Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats.

1. Net glycogen accumulation was measured in sequentially removed samples during perfusion of the liver of starved streptozotocin-diabetic rats, and shown to be significantly impaired, compared with rates in normal (starved) rats. 2. In perfusions of normal livers with glucose plus C3 substrates, there was an increase in the proportion of glycogen synthetase 'a', compared with that in the absence of substrates. This response to substrates, followed in sequential synthesis and enzymic sensitivity in the perfused liver of diabetic rats were reversed by pretreatment in vivo with glucose plus fructose, or insulin. Glucose alone did not produce this effect. 4. Glucose, fructose, insulin or cortisol added to e perfusion medium (in the absence of pretreatment in vivo) did not stimulate glycogen synthesis in diabetic rats. 5. In intact diabetic rats, there was a decline in rates of net hepatic glycogen accumulation, and the response of glycogen synthetase to substrates. The most rapid rates of synthesis were obtained after fructose administration. 6. These results demonstrate that there is a marked inherent impairment in hepatic glycogen synthesis in starved diabetic rats, which can be rapidly reversed in vivo but no in perfusion. Thus hepatic glycogen synthesis does not appear to be sensitive to either the short-term direct action of insulin (added alone to perfusions) of to long-term insulin deprivation in vivo. The regulatory roles of substrates, insulin and glycogen synthetase in hepatic glycogen accumulation are discussed.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells.

Rats trained to the "8 + 16" controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from liver containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase a. Attempts of influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat.

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9alpha-fluoro-11beta, 21-dihydroxy-16alpha,17alpha-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells

Rats trained to the “8 + 16” controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from livers containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase $\text{a}$. Attempts to influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.     

Short-term stimulation of net glycogen production by insulin in rat hepatocytes

Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.     

Fatty acid synthesis in the perfused liver of adrenalectomized rats.

1. Fatty acid synthesis, measured in the perfused liver of fed adrenalectomized rats with 3H2O and 14C-labelled precursors, was less than in control sham-operated rats. 2. This defect was more extensive for synthesis of fatty acids incorporated into triacylglycerols than into phospholipids. 3. There was impairment in desaturation and export of newly synthesized fatty acid. 4. Fatty acid synthesis and desaturation were restored to normal rates 5h after treatment with cortisol in vivo. 5. Fatty acid synthesis was seasonally variable, being highest in the winter; the impairment after adrenalectomy was observed in all seasons. 6. In perfusions with oleate (0.7 mM), no further impairment in fatty acid synthesis was discerned in livers from adrenalectomized rats, in which the rate resembled that in control livers. 7. No defect in the incorporation of oleate into glycerides was discerned in livers from adrenalectomized rats. 8. Cortisol exerted no stimulatory effect on fatty acid synthesis when added to perfusion media. 9. The impairment in hepatic lipogenesis, demonstrable after adrenalectomy, shows that adrenal glucocorticoids promote hepatic capacity for fatty acid synthesis de novo, at least in intact non-diabetic rats. It is suggested that this effect is mediated by insulin, perhaps through direct action on the liver.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Effect of a meal feeding schedule on hepatic glycogen synthesis and gluconeogenesis in rats

We investigated the effect of a meal feeding schedule (MFS) on food intake, hepatic glycogen synthesis, hepatic capacity to produce glucose and glycemia in rats. The MFS comprised free access to food for a 2-hour period daily at a fixed mealtime (8.00-10.00 a.m.) for 13 days. The control group was composed of rats with free access to food from day 1 to 12, which were then starved for 22 h, refed with a single meal at 8.00-10.00 a.m. and starved again for another 22 h. All experiments were performed at the meal time (i.e. 8.00 a.m.). The MFS group exhibited increased food intake and higher glycogen synthase activity. Since gluconeogenesis from L-glutamine or L-alanine was not affected by MFS, we conclude that the increased food intake and higher glycogen synthase activity contributed to the better glucose maintenance showed by MFS rats at the fixed meal time.     

The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat.

Hepatocytes from adrenalectomized 48 h-starved rats responded to increasing glucose concentrations with a progressively more complete inactivation of phosphorylase. Yet no activation of glycogen synthase occurred, even in a K+-rich medium. Protein phosphatase activities in crude liver preparations were assayed with purified substrates. Adrenalectomy plus starvation decreased synthase phosphatase activity by about 90%, but hardly affected phosphorylase phosphatase activity. Synthase b present in liver extracts from adrenalectomized starved rats was rapidly and completely converted into the a form on addition of liver extract from a normal fed rat. Glycogen synthesis can be slowly re-induced by administration of either glucose or cortisol to the deficient rats. In these conditions there was a close correspondence between the initial recovery of synthase phosphatase activity and the amount of synthase a present in the liver. The latter parameter was strictly correlated with the measured rate of glycogen synthesis in vivo. The decreased activity of synthase phosphatase emerges thus as the single factor that limits hepatic glycogen deposition in the adrenalectomized starved rat.     

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

Insulin infusion through the portal vein immediately after a pulse of [3-14C]pyruvate in 24 hr starved rats enhanced the appearance of [14C]glucose at 2, 5 and 10 min and glucose specific activity at 1, 2 and 20 min in blood collected from the cava vein at the level of the suprahepatic veins. Insulin infusion for 5 min decreased liver pyruvate concentration and enhanced both liver and plasma lactate/pyruvate ratio, and it decreased the plasma concentration of all amino acids. When insulin was infused together with glucose, [14C]glucose levels and glucose specific activity decreased in blood but there was a marked increase in liver [14C]glycogen, glycogen specific activity and glycogen concentration, and an increase in liver lactate/pyruvate ratio. The effect of insulin plus glucose infusion on plasma amino acids concentration was smaller than that found with insulin alone. It is proposed that insulin effect enhancing liver gluconeogenesis is secondary to its effect either enhancing liver glycolysis which modifies the liver's cytoplasmic oxidoreduction state to its more reduced form, increasing liver amino acids consumption or both. In the presence of glucose, products of gluconeogenesis enhanced by insulin are diverted into glycogen synthesis rather than circulating glucose. This together with results of the preceding paper (Soley et al., 1985), indicates that glucose enhances liver glycogen synthesis from C3 units in the starved rat, the process being further enhanced in the presence of insulin.     

Stimulation by vasopressin of glycogen breakdown and gluconeogenesis in the perfused rat liver

1. Vasopressin (anti-diuretic hormone, [8-arginine]vasopressin) stimulated the breakdown of glycogen in perfused livers of fed rats, at concentrations (50-600muunits/ml) that have been reported in the blood of intact rats, especially during acute haemorrhagic shock. 2. In perfused livers from starved rats, vasopressin (30-150muunits/ml) stimulated gluconeogenesis from a mixture of lactate, pyruvate and glycerol. 3. Vasopressin prevented accumulation of liver glycogen in the perfused liver of starved rats, or in starved intact rats. 4. The action of vasopressin on hepatic carbohydrate metabolism thus resembles that of glucagon; the minimum effective circulating concentrations of these hormones are of the same order (100pg/ml). 5. The stimulation of hepatic glucose output by vasopressin is discussed in connexion with the release of glucose and water from the liver.     

Nucleotide sequences of genes encoding the type II chloramphenicol acetyltransferases of Escherichia coli and Haemophilus influenzae, which are sensitive to inhibition by thiol-reactive reagents.

A gastric [U-14C]glucose load (4.8 mg/g body wt.) was delivered to unrestrained post-absorptive or 30 h-starved rats bearing peripheral and portal vein catheters and continuously perfused with [3-3H]glucose, in order to compare their metabolic and hormonal responses. In the basal state, portal and peripheral glycaemia were less in starved rats than in rats in the post-absorptive period (P less than 0.01), whereas blood lactate was similar. Portal insulinaemia (P less than 0.05) and protal glucagonaemia (P less than 0.005) were lower in starved rats, but insulin/glucagon ratio was higher in post-absorptive rats (P less than 0.005). The glucose turnover rate was decreased by starvation (P less than 0.005). After glucose ingestion, blood glucose was similar in post-absorptive and starved rats. A large portoperipheral gradient of lactate appeared in starved rats. Portal insulinaemia reached a peak at 9 min, and was respectively 454 +/- 68 and 740 +/- 65 mu-units/ml in starved and post-absorptive rats. Portal glucagonaemia remained stable, but was higher in post-absorptive rats (P less than 0.05). At 60 min after the gastric glucose load, 30% of the glucose was delivered at the periphery in both groups. The total glucose appearance rate was higher in starved rats (P less than 0.05), as was the glucose utilization rate (P less than 0.05), whereas the rate of appearance of exogenous glucose was similar. This was due to a non-suppressed hepatic glucose production in the starved rats, whereas it was totally suppressed in post-absorptive rats. At 1 h after the glucose load, the increase in both liver and muscle glycogen concentration was greater in starved rats. Thus short-term fasting induces an increased portal lactate concentration after a glucose load, and produces a state of liver insulin unresponsiveness for glucose production, whereas the sensitivity of peripheral tissues for glucose utilization is unchanged or even increased. This might allow preferential replenishment of the peripheral stores of glycogen.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Glucose turnover in the post-absorptive rat and the effects of halothane anaesthesia.

1. Rates and rate coefficients of glucose utilization and replacement in post-absorptive rats, either conscious or under halothane anaesthesia, were determined in a thermoneutral environment by using [5-3H]- and [U-14C]glucose. Label was not injected into rats under halothane until about 0.5h after anaesthesia was initiated. 2. Comparison with the results for 24h-starved rats in the preceding paper [Heath et al. (1977) Biochem. J. 162, 643-651] showed that insulin concentrations were considerably higher but rate coefficients for glucose utilization were little altered in post-absorptive rats. Sensitivity to insulin was thus considerably increased by a 24h period of starvation in the rat. 3. Fractional recycling of glucose carbon in post-absorptive rats was under one-half of that in starved rats, reflecting the larger contribution of liver glycogenolysis to glucose production in the former. 4. In post-absorptive rats halothane decreased the mean rate of glucose utilization by about 17%. This decrease was associated with an increase in mean plasma insulin concentration, showing that halothane decreased sensitivity to insulin. 5. Recycling was slightly increased by halothane, indicating that the contribution of liver glycogen to the total glucogenic rate was decreased, probably because liver glycogen concentration were about 40% lower throughout the rate determinations in halothane. 6. Comparison of our results with earlier work shows that during and shortly after induction of halothane anaesthesia glucose turnover must have been greatly increased whereas from about 0.5h after induction it was decreased.     

Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

Glycogen synthesis by hepatocytes from diabetic rats.

Hepatocytes prepared from streptozotocin- and alloxan-diabetic rats starved for 24 h contain 0.5--2% wet wt. of glycogen. Glycogen synthesis in the hepatocytes from such rats, after prior depletion of the glycogen by glucagon injection, was studied. As distinct from cells from normal animals, there was no glycogen synthesis from glucose as sole substrate, even at concentrations of 60 mM. When supplied with glucose, a gluconeogenic precursor (lactate, dihydroxyacetone or fructose), and with glutamine there was concurrent synthesis of glucose and of glycogen. Without glutamine there was little or no glycogen synthesis. The rate of glycogen formation was in the same range as for cells from control rats. Glutamine addition markedly activated glycogen synthase in cells of starved diabetic rats, but there was no effect on phosphorylase. We obtained very little synthesis of glycogen with hepatocytes from fed diabetic rats, whereas with normal animals, synthesis by such cells equals or exceeds that obtained from starved rats. The conversion of synthase b (inactive) into the active form was studied in rat liver homogenates. The activation of the synthase in cells from starved diabetic rats is somewhat less than that from normal animals, but that from fed diabetic rats is markedly decreased compared with that in livers of fed control animals or that of starved diabetic animals.     

Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes.

Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.     

Glycogen synthesis in the perfused liver of the starved rat

1. In the isolated perfused liver from 48h-starved rats, glycogen synthesis was followed by sequential sampling of the two major lobes. 2. The fastest observed rates of glycogen deposition (0.68–0.82μmol of glucose/min per g fresh liver) were obtained in the left lateral lobe, when glucose in the medium was 25–30mm and when gluconeogenic substrates were present (pyruvate, glycerol and serine: each initially 5mm). In this situation there was no net disappearance of glucose from the perfusion medium, although ¹⁴C from [U-¹⁴C]glucose was incorporated into glycogen. There was no requirement for added hormones. 3. In the absence of gluconeogenic precursors, glycogen synthesis from glucose (30mm) was 0–0.4μmol/min per g. 4. When livers were perfused with gluconeogenic precursors alone, no glycogen was deposited. The total amount of glucose formed was similar to the amount converted into glycogen when 30mm-glucose was also present. 5. The time-course, maximal rates and glucose dependence of hepatic glycogen deposition in the perfused liver resembled those found in vivo in 48h-starved rats, during infusion of glucose. 6. In the perfused liver, added insulin or sodium oleate did not significantly affect glycogen synthesis in optimum conditions. In suboptimum conditions (i.e. glucose less than 25mm, or with gluconeogenic precursors absent) insulin caused a moderate acceleration of glycogen deposition. 7. These results suggest that on re-feeding after starvation in the rat, hepatic glycogen deposition could be initially the result of continued gluconeogenesis, even after the ingestion of glucose. This conclusion is discussed, particularly in connexion with the role of hepatic glucokinase, and the involvement of the liver in the glucose intolerance of starvation.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [¹⁴C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. (1) Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. (2) Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. (3) Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. (4) Regulation of insulin-independent glycogenesis in response to an increase from 5 to 10 mM glucose, and of insulin-dependent glycogen synthesis were different. Since the effects of this ‘physiological’ increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.