Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

Glycogen Metabolism in Bovine Adrenal Medulla

Abstract: Glycogen content was determined both in whole adrenal medullary tissue and in isolated adrenal chromaffin cells, in which it responds to glucose deprivation and restoration. [¹⁴C]glucose incorporation into glycogen in isolated adrenal chromaffin cells is increased by previous glucose deprivation ("fasting"). Total glycogen synthase activities are 452 ± 66 mU/g in whole tissue and 305 ± 108 mU/g in isolated cells. The K _m of glycogen synthase for UDP-glucose is 0.67 mM with 13 m m glucose-6-phosphate and 1 m m without this effector. The in vitro inactivation process of glycogen synthase a has been found to be mainly cyclic AMP-dependent, but it also responds to Ca²⁺. Total glycogen phosphorylase activities are 8.69 ± 1.26 U/g in whole tissue and 2.38 ± 0.30 U/g in isolated cells. The requirements for interconversion in vitro of both glycogen synthase and phosphorylase suggest a system similar to that of other tissues. During incubation of isolated adrenal chromaffin cells with 5 m m -glucose, phosphorylase a activity decreases and synthase a activity increases; these changes are more marked in "fasted" cells. Glycogen content and glycogen synthase and phosphorylase activities are higher in the adrenal medulla than in the brain, suggesting a greater metabolic role of glycogen in the adrenal medulla.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Insulin regulation of hepatic glycogen synthase and phosphorylase.

The relative roles of insulin and glucose in the regulation of hepatic glycogen synthase and phosphorylase were studied in hepatocytes from fed rats. Elevation of extra-cellular glucose led to a rapid decrease in phosphorylase a activity followed by a slower increase in glycogen synthase I activity. A reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose was observed; following initial glucose-induced inactivation of phosphorylase, there was a highly significant linear inverse relationship between residual phosphorylase activity and glycogen synthase activation. Insulin led to a further decrease in phosphorylase activity and a 30-50% additional increase in glycogen synthase activity over that caused by glucose. The effects of insulin required the presence of glucose and served to augment acute glucose stimulation of glycogen synthase and inhibition of phosphorylase. Insulin did not perturb the reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose. The results suggest that the ability of insulin to activate hepatic glycogen synthase can be entirely accounted for by its ability to inactivate phosphorylase.     

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

Glycogen supercompensation in rat soleus muscle during recovery from nonweight bearing

The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing.     

Glycogen-storage disease in rats, a genetically determined deficiency of liver phosphorylase kinase.

Rats from an inbred strain (NZR/Mh) were found to have high concentrations of glycogen in their livers, even after 24 h of starvation. Despite this, blood glucose concentrations were well maintained on starvation for up to 72 h. The primary defect is a deficiency of liver phosphorylase kinase, causing a lack of active glycogen phosphorylase, although total phosphorylase is normal. The intravenous injection of glucagon caused a rapid activation of cyclic AMP-dependent protein kinase in the liver, but no increase in either phosphorylase kinase or phosphorylase a activity. Although total glycogen synthase activity in the livers of affected rats was higher than normal, glycogen synthase in the active form was very low, presumably as a result of the high liver glycogen content. The condition is transmitted as autosomal recessive and, apart from hepatomegaly, the affected rats appear healthy.     

Glycogen synthesis by rat hepatocytes.

1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat.

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Rat liver glycogen metabolism in the perinatal period.

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen synthase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximum on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose or AMP on the respective phosphatases was considered. It was found that the Km of phosphorylase phosphatase for glucose in the prenatal period was 5--7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6--15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time. Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver. It is suggested that the rapid changes observed immediately after birth are due to an effect of gypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Stimulating role of potassium ions and ouabain on glycogen synthesis in adipose tissue.

1. Exposure of fat-pads to increasing concentrations of K+ in the presence of insulin stimulates the incorporation of labelled glucose into glycogen. In the absence of hormone, only a slight incorporation of glucose into glycogen and slight glucose oxidation were detectable. 2. Ouabain alone, up to 100 microM, had no effect on synthesis of glycogen. Ouabain reinforced the effect of insulin on the conversion of glucose into glycogen in a Na+ medium and in a equimolar Na+-K+ medium, but not in a K+ medium. In addition, ouabain modified the optimal K+/Na+ ratio for glycogen synthesis. 3. The proportion of glycogen synthase in the active form was increased in a K+ medium, and a faster rate of conversion of synthase b into a was observed under these conditions. No difference was detected in the rate of inactivation of phosphorylase in a K+ or a Na+ medium. 4. Even though these results, taken together, are consistent with the proposed role of phosphorylase a in the regulation of synthase activation, the molecular mechanism of action of K+ in adipose tissue in increasing synthesis of glycogen cannot be explained simply by a faster inactivation of phosphorylase a. It is concluded that some undetermined effector(s) or signal could itself be a primary determinant for the greater activation of synthase observed in a K+ medium.     

Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes

Protein phosphatase-1 (PP1) plays an important role in the regulation of glycogen synthesis by insulin. Protein targeting to glycogen (PTG) enhances glycogen accumulation by increasing PP1 activity against glycogen-metabolizing enzymes. However, the specificity of PTG's effects on cellular dephosphorylation and glucose metabolism is unclear. Overexpression of PTG in 3T3-L1 adipocytes using a doxycycline-controllable adenoviral construct resulted in a 10-20-fold increase in PTG levels and an 8-fold increase in glycogen levels. Inclusion of 1 microg/ml doxycycline in the media suppressed PTG expression, and fully reversed all PTG-dependent effects. Infection of 3T3-L1 adipocytes with the PTG adenovirus caused a marked dephosphorylation and activation of glycogen synthase. The effects of PTG seemed specific, because basal and insulin-stimulated phosphorylation of a variety of signaling proteins was unaffected. Indeed, glycogen synthase was the predominant protein whose phosphorylation state was decreased in 32P-labeled cells. PTG overexpression did not alter PP1 protein levels but increased PP1 activity 6-fold against phosphorylase in vitro. In contrast, there was no change in PP1 activity measured using myelin basic protein, suggesting that PTG overexpression specifically directed PP1 activity against glycogen-metabolizing enzymes. To investigate the metabolic consequences of altering PTG levels, glucose uptake and storage in 3T3-L1 adipocytes was measured. PTG overexpression did not affect 2-deoxy-glucose transport rates in basal and insulin-stimulated cells but dramatically enhanced glycogen synthesis rates under both conditions. Despite the large increases in cellular glucose flux upon PTG overexpression, basal and insulin-stimulated glucose incorporation into lipid were unchanged. Cumulatively, these data indicate that PTG overexpression in 3T3-L1 adipocytes discretely stimulates PP1 activity against glycogen synthase and phosphorylase, resulting in a marked and specific increase in glucose uptake and storage as glycogen.     

Activity of glycogen metabolizing enzymes in glucose deprived HT 29 adenocarcinoma cell-line

When deprived of glucose, the cultured HT 29 adenocarcinoma cells are able to mobilize their glycogen within 4 hours. Glycogen phosphorylase is strongly activated during the first hour of glucose starvation. Then, while the a/a + b ratio for phosphorylase is declining, glycogen synthase is partially converted into the a form; this conversion does occur although glycogen phosphorylase is far from being totally inactivated. After 4 hours, activity of both a and total forms of glycogen synthase decrease. Cell UDP-glucose and glucose-6-P levels are declining during the 24 hours period of glucose starvation. Cell ATP content decreases by only 50 percent over the same period of time.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3′,5′-monphosphate (cylic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation.