Glycogen synthesis from pyruvate in the periportal and from glucose in the perivenous zone in perfused livers from fasted rats

The isolated liver of 24 h fasted rats was perfused in a non-recirculating manner in the orthograde or retrograde direction with media containing glucose and/or gluconeogenic precursors. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the only exogenous substrate glycogen was formed exclusively in the perivenous area during both orthograde and retrograde perfusion. With gluconeogenic precursors as the exogenous substrates glycogen was deposited in the periportal zone during orthograde perfusion and in the intermediate zone during retrograde perfusion. Supply of glucose and gluconeogenic substrates initiated glycogen synthesis only in the upstream region, i.e. in the periportal zone during orthograde and in the perivenous zone during retrograde perfusion. This localization of glycogen synthesis was probably due to an unavoidable, insufficient oxygen supply of the respective downstream area. In general, the results confirm the hypothesis that periportal and perivenous glycogen was synthesized from different substrates.     

N-acetylglucosamine-insensitive glucose phosphorylation in isolated hepatocytes from rats fasted for 48 h

Glucose phosphorylation in isolated hepatocytes was studied by the release of 3H from D-[2-3H]glucose. Glucokinase activity was decreased by fasting rats for 48 h and was further reduced in cells by adding 30 mM GlcNAc, a potent competitive inhibitor. Although this treatment resulted in the loss of more than 97% of glucokinase activity in hepatocytes, glucose phosphorylation proceeded at an appreciable rate. These observations demonstrate the involvement of a high -K0.5 enzyme system in addition to glucokinase in hepatocyte glucose phosphorylation.     

Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers

Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition.     

Decreased response to cAMP in the glucose and glycogen catabolism in perfused livers of Walker-256 tumor-bearing rats

The hepatic response to cyclic adenosine monophosphate (cAMP) and N6-monobutyryl-cAMP (N6-MB-cAMP) in the glucose and glycogen catabolism and hepatic glycogen levels were evaluated in Walker-256 tumor-bearing rats, on days 5 (WK5), 8 (WK8), and 11 (WK11) after the implantation of tumor. Rats without tumor fed ad libitum (fed control rats) or that received the same daily amount of food ingested by anorexics tumor-bearing rats (pair-fed control rats) or 24 h fasted (fasted control rats) were used as controls. Glucose and glycogen catabolism were measured in perfused liver. Hepatic glycogen levels were lower (p < 0.05) in WK5, WK8, and WK11 rats in comparison with fed control rats, but not in relation to the pair-fed control rats. However, the stimulatory effect of cAMP (3 and 9 μM) in the glycogen catabolism was lower (p < 0.05), respectively, in WK5 and WK8 rats compared to the pair-fed and fed control rats. Accordingly, the suppressive effect of cAMP (6 μM) in the glucose catabolism, under condition of depletion of hepatic glycogen (24 h fast), was lower (p < 0.05) in WK5 and WK11 rats than in fasted control rats. Similarly, the suppressive effect of N6-MB-cAMP (1 μM), a synthetic analogue of cAMP that it is not degraded by phosphodiesterase 3B (PDE3B), in the glucose catabolism was lower (p < 0.05) in WK5 rats compared to fasted control rats. In conclusion, livers of Walker-256 tumor-bearing rats showed lower response to cAMP in the glucose and glycogen catabolism in various stages of tumor development (days 5, 8 and 11), which was probably not due to the lower hepatic glycogen levels nor due to the increased activity of PDE3B.     

Insulin stimulates triacylglycerol secretion by perfused livers from fed rats but inhibits it in livers from fasted or insulin-deficient rats implications for the relationship between hyperinsulinaemia and hypertriglyceridaemia.

We determined whether the direction of the acute effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state or on the in vitro experimental system used. The effect of insulin on triacylglycerol secretion was studied using perfused livers isolated from rats under three metabolic conditions: fed normo-insulinaemic, 24-h fasted and fed, streptozotocin-diabetic (insulin-deficient). Insulin acutely activated triacylglycerol secretion (by 43%) in organs from fed, normo-insulinaemic animals, whereas it inhibited triacylglycerol secretion in livers isolated from fasted or insulin-deficient rats (by 30 and 33%, respectively). By contrast, in 24-h-cultured hepatocytes insulin invariably acutely inhibited triacylglycerol secretion irrespective of the metabolic state of the donor animals. It is concluded that the use of perfused livers enables the observation of a switch in the direction of insulin action on hepatic triacylglycerol secretion from stimulatory, in the normo-insulinaemic state, to inhibitory in the fasting or insulin-deficient state. The possible implications of this switch for the relationship between hyperinsulinaemia, increased hepatic very-low-density lipoprotein-triacylglycerol secretion and hypertriglyceridaemia observed in vivo are discussed.     

Circadian rhythm of blood glucose and tissue glycogen in fed and fasted rats

Blood glucose and tissue glucogen circadian rhythms were determined in male Wistar rats adapted 3 weeks to an artificial lighting regimen of 12 hours' light and 12 hours' darkness. Over a period of 24 hours we examined at 3-hour intervals the blood glucose concentration and the glycogen content of the liver, heart, skeletal muscle (quadriceps femoris) and white (epididymal) and brown (interscapular) adipose tissue of fed rats and rats fasted for 24 hours. The experiments were carried out in the autumn and the results were evaluated statistically by an analysis of variance and the cosinor test. The blood glucose level and the glycogen concentration in all the given tissues, in both fed and starved rats, displayed rhythmic oscillations with a 24- or 12-hour period in the course of the day, with the exception of glycogen in the white adipose tissue of fed rats, in which cosinor analysis failed to demonstrate any rhythm. One day's fasting did not affect the character of circadian rhythm.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.     

Fructose administration stimulates glucose phosphorylation in the livers of anesthetized rats

A method allowing one to measure the rate of glucose phosphorylation in the livers of anesthetized rats is described. Upon injection of [2-3H]glucose into the portal vein, about 90% of the radioactivity remained in the liver for approximately 30 s. The proportion of radioactivity accounted for by tritiated water increased linearly at a rate of about 5%/min in control animals. Fructose injected into the penile vein stimulated this rate up to 2.2- and 2.7-fold in fed and overnight starved rats respectively, a maximal effect being observed at a dose of 50 mg/kg under both conditions. Fructose was also active when administered by intragastric infusion. The ketose caused increases in the concentration of fructose 1-phosphate, which reached values known to relieve the inhibition exerted on glucokinase by its regulatory protein.     

Multiple requirements for glycogen synthesis by hepatocytes isolated from fasted rats

Glycogen synthesis from various combinations of substrates by hepatocytes isolated from rats fasted 24 h was studied. As reported by Katz et al. (Katz, J., Golden, S., and Wals, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3433-3437), appreciable rates of glycogen synthesis occurred only in the presence of gluconeogenic precursors and one of several amino acids, which includes L-glutamine. L-Leucine had negligible effects on glycogen synthesis from 20 mM dihydroxyacetone and/or 15 mM glucose when L-glutamine was not added to the medium. In the presence of 10 mM L-glutamine, L-leucine greatly increased glycogen synthesis from these substrates. alpha-Ketoisocaproate was ineffective, as was oleate. NH4Cl depressed glycogen synthesis from 10 mM glucose plus 20 mM dihydroxyacetone in the absence of added L-glutamine and enhanced that in its presence, but these effects were weak compared to those of L-leucine. The amino acid analogues L-norvaline and L-norleucine exerted effects that were similar to those exerted by L-leucine. Under all conditions studied, cycloheximide and puromycin inhibited net glycogen synthesis. Cycloheximide did not stimulate gluconeogenesis from dihydroxyacetone, or phosphorylase in hepatocytes from starved rats, or glycogenolysis in hepatocytes from fed rats. Puromycin, however, stimulated glycogenolysis in hepatocytes from fed rats. Glycogen synthesis from 20 mM dihydroxyacetone proceeds with a pronounced initial lag phase that can be shortened by incubation of cells with glutamine plus leucine before addition of dihydroxyacetone. Concurrent measurements of glycogen synthesis, glycogen synthase, and gluconeogenesis under different conditions reveal that in addition to protein synthesis, activation of glycogen synthase, which must occur to allow glycogen synthesis in hepatocytes, requires a second component which can be satisfied by addition of dihydroxyacetone or fructose to the cells.     

Fatty acid metabolism of the perfused caudate lobe from livers of fed and fasted non-pregnant and fasted late pregnant ewes

1. Fatty acid metabolism has been compared in perfused liver lobes from fed and fasted non-pregnant sheep and fasted pregnant sheep to provide further information on the control of ketogenesis in this species. 2. Ketogenesis from exogenous palmitate was greatest in lobes from fasted pregnant sheep and least in lobes from fed non-pregnant sheep, whereas rates of ketogenesis from exogenous octanoate (0.4 mM) were similar in lobes from sheep in all three states. 3. High rates of ketogenesis from endogenous fatty acids occurred in perfused lobes from fasted pregnant sheep, apparently owing to enhanced lipolysis. 4. Activities of glycerol-3-phosphate acyltransferase, carnitine palmitoyl transferase (CPT) and other enzymes involved in ketone production did not change with physiological state; sheep differ markedly from rats in this respect. 5. The results suggest that the primary point of control of ketogenesis within the liver of sheep is at the level of CPT; the lack of change in maximum CPT activity suggests that control by modulators of this enzyme activity is even more important in sheep than in rats.     

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rate is blocked by protein synthesis inhibitor

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3′,5′-monosphosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity.In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.