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.     

Swelling of rat hepatocytes stimulates glycogen synthesis

In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following observations. 1) The extent of stimulation of glycogen synthesis by both metabolizable and nonmetabolizable amino acids was directly proportional to their ability to increase cell volume, except for proline, which stimulated glycogen synthesis more than could be accounted for by the increase in cell volume. 2) Both cell swelling and stimulation of glycogen synthesis by amino acids were prevented when hepatocytes were incubated in hyperosmotic media containing sucrose or raffinose. 3) Increasing the cell volume by incubating hepatocytes in Na(+)-depleted media in the absence of amino acids also stimulated glycogen synthesis. 4) Stimulation of glycogen synthesis by Na+ depletion was prevented by restoring the normal osmolarity with sucrose, but not with choline chloride which, by itself, stimulated glycogen synthesis and increased the cell volume. It is concluded that stimulation of glycogen synthesis by amino acids is due, at least in part, to an increase in hepatocyte volume resulting from amino acid uptake, and that hepatocyte swelling per se stimulates glycogen synthesis.     

Zonal distribution of glycogen synthesis in isolated rat hepatocytes

Incubation of hepatocytes isolated from fasted rats with [14C]glucose for short periods of time showed that the initial stages of glycogen synthesis occur near the plasma membrane. Incubation with [14C]glucose followed by cold glucose demonstrated that glycogen synthesis is always active at the hepatocyte periphery and that previously synthesised glycogen moves towards the centre of the cell, while its place is filled by newly synthesised molecules. However, the reverse experiment, incubation with cold glucose before addition of [14C]glucose, showed that, as glycogen synthesis progresses, it also becomes gradually active in more internal sites of the hepatocyte. These results indicate a spatial order in the synthesis of hepatic glycogen.     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

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.     

Stabilization of glycogen stores and stimulation of glycogen synthesis in hepatocytes by phenacyl imidazolium compounds

LY177507 is representative of a series of phenacyl imidazolium compounds that cause marked lowering of blood glucose levels in animal models of noninsulin-dependent diabetes mellitus. In studies conducted with isolated rat hepatocytes, LY177507 inhibited net glucose production from a variety of substrates, inhibited glycolysis from exogenous glucose and endogenous glycogen, inhibited glycogenolysis, and stimulated glycogenesis. These effects of LY177507 appear to be the consequence of activation of glycogen synthase and inactivation of glycogen phosphorylase. In vivo studies with normal fed rats demonstrated a decrease in blood glucose, an increase in hepatic glycogen stores, and an inactivation of glycogen phosphorylase. Phenacyl imidazolium compounds appear to lower blood glucose levels and affect hepatic carbohydrate metabolism by a mechanism unlike other known hypoglycemic compounds.     

Indirect versus direct routes of hepatic glycogen synthesis

During refeeding after a brief period of starvation, glucose carbon is deposited into hepatic glycogen by both a direct and an indirect route. In the indirect route glucose is first metabolized to 3-carbon precursors, which then transverse the gluconeogenic pathway before being deposited into glycogen. Recent studies have yielded widely different estimates of the percentage of glucose carbon that follows the indirect route. Work summarized here demonstrates that the relative contributions of glucose carbon to hepatic glycogen formation by the indirect and direct pathways are greatly dependent on experimental design, and at least in vitro, are possibly dependent on the extent of glycogen/glucose 1-P recycling. Under physiological refeeding conditions in vivo, both pathways are used, each contributing approximately 50% of the amount of carbon appearing in glycogen. The level of glucokinase activity does not appear to be responsible for poor glucose utilization in liver. Poor glucose utilization in isolated liver preparations may result from the absence of a neurophysiological feedback loop that senses the arterial/portal glucose gradient and then regulates whole liver glucose uptake.     

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.     

Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary culture

Glycogen synthesis was examined in primary cultures of adult rat hepatocytes that had been isolated from rats following a 24-h fast. Glycogen synthesis was dependent on the concentration of glucose in the culture medium and also required the presence of insulin. The addition of dexamethasone to the culture medium also increased the amount of glycogen synthesis. When the culture medium was supplemented with [U-14C,3-3H]glucose, it was found that approximately 60% of the glucose incorporated into glycogen was not derived from the pool of labeled glucose. In addition, the relative ratio of 3H/14C in the newly synthesized glycogen was approximately 50% of the ratio of the two isotopes in glucose in the culture medium, indicating that the glucose had undergone metabolism prior to its incorporation into glycogen. However, when hepatocytes were isolated from rats that had been fed ad libitum and the synthesis of glycogen from [U-14C,3-3H]glucose was followed, the relative ratio of the two isotopes in glycogen was similar to that measured for glucose in the culture medium, indicating that the glucose was directly incorporated into glycogen without any apparent metabolism. These results indicate that the synthesis of glycogen from glucose may, at least in part, follow an indirect pathway whereby glucose is metabolized prior to incorporation of the carbon into glycogen, but that the pathway followed for the synthesis of glycogen is dependent on the prior metabolic state of the animal.     

Inhibition of glycogen synthesis in rat hepatocytes by medium Zn2+

In hepatocytes from fasted rats, Zn2+ in the range from 0 to 500 microM has relatively minor effects on gluconeogenesis from most substrates, or on ureagenesis from NH3. In hepatocytes from fed rats, Zn2+ does not affect glycogenolysis. In hepatocytes from fasted rats, in which glycogen is being actively synthesized using the substrate combination (Katz et al. (1976) Proc. Natl.Acad.Sci.USA 73,3433-3437) of glucose, lactate and glutamine (all 10mM), Zn2+ markedly inhibits glycogen synthesis, with total inhibition at 500 microM, and a half maximal effect in the range from 50 to 100 microM. Dipicolinate (pyridine 2,6-dicarboxylate), a zinc chelator, is about as effective as L-glutamine in activating glycogen synthesis with the substrate combination of dihydroxyacetone, lactate and glucose (all 10mM). This suggests the possible hypothesis that endogenous Zn2+ might control the rate of glycogen synthesis in vivo. However, alternate explanations such as metabolite accumulation are also possible, since dipicolinate causes inhibition of gluconeogenesis from L-lactate.     

Resistance of hepatic glycogen to depletion in obese Zucker rats

Glycogen stores (liver and carcass) have been studied in lean and obese Zucker rats. The animals were submitted to one of three feeding conditions: ad libitum, a 48-h fast, or a 48-h fast and food ad libitum for 24 h, and to two environmental conditions, either thermoneutrality or an acute cold exposure (2 days at 4-7 degrees C). After a 2-day fast at 25 degrees C, the liver glycogen store was reduced by 45 times in the lean rats, while it was decreased by only 3 times in the obese rats. Under these conditions, the liver glycogen store was 45 times higher in the obese than in the lean rats. After 2 days in the cold, liver glycogen store was 4.4 times higher in obese rats than in lean rats. After a 2-day fast in the cold, the liver glycogen store in the obese rats was 30 times higher than in the lean rats. In comparison to fasting at thermoneutrality, fasting in the cold did not lead to a further reduction in hepatic glycogen in obese Zucker rats. The differences observed in the mobilization of the hepatic glycogen store between obese and lean rats have not been found in the mobilization of the carcass glycogen store. Drastic conditions, such as a 2-day fast in the cold, did not exhaust the glycogen store in obese Zucker rats. The present observations point out that obese Zucker rats cannot mobilize the entire hepatic glycogen store, as seen in lean control rats. The role of this abnormality in the high hyperlipogenesis that maintains the obese state is still to be evaluated.     

Substrate control of glycogen levels in isolated hepatocytes from fed rats.

Isolated parenchymal cells from fed rat liver rapidly lose glycogen when incubated with glucose. The addition of glycerol or fructose but not insulin prevents much of the breakdown. When cells are incubated with glycerol and glucose, more glycogen is retained with the further addition of xylitol than of fructose or pyruvate. Oleate stimulates glycogen breakdown. The results indicate that glycerol may play an important physiological role in the control of glycogen synthesis in the liver, possibly by esterifying fatty acids. Furthermore, the results support the concept that the main effect of insulin on liver glycogen levels $\text{in vivo}$ may be the results of diminished flow of free fatty acids to the liver.     

Use of deuterium labelled glucose in evaluating the pathway of hepatic glycogen synthesis

Deuterium labelled glucose has been used to study the pathway of hepatic glycogen synthesis during the fasted-refed transition in rats. Deuterium enrichment of liver glycogen was determined using nuclear magnetic resonance as well as mass spectroscopy. Sixty minutes after oral administration of deuterated glucose to fasted rats, the portal vein blood was fully enriched with deuterated glucose. Despite this, less than half of the glucose molecules incorporated into liver glycogen contained deuterium. The loss of deuterium label from glucose is consistent with hepatic glycogen synthesis by an indirect pathway requiring prior metabolism of glucose. The use of deuterium labelled glucose may prove to be a useful probe to study hepatic glycogen metabolism. Its use may also find application in the study of liver glycogen metabolism in humans by a noninvasive means.     

Pancreatic and hepatic glycogen content in normoglycemic and hyperglycemic rats

As judged from morphological criteria, glycogen accumulates to a larger extent in insulin-producing B-cells than in acinar cells of the pancreas in situations of sustained hyperglycemia. In the present study, the glycogen content of the pancreatic gland and liver was measured in either euglycemic or glucose-infused hyperglycemic control rats, as well as in streptozotocin-induced diabetic rats. Whilst the glycogen content of the pancreas was significantly higher in STZ rats than in control euglycemic rats, it was further enhanced in glucose-infused control rats, despite the fact that the latter animals were not more severely hyperglycemic and for a shorter time than STZ rats. From these measurements, it was estimated that, relative to wet weight, the glycogen content was, under the present experimental conditions, about 75 times higher in insulin-producing than other pancreatic cells. Moreover, it is proposed that the intravenous administration of glucagon may help in distinguishing between the glycogen present in the endocrine and exocrine moieties of the pancreatic gland, this hormone being apparently unable to provoke glycogenolysis in the exocrine pancreas, at variance with the situation prevailing in isolated pancreatic islets.     

Cytofluorimetric study of the glycogen content in hepatocytes of varying ploidy in adult rats]

A combined method, that allows measuring glycogen and DNA contents in one of the same cell, was applied for quantitative determination of these in mono- and binucleate hepatocytes with different ploidy obtained from adult rats. The mean glycogen content was shown to increase proportionally to the genome number within the changes of the hepatocyte ploidy from 2 to 8c.     

Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes.

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.     

Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes

In Type 2 diabetes, increased glycogenolysis contributes to the hyperglycaemic state, therefore the inhibition of GP (glycogen phosphorylase), a key glycogenolytic enzyme, is one of the possibilities to lower plasma glucose levels. Following this strategy, a number of GPis (GP inhibitors) have been described. However, certain critical issues are associated with their mode of action, e.g. an impairment of muscle function. The interaction between GP and the liver glycogen targeting subunit (termed G(L)) of PP1 (protein phosphatase 1) has emerged as a new potential anti-diabetic target, as the disruption of this interaction should increase glycogen synthesis, potentially providing an alternative approach to counteract the enhanced glycogenolysis without inhibiting GP activity. We identified an inhibitor of the G(L)-GP interaction (termed G(L)-GPi) and characterized its mechanism of action in comparison with direct GPis. In primary rat hepatocytes, at elevated glucose levels, the G(L)-GPi increased glycogen synthesis similarly to direct GPis. Direct GPis significantly reduced the cellular GP activity, caused a dephosphorylation of the enzyme and decreased the amounts of GP in the glycogen-enriched fraction; the G(L)-GPi did not influence any of these parameters. Both mechanisms increased glycogen accumulation at elevated glucose levels. However, at low glucose levels, only direct GPis led to increased glycogen amounts, whereas the G(L)-GPi allowed the mobilization of glycogen because it did not block the activity of GP. Due to this characteristic, G(L)-GPi in comparison with GPis could offer an advantageous risk/benefit profile circumventing the potential downsides of a complete prevention of glycogen breakdown while retaining glucose-lowering efficacy, suggesting that inhibition of the G(L)-GP interaction may provide an attractive novel approach for rebalancing the disturbed glycogen metabolism in diabetic patients.     

Cytoarchitectonics of different areas of the thymus in rats

Correlation of various parts in the gland of white non-inbred 3-month-old male rats has been studied morphometrically. Five zones have been distinguished in the lobe: in the cortex--subcapsular, middle and border-like with medullary substance, in the medulla--border-like with cortical substance, deep zones. A high level of dividing and capable for division poorly differentiated cell forms, especially in the thymic cortex are noted in the gland's contents.