or of a pathway from glucose to glycogen without glucose 6-phosphate as an intermediate. Galactose carbon, relative to pyruvate carbon, was preferentially incorporated into liver glycogen, so that a substrate converted in liver into glycogen without glucose 6-phosphate as an intermediate could be detected by this approach.     

Dilantin and salicylate effects on hepatic thyroxine bio-availability and dialyzable thyroxine

Earlier studies have shown that drugs such as dilantin inhibit T4 binding by thyroid hormone binding globulin (TBG) and cause a displacement of T4 from TBG to prealbumin with no change in the albumin-bound T4 fraction. Since recent studies have shown albumin-bound T4 is freely transported into liver, the present studies are designed to investigate drug effects on T4 transport in liver. The effect of salicylate and diphenylhydantoin (Dilantin) on T4 in human serum were examined both in vitro by using equilibrium dialysis and in vivo in the rat liver by using a tissue sampling single injection technique. Serum was obtained from 6 healthy normal volunteers and was made either 0 or 0.5 mM Dilantin and either 0 or 10 mM sodium salicylate. The portal vein injection vehicle contained 125I-T4/3H-water (highly diffusible internal reference) mixed with either a) Ringer's (0.1 g/dl albumin), b) 5% T4 antiserum, or c) 80% human serum. The free dialyzable fraction in vitro was raised by 40 and 125% after the addition of Dilantin and salicylate respectively. However, the percent of total T4 that was transported into liver on one pass, 17 +/- 1%, was not different in the control, the salicylate treated, or the Dilantin-treated sera. Therefore, in contrast to the in vitro dialyzable measurement of free T4, which is elevated by toxic concentrations of Dilantin or salicylate, the bio-available fraction of T4 as determined by the single pass perfusion technique, is unchanged in rat liver in vivo. These drug-induced changes in free T4 in vitro and bio-available T4 in vivo are similar to the ones reported previously in non-thyroidal illness.     

The use of tritiated water to measure absolute rates of hepatic glycogen synthesis.

Glucogen synthesis in rat liver in vivo was measured by the incorporation of 3H from 3H2O into glycogen. In meal-fed rats incorporation and the incorporation of 3H into glycogen was linear up to 100 min. Before feeding glycogen concentration and the incorporation of 3H were both low; and both rose on feeding to give maximal values after 2-3h. The glycogen concentration was maintained for a further 5h but the incorporation of 3H rapidly declined to pre-feeding values. This shows that glycogen turnover was low in the post-prandial rat. Streptozotocin diabetes decreased the rise in glycogen concentration on feeding and had a similar effect on 3H2O incorporation. Both effects were reversed by insulin administration. The number of 3H atoms incorporated per glycogen glucose moiety formed in biosynthetic experiments (2.84 +/- 0.47) was relatively constant and allowed absolute biosynthetic rates to be calculated. Degradation of glucose from glycogen labelled by 3H2O showed that most of the 3H was located at C-2 and C-5. The incorporation would arise by rapid equilibration of hexose phosphates through phosphoglucose isomerase, transaldolase and triose phosphate isomerase.     

Pregnancy and pentobarbital anaesthesia modify hepatic synthesis of acylglycerol glycerol and glycogen from gluconeogenic precursors during fasting in rats.

1. Incorporation of gluconeogenic precursors into blood glucose and hepatic glycogen and acylglycerol glycerol was examined in 24 h-fasted virgin rats by using a flooding procedure for substrate administration. At 10 min after their intravenous injection, the conversion of alanine or glycerol into liver glycogen or acylglycerol glycerol was proportional to glucose synthesis. 2. In 24 h-fasted 21-day-pregnant rats, the incorporation of alanine and glycerol into hepatic acylglycerol glycerol was markedly enhanced compared with the control group. In addition, during fasting at late pregnancy, the proportion of substrates directed to acylglycerol glycerol as compared with the fraction incorporated into glucose was augmented. 3. In pentobarbital-treated fasted rats, the incorporation of both alanine and pyruvate into circulating glucose and into hepatic glycogen and acylglycerol glycerol was increased. Pentobarbital treatment increased the proportion of substrates incorporated into liver glycogen, compared with the fraction appearing in circulating glucose. These changes were concomitant with a marked accumulation of glycogen. 4. The data indicate that, during fasting, gluconeogenesis provides glucose as well as hepatic glycogen and acylglycerol glycerol, independently of whether the substrates enter gluconeogenesis at the level of pyruvate or dihydroxyacetone phosphate.     

Carbohydrate metabolism in liver from foetal and neonatal sheep

1. During development of the sheep, the activities of UDP-glucose–α-glucan glucosyltransferase and UDP-glucose pyrophosphorylase and the glycogen content are highest in the liver of lambs 2 weeks old and considerably lower in liver from adult sheep. 2. The activity of hexokinase and the rate of incorporation of [¹⁴C]-glucose into glycogen are much lower in liver from postnatal sheep than in rat liver. 3. The activities of hexose diphosphatase and glucose 6-phosphatase and the rates of incorporation of [¹⁴C]pyruvate and [¹⁴C]propionate into glycogen increase from low levels in the liver of foetal sheep to maxima a few weeks after birth. The activities in the liver of adult sheep are slightly lower. 4. The incorporation rate of [¹⁴C]pyruvate into glucose has been measured in liver slices from rats, sheep and chick embryos at several ages of these animals. This pathway is active in liver from foetal sheep, embryonic chicks and postnatal rats or sheep, but is absent from the liver from foetal rats. 5. Fructose metabolism, as measured by the rates of incorporation of [¹⁴C]fructose into glycogen and glucose in liver slices and by assays of liver ketohexokinase, is barely detectable in the liver of foetal sheep and appears soon after birth. 6. During development of the sheep, the incorporation rate of [¹⁴C]galactose into glycogen in liver slices is highest in foetal sheep and decreases with increasing age of the animal. 7. These findings are discussed with reference to the changing pattern of carbohydrate metabolism during neonatal development of liver in the sheep.     

Changes in Amino Acid Metabolism in vivo with Time after Feeding

Changes in the metabolism in vivo of amino acids with the lapse of time after feeding a diet were investigated by measuring the incorporation of ¹⁴C into some body components one hour after injection with ¹⁴C-amino acid mixture.

The incorporation of ¹⁴C into protein in the liver and carcass was rather constant, but that into blood sugar, liver glycogen, and lipids in the liver and carcass showed a change with the lapse of time after feeding a 25% casein diet or a protein-free diet. The incorporation of ¹⁴C into liver glycogen was stimulated shortly after feeding, but it was reduced at 7 hr, when a large amount of glycogen was still in the liver. On the contrary, the specific activity of blood sugar increased with the lapse of time after feeding. The conversion of ¹⁴C-amino acids into lipids in the liver and carcass was stimulated shortly after feeding.

The incorporation of ¹⁴C into protein was higher in the rats fed the protein-free diet than in those fed the 25% casein diet, and the higher incorporation was partly counterbalanced by the lower incorporation of ¹⁴C into lipids and glycogen in the rats fed the protein-free diet.     

Increased gluconeogenesis in rats exposed to hyper-G stress

The role of gluconeogenesis on the increase in plasma glucose and liver glycogen of rats exposed to hyper-G (radial acceleration) stress was determined. Overnight-fasted, male Sprague-Dawley rats (250-300 g) were injected i.p. with uniformly labeled 1 4C lactate, alanine, or glycerol (5 microCi/rat) and immediately exposed to 3.1G for 0.25, 0.50, and 1.0 hr. 1 4C incorporation of the labeled substrates into plasma glucose and liver glycogen was measured and compared to uncentrifuged control rats injected in a similar manner. Significant increases in 1 4C incorporation of all three labeled substrates into plasma glucose were observed in centrifuged rats at all exposure periods; 1 4C incorporation into liver glycogen was significantly increased only at 0.50 and 1.0 hr. The i.p. administration (5 mg/100-g body wt) of 5-methoxyindole-2-carboxylic acid, a potent gluconeogenesis inhibitor, prior to centrifugation blocked the increase in plasma glucose and liver glycogen during the first hour of centrifugation. The increase in plasma glucose and liver glycogen was also abolished in adreno-demedullated rats exposed to centrifugation for 1.0 hr. Propranolol, a beta-adrenergic blocker, suppressed the increase in plasma glucose of rats exposed to centrifugation for 0.25 hr. From the results of this study, it is concluded that the initial, rapid rise in plasma glucose as well as the increase in liver glycogen of rats exposed to hyper-G stress can be attributed to an increased rate of gluconeogenesis, and that epinephrine plays a dominant role during the early stages of exposure to centrifugation.     

Rapid effects of insulin and glucose on the hepatic incorporation of gluconeogenic substrates into glyceride glycerol and glycogen

1. The hepatic utilization of gluconeogenic substrates was investigated shortly after portal infusion of either insulin or glucose in fasted rats. 2. After 20 min of insulin infusion blood glucose concentration decreased. However, neither glucose generation from precursors such as alanine or pyruvate nor their incorporation into fatty acids was modified. Under these conditions, insulin rapidly increased the incorporation of gluconeogenic substrates into the hepatic glyceride glycerol fraction. Insulin treatment led to a decrease in substrate incorporation into liver glycogen. 3. After 20 min of portal glucose infusion both plasma insulin and glucose concentrations increased and the incorporation of pyruvate into hepatic glyceride glycerol and into glycogen was also stimulated. 4. A close relationship was observed between blood glucose concentrations and the level of incorporation of gluconeogenic substrates into liver glycogen. 5. In conclusion, during fasting insulin stimulates the incorporation of gluconeogenic substrates into the glycerol moiety of hepatic glycerides, which may be the preferential mechanism through which fatty acid esterification is accomplished during refeeding. This effect of insulin is rapid and detected even before other classical modifications induced by the hormone such as gluconeogenesis inhibition or lipogenesis activation. Furthermore, the effect is not related to insulin-induced hypoglycemia since glucose infusion mimics insulin action on glyceride glycerol synthesis.     

The distribution of glucosamine in mammalian glycogen from different species, organs and tissues

The reasons for the occurrence of trace amounts of glucosamine in animal liver glycogens have been explored. Human liver glycogen is now shown to contain this amino sugar. Galactosamine, known to be the source of the incorporated glucosamine, is found to give rise to glucosamine in glycogen when administered orally, or as the N-acetyl derivative. The rabbit can also incorporate glucosamine into kidney glycogen but not into glycogen in heart or skeletal muscle. These experiments led to the discovery that glucosamine is incorporated into rabbit liver glycogen in such a way that there is intermolecular heterogeneity in the content of glucosamine, suggesting that there exists more than one pool of liver glycogen.     

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Glycogen synthesis stimulation by adenylate cyclase inhibition in rat epididymal adipocytes

The effects of adenylate cyclase inhibition on the transport of glucose and fructose and their incorporation into glycogen were investigated in order to assess the extent to which lowered cAMP levels can take part in the various components of glycogen synthesis regulation in isolated rat epididymal adipocytes. The dose-response characteristics of (R)-N-(2-phenylisopropyl)adenosine (PIA), a potent and specific adenylate cyclase inhibitor, on glycogen synthesis were compared with those effectively inhibiting lipolysis, a measure of functional cAMP levels. PIA had no effect on basal glucose or fructose transport but stimulated glucose and fructose incorporation into glycogen. Their respective incorporation was 10 and 69% of that achieved in the presence of insulin. These effects of PIA were shown to be in part the result of increased glycogen synthase I activity. PIA was 20% as effective as insulin in this action. Thus, were insulin to lower cAMP levels and/or inhibit cAMP-dependent protein kinase, this action would be irrelevant to glucose transport but would contribute to the stimulation of glycogen metabolism. However, an additional mechanism(s) involving neither increased glucose transport nor lowered cAMP levels is required to account for the full action of insulin. Fat cells in the absence of medium glucose and in the presence of 10(-7) M PIA and adenosine deaminase constitute a system functionally depleted of cAMP where this mechanism can be studied in isolation.     

Effect of insulin on glycogen metabolism in isolated catfish hepatocytes

Insulin effect on carbohydrate metabolism in catfish hepatocytes consisted of a significant decrease of cell glycogen concentration both in the absence and in the presence of glucose in the medium. The hormone did not influence either the output of glucose from the cell or the intracellular glucose level. Experiments with radioactive glucose showed a very low uptake of the sugar by the hepatocytes; correspondingly the incorporation of radioactivity into glycogen was very low and not influenced by insulin. The glycogen content in catfish liver cells was influenced by the hormone in the opposite way to rat liver cells.     

Effects of fasting and refeeding on the in vitro insulin sensitivity of rat aorta

Rat aorta responds to refeeding after a fast in a manner similar to adopose tissue and liver by developing an enhanced capacity for lipogenesis and glycogen synthesis from glucose. The in vitro incorporation of D-U-14C-glucose into aortic triglycerides and glycogen was two- to four-fold higher in rats refed for three to five days after a three day fast than in ad libitum fed controls. Insulin significantly stimulated this incorporation only during refeeding for three days after a three-day fast. The glycogen synthesizing system appeared to be stimulated and to become sensitive to insulin earlier in the refeeding process than did the lipogenic system. The in vitro incorporation of 14C-glucose into aortic phospholipids was less affected by the nutritional state of the animal, and was not stimulated by insulin at any stage of the experiment. Possible mechanisms for the development of insulin supersensitivity and the implications for lipid accumulation in the artery wall are discussed.     

Elucidation of the role of fructose 2,6-bisphosphate in the regulation of glucose fluxes in mice using in vivo (13)C NMR measurements of hepatic carbohydrate metabolism.

Fructose 2,6-bisphosphate (Fru-2,6-P2) plays an important role in the regulation of major carbohydrate fluxes as both allosteric activator and inhibitor of target enzymes. To examine the role of Fru-2,6-P2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru-2,6-P2 levels were elevated in ADM mice with adenovirus-mediated overexpression of a double mutant bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (n = 6), in comparison to normal control mice (control, n = 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13C NMR including 3D localization in conjunction with [1-13C]glucose infusion. In addition to glycogen C1, the C6 and C2-C5 signals were measured simultaneously for the first time in vivo, which provide the basis for the estimation of direct and indirect synthesis of glycogen in the liver. The rate of label incorporation into glycogen C1 was not different between the control and ADM group, whereas the rate of label incorporation into glycogen C6 signals was in the ADM group 5.6 +/- 0.5 micro mol.g-1.h-1, which was higher than that of the control group of 3.7 +/- 0.5 micro mol.g-1.h-1 (P < 0.02). The rates of net glycogen synthesis, determined by the glycogen C2-C5 signal changes, were twofold higher in the ADM group (P = 0.04). The results provide direct in vivo evidence that the effects of elevated Fru-2,6-P2 levels in the liver include increased glycogen storage through indirect synthesis of glycogen. These observations provide a key to understanding the mechanisms by which elevated hepatic Fru-2,6-P2 levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus.     

Hormonal effects on gluconeogenesis from (U-14C)glutamate in rainbow trout (Salmo gairdneri)

Blood glucose was significantly decreased by insulin (4 I.U./kg). Glucagon (1 mg/kg) and Cortisol (5 mg/kg) administration produced a significant hyperglycaemia. Insulin administration did not modify liver glycogen levels. Glucagon showed a marked liver glycogen mobilization. Cortisol stimulated liver glycogen deposition. Insulin and Glucagon showed a significant inverse effect on gluconeogenesis from (U-14C)glutamate, decreasing and increasing 14C-glucose formation respectively. Hormonal treatments did not influence the very low levels of incorporation of (U-14C)glutamate into liver and muscle glycogen.     

Metabolic heterogeneity in rabbit brain glycogen

Glycogen has been carefully isolated from rabbit brain tissue and found to be of significantly greater molecular size (up to approx. 100 MDa) and heterogeneity than previously reported. The incorporation of radioisotope from glucose, pyruvate or acetate precursor has been shown to be non-uniform, being similar to the metabolic inhomogeneity observed in other tissues. Physicochemical studies have shown the gross hydrodynamic structure of the glycogen to be inhomogeneous and to differ significantly from that of liver glycogen.     

Inhibitory effect of salicylate on the incorporation of l-[U-14C]leucine into the protein of rat tissue preparations in vitro

1. The inhibitory effect of salicylate, in concentrations ranging from 0.1 to 20mm, on the incorporation of radioactivity from l-[U-(14)C]leucine into the protein of isolated rat diaphragm muscle and of cell-free systems from rat liver was studied. 2. The lowest salicylate concentrations producing significant inhibitions of amino acid incorporation were as follows: isolated rat diaphragm, 0.1mm; rat-liver mitochondrial-microsomal system, 0.1mm; rat-liver microsomal system, 0.3mm. 3. Salicylate concentrations of 2.5mm and above were found to inhibit creatine-kinase activity in vitro.     

STUDIES OF NATIVE GLYCOGEN ISOLATED FROM SYNCHRONIZED TETRAHYMENA PYRIFORMIS (HSM)

Native glycogen was isolated from Tetrahymena pyriformis (HSM) by isopycnic centrifugation in cesium chloride density gradients. A density of 1.62 to 1.65 was isopycnic for glycogen. Most of the banded glycogen existed as 35 to 40 mµ particles which had a sedimentation coefficient of 214. These particles were composed of aggregates of 2 to 3 mµ spherical particles. Extraction of glycogen with hot alkali reduced the sedimentation coefficient of native glycogen from 214 to 64.7 and the particle diameter from approximately 40 to 20 mµ and smaller. Cell division was synchronized by a repetitive 12-hour temperature cycle, and glycogen was measured at several times during the cell cycle. The temperature cycle consisted of 9.5 hours at 12°C and 2.5 hours at 27°C. Approximately 90 per cent of the cells divided during the last 1.5 hours of the warm period. The carbohydrate/protein ratio of cells at the end of the cold period was 0.27 and was reduced slightly during the warm period. Glucose was incorporated into glycogen during both periods, although the rate of incorporation was greater during the warm period. No preferential incorporation on the basis of particle size was noted. Incorporation was measured in both native glycogen and KOH-extracted glycogen. Tetrahymena glycogen is compared with rat liver glycogen previously isolated by similar procedures, and the significance of using combined rate-zonal and isopycnic centrifugation for isolating native glycogen is discussed.