The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid

The biosynthesis of N-glycoloylneuraminic acid in fractionated porcine submandibular glands was investigated. The following substrates: [3H]N-acetylmannosamine, free [14C]N-acetylneuraminic acid, CMP-[14C]N-acetylneuraminic acid, [14C]N-acetylneuraminic acid linked alpha(2----3) to galactose residues, or alpha(2----6) to Gal-beta(1----4)-GlcNAc residues of porcine submandibular mucin and [14C]N-acetylneuraminic acid linked alpha(2----6) to GalNAc residues of ovine submandibular gland mucin were incubated, in the presence of cofactors, with the soluble protein, heavy membrane and microsomal fractions of porcine submandibular glands. Radio thin-layer chromatographic analysis revealed that only one substrate, CMP-[14C]N-acetylneuraminic acid, was hydroxylated. The product was identified as CMP-[14C]N-glycoloylneuraminic acid by (i) co-chromatography with non-radioactive CMP-N-glycoloylneuraminic acid standard, (ii) acid hydrolysis to free [14C]N-glycoloylneuraminic acid, (iii) alkaline hydrolysis to yield N-glycoloylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid and (iv) transfer of [14C]N-glycoloylneuraminic acid to asialo-fetuin by sialyltransferase. 85% of CMP-N-acetylneuraminic acid hydroxylase activity was present in the soluble protein fraction, with small amounts of activity in the two particulate fractions. The CMP-N-acetylneuraminic acid hydroxylase in the soluble protein fraction had an absolute requirement for Fe2+ ions and a reducing cofactor. NADPH and NADH were by far the most effective cofactors, smaller amounts of hydroxylation could, however, be supported by ascorbic acid and 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin.     

Evidence for the glycoprotein nature of retina glycogen

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.     

The metabolic significance of pentose cycle measurements in perfused liver

The controversial dissension concerning the nature of the pentose cycle in liver is investigated. The metabolism of [2-14C]Glc and [1-14C]Rib in chronically perfused normal and regenerating rabbit liver and acutely perfused rat liver are used to test the mechanistic predictions and contribution of the F-type pentose cycle. 14C was traced in Glc, Glc 6-P, Fru 6-P, glycogen and Rib 5-P. None of the data complied with the critical theoretical limits set for the C-1/C-3 ratio (the identity badge of the F-type pentose cycle or pathway) for all values of F-type PC from 0-100%. Thus apparent F-type PC measurements using the Katz & Wood method gave a wide scatter of calculated values. The 14C distributions in Rib 5-P do not conform with the predictions of the F-type PC but are in agreement with the many previous results of similar experiments reported by Hiatt and co-workers. In perfused rat liver the C-1/C-3 constants in Glc 6-P and glycogen also failed to conform with F-PC theory following the metabolism of [2-14C]Glc. The metabolism of [5-14C]Glc and distribution of 14C in Glc 6-P and glycogen showed that L-type PC was 18%, in close agreement with a previous published value of 22% for rat hepatocytes. Metabolism of [6-14C]Glc and [4-14C]Glc (as [4,5,6-14C]Glc) showed that Pyruvate Recycling was active in perfused rat liver. None of the data from these comprehensive investigations can confirm the results of the recent study reported by the Landau laboratory on the pentose pathway metabolism of Glc and Rib in perfused rat liver.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

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.     

Characteristics of glycogen synthesis from [1-14C]glucose and its labeled precursors in the piglet liver

The in vivo experiments have established that the rapid decrease in the glycogen content in the liver of piglets during the first 24 hours after birth is associated with the reduction of the degree of label inclusion from [1-14C]glucose into polysaccharide. The level of label inclusion from [1-14C]pyruvate and [1-14C]lactate into the liver glycogen in new-born piglets is higher than from [1-14C]alanine and [1-14C]glutamic acid. During the days immediately after birth the extension of the pool of glucogenic substrates occurs at the expense of alanine and other amino acids during catabolism of which pyruvate is formed. The degree of label inclusion from the investigated substrates into the liver glycogen of piglets of early age decreases in the series: [1-14C]glucose greater than [1-14C]lactate greater than [1-14C]pyruvate greater than [1-14C]alanine. Glutamic acid in the liver of piglets of early age is not a glucogenic substrate.     

Glucose infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

After a pulse of [3-14C]pyruvate, 24 hr starved rats were infused through the portal vein with two different doses of glucose (7.8 or 20.8 mg/min) or the medium, and blood was collected from the inferior cava vein at the level of the suprahepatic veins. The highest dose of glucose enhanced the appearance of [14C]glucose in blood from the 2nd to the 20th min after tracer delivery. It also enhanced production of [14C]glycogen and concentration of glycogen in the liver after 5 and 20 min. At 20 min of glucose infusion the appearance of [14C]glyceride glycerol in liver as well as liver lactate concentration and lactate/pyruvate ratio were increased. The low dose of glucose used enhanced liver values of [14C]glycogen, [14C]glycogen specific activity and glycogen concentration. Our results support the hypothesis that in the starved rat glucose is converted into C3 units prior to being deposited as liver glycogen and based on the liver zonation model (Jungermann et al., 1983) it is proposed that glucose stimulated gluconeogenesis by shifting the liver to the cytosolic redox state as a secondary consequence of increased glycolytic activity.     

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.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

Adrenergic inhibition of branched-chain 2-oxo acid dehydrogenase in rat diaphragm muscle in vitro.

In theory, the complete oxidation to CO2 of amino acids that are metabolized by conversion into tricarboxylic acid-cycle intermediates may proceed via their conversion into acetyl-CoA. The possible adrenergic modulation of this oxidative pathway was investigated in isolated hemidiaphragms from 40 h-starved rats. Adrenaline (5.5 microM), phenylephrine (0.49 mM) and dibutyryl cyclic AMP (10 microM) inhibited 14CO2 production from 3 mM-[U-14C]valine by 35%, 28% and 19% respectively. At the same time, these agents stimulated glycogen mobilization (measured as a decrease in glycogen content) and glycolysis (measured as lactate release). Adrenaline, phenylephrine and dibutyryl cyclic AMP did not inhibit 14CO2 production from 3 mM-[U-14C]aspartate or 3 mM-[U-14C]glutamate, although, as in the presence of valine, the agents stimulated glycogen mobilization and glycolysis. The rate of proteolysis (measured as tyrosine release in the presence of cycloheximide) was not changed by adrenaline. The data indicate that the adrenergic inhibition of 14CO2 production from [U-14C]valine was not a consequence of radiolabel dilution. Inhibition was apparently specific for branched-chain amino acid metabolism in that the adrenergic agonists also inhibited 14CO2 production from [1-14C]valine, [1-14C]leucine and [U-14C]isoleucine. Since 14CO2 production from the 1-14C-labelled substrates is a specific measure of decarboxylation in the reaction catalysed by the branched-chain 2-oxo acid dehydrogenase complex, it is at this site that the adrenergic agents are concluded to act.     

The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.     

A 42,000-Da protein in rabbit tissues and in a glycogen synthase preparation cross-reacts with antibodies to glycogenin

Rabbit skeletal muscle glycogen previously has been shown to be covalently bound to a 40,000-Da protein ("glycogenin") via a novel glucosyl-tyrosine linkage [I.R. Rodriguez and W.J. Whelan (1985) Biochem. Biophys. Res. Commun. 132, 829-836]. Antibodies raised against rabbit skeletal muscle glycogenin cross-react with a similar protein present in rabbit heart and liver glycogens, as well as with a 42,000-Da "acceptor protein" present in high-speed supernatants of rabbit muscle, heart, retina, and liver. This 42,000-Da protein incorporates [U-14C]Glc when an ammonium sulfate fraction prepared from the tissue supernatants is incubated with UDP-[U-14C]Glc. The [U-14C]Glc incorporated can be removed quantitatively by treatment with amylolytic enzymes, indicating that the [U-14C]Glc incorporation represents elongation of a preexisting glucan attached to the acceptor protein. Furthermore, a commercial preparation of rabbit skeletal muscle glycogen synthase contains this 42,000-Da protein. We propose that the 42,000-Da protein represents the free form of glycogenin in tissues, with its covalently attached glucan chain(s) providing a "primed" elongation site for glycogen synthesis.     

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.     

Glucose metabolism in the newborn rat. Temporal studies in vivo

1. The concentrations of plasma d-glucose, l-lactate, free fatty acids and ketone bodies and of liver glycogen were measured in caesarian-delivered newborn rats at time-intervals up to 4h after delivery. Glucose and lactate concentrations decreased markedly during the first hours after delivery, but there was a delay of 60-90min before significant glycogen mobilization occurred. 2. The specific radioactivity of plasma d-glucose was measured as a function of time for up to 75min after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into caesarian-delivered rats at 0, 1 and 2h after delivery. Calculations revealed that there was an appreciable rate of glucose formation at all ages studied, but immediately after delivery this was exceeded by the rate of glucose utilization. Around 2h post partum the rate of glucose utilization decreased dramatically and this coincided with a reversal of the immediately postnatal hypoglycaemia. 3. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose and liver glycogen was measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into rats immediately after delivery. The logarithm of the specific radioactivity of plasma l-[U-(14)C]lactate decreased linearly with time for at least 60min after injection and the calculated rate of lactate utilization exceeded the rate of lactate formation. 4. (14)C incorporation into plasma d-glucose was maximal from 30-60min after injection of l-[U-(14)C]lactate and the amount incorporated at 60min was 23% of that present in plasma lactate. Although (14)C was also incorporated into liver glycogen the amount was always less than 3% of that present in plasma glucose. 5. The results are discussed in relationship to the adaptation of the newly born rat to the extra-uterine environment and the possible involvement of gluconeogenesis at this time before feeding is established.     

The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists.

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.     

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle)

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs-Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 microM palmitate. Palmitate increased the insulin-stimulated [(14)C]glycogen synthesis, decreased lactate production, and did not alter D-[U-(14)C]glucose decarboxylation and 2-deoxy-D-[2,6-(3)H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1-(14)C]pyruvate decarboxylation and increased (14)CO(2) produced from [2-(14)C]pyruvate. Palmitate reduced insulin-stimulated phosphorylation of insulin receptor substrate-1/2, Akt, and p44/42 mitogen-activated protein kinases. Bromopalmitate, a non-metabolizable analogue of palmitate, reduced [(14)C]glycogen synthesis. A strong correlation was found between [U-(14)C]palmitate decarboxylation and [(14)C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6-phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin-stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin-stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.     

A rapid effect of thyroxine on accumulation of diacylglycerols and on activation of protein kinase C in liver cells

L-Thyroxine rapidly stimulated the accumulation of diacylglycerols in isolated hepatocytes and in liver when lipids were prelabeled with [14C]oleic acid or with [14C]CH3COONa. Perfusion of the liver of hypothyroid animals with L-thyroxine-containing solution or incubation of liver fragments with the hormone increased the content of diacylglycerols in the liver cells. The increase in [14C]diacylglycerol level in the liver cells was accompanied by a decrease in the level of [14C]phosphatidylcholine, whereas contents of other 14C-labeled phospholipids, such as phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4P), and phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P2), and of 14C-labeled fatty acids were the same as in the control. The L-thyroxine-induced accumulation of diacylglycerols in hepatocytes was not affected by neomycin but was inhibited by propranolol. Incubation of hepatocytes prelabeled with [14C]oleic acid with L-thyroxine and ethanol (300 mM) was accompanied by generation and accumulation of [14C]phosphatidylethanol that was partially suppressed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). L-Thyroxine was responsible for the translocation of protein kinase C from the cytosol into the membrane fraction and for a many-fold activation of the membrane-bound enzyme. D-Thyroxine failed to affect the generation of diacylglycerols in hepatocytes and the activity of protein kinase C.     

Effect of L-alanine infusion on gluconeogenesis and ketogenesis in the rat in vivo.

1. In 48 h-starved 6-week-old rats the 14C incorporation in vivo into blood glucose from a constant-specific-radioactivity pool of circulating [14c]actateconfirmed that lactate is the preferred gluconeogenic substrate. 2. Increasing the blood [alanine] to that occurrring in the fed state increased 14C incorporation into blood glucose 2.3-fold from [14c]alanine and 1.7-fold from [14c]lactate. 3. When the blood [alanine] was increased to that in the fed state, the 14C incorporation into liver glycogen from circulating [14c]alanine or [14c]lactate increased 13.5- and 1.7-fold respectively. 4. The incorporation of 14C into blood acetoacetate and 3-hydroxybutyrate from a constant-specific-radioactivity pool of circulating [14c]oleate was virtually abolished by increasing the blood [alanine] to that existing in the fed state. However, the [acetoacetate] remained unchanged, whereas [3-hydroxybutyrate] decreased, although less rapidly than did its radiochemical concentration. 5. It is concluded that during starvation in 6-week-old rats, the blood [alanine] appears to influence ketogenesis for circulating unesterfied fatty acids and inversely affects gluconeogenesis from either lactate or alanine. A different pattern of gluconeogenesis may exist for alanine and lactate as evidenced by comparative 14C incorporation into liver glycogen and blood glucose.     

N-acetylglucosaminyltransferase III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct. Application of a sensitive and specific assay by use of high-performance liquid chromatography

A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three transferase products formed from the common acceptor oligosaccharide substrate GlcNAc beta 1----2Man alpha 1----3(GlcNAc beta 1----2Man alpha 1---- 6)Man beta 1----4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breakdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.