Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.     

Storage of glycogen in rat colonic epithelium during induction of secretion and absorption in vitro

Summary Changes induced in the ultrastructure of the epithelium of the rat colon descendens by long-term electric field stimulation (EFS) in an Ussing chamber were investigated. The anion secretion, which was induced by EFS and was measured by the short-circuit current, fell continuously during a 5 h stimulation. At the end of the stimulation period, small particles were observed in the epithelium; these did not appear in unstimulated control tissue. They were localized predominantly in the apical part of the cell. By staining with periodic acidthiosemicarbazide-silver proteinate and because of their sensitivity to -amylase, they were identified as glycogen deposits. This storage of glycogen was time-dependent and was first visible after an EFS of 2 h. It did not appear if glucose was substituted in the bathing solution by sodium butyrate. Glycogen particles were also observed after addition of forskolin, which in contrast to EFS causes a high secretory activity that is stable over several hours. The surface cells contained significantly more glycogen than the crypt cells when secretion was stimulated by EFS or forskolin. The formation of glycogen during EFS was not prevented by tetrodotoxin (TTX). In contrast, TTX itself, which causes maximal absorptive activity by blocking secretomotor neurons, induced the appearance of glycogen in the enterocytes without EFS. However, in the presence of TTX, the amount of glycogen was the same in surface and crypt cells. The results demonstrate that the capacity to synthesize and store glycogen, which has up to now only been observed in embryonic or tumor epithelial cells, is still present in adult colonic mucosa. Procedures carried out to change the functional state of the epithelium seem to induce, at least in vitro, a disinhibition of this capacity.     

Transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia

1. The utilization and control of glycogen stores were studied in the isolated segmental ganglia of the horse leech, Haemopis sanguisuga. The glycogen in the ganglia was extracted and assayed fluorimetrically and its cellular localization and turnover studied by autoradiography in conjunction with [3H] glucose. 2. The glycogen levels were measured after incubation with different neurotransmitters for 60 min at 28 degrees C. The results for each experimental ganglion were compared to a paired control ganglion, and the results analysed by paired t-tests. 3. Several transmitter substances (5-HT, octopamine, dopamine, noradrenaline, histamine) produced reductions in glycogen (glycogenolysis); other transmitters (glutamate, GABA) produced increases in glycogen (gluconeogenesis); others (adenosine, glycine) produced reductions or increases, depending on concentration. Acetylcholine had no effect on the glycogen levels. 4. Most of the glycogen in the ganglia is localized in the packet glial cells, which surround the neuron perikarya. Autoradiographic analysis demonstrated that the effects of histamine and dopamine were principally on the glycogen in the glial cells. 5. Adenylate cyclase was demonstrated by electron microscope histochemistry to be localized on the plasma membranes of the glial cells, and to a lesser extent on the neuronal membranes. 6. It is concluded that the changes in glycogen in the glial cells may be party controlled by transmitters via adenylate cyclase. This may provide a sensitive mechanism for coupling neuronal activity with energy metabolism.     

The effect of cortisol on glycogen and fructose-1,6-bisphosphatase in baby hamster kidney cells infected with Chlamydia trachomatis

This study was designed to assay changes in glycogen synthesis which may occur as a result of cortisol treatment of chlamydial-infected cells. Monolayers of baby hamster kidney (BHK) cells, unlabeled and prelabeled with [6-14C]glucose, were treated with various concentrations of cortisol before (pretreated) or during (post-treated) infection with Chlamydia trachomatis. At designated times after absorption, the cells were harvested and assayed for total glycogen and 14C accumulation in glycogen. The total amount of glycogen accumulated in cells during the period of greatest chlamydial glycogen synthesis (36 h) was not affected by cortisol treatment. Cortisol treatment appeared to have retarded the accumulation of glycogen in treated infected cells until 30 h after infection. Treated infected cells prelabeled with [6-14C]glucose accumulated a greater amount of 14C in glycogen than untreated infected cells. All cortisol-treated, infected cells exhibited elevated levels of fructose-1,6-bisphosphatase activity, whereas untreated infected cells did not. The hypothesis that cortisol affects chlamydia multiplication by altering the intracellular environment of the host cell is compatible with the results obtained.     

Glycogen in pancreatic islets of steroid diabetic rats

Summary In the islets of the rat pancreas, steroid diabetes induced by triamcinolon-acetonid leads to degranulation of the B cells and glycogen infiltration. The glycogen cannot be satisfactorily detected using methods like the chromic acid technique according to Bauer, staining with Best's carmine, or the usually applied periodic acid-Schiff (PAS) reaction. Glycogen detection is improved, however, when lead tetraacetate is used in place of periodic acid as oxidizing agent. When combining the carbohydrate detection method with the peroxidase — antiperoxidase (PAP) method used for immunocytochemical detection of the various pancreatic islet hormones, paraffin sections reveal that glycogen is primarily localized in granulated B cells; the degranulated B cells also contain glycogen, though in smaller amounts. In contrast, the islet cells containing somatostatin, glucagon and pancreatic polypeptide are nearly free of glycogen.This study was supported by the Deutsche Forschungsgemeinschaft K1 426/2     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during cell differentiation

1. The variation in cellular glycogen content of differentiating cells derived from myxamoebae that initially contained a wide range of glycogen contents (0.047-5.56mg of glycogen/10(8) myxamoebae) has been studied. 2. Myxamoebae that initially contained 0.047-3.62mg of glycogen/10(8) myxamoebae all gave rise to fruiting bodies that contained similar amounts of glycogen (0.06-0.11mg of glycogen/10(8) cells) but myxamoebae that initially contained 5.56mg of glycogen formed fruiting bodies containing 0.5mg of glycogen/10(8) cells. 3. Despite the high net rate of glycogen disappearance (during cell differentiation) from cells that contained more than 2mg of glycogen/10(8) cells initially, there were still significant variations in the rate of glycogen synthesis. The rate of glycogen synthesis reached a peak at the aggregation stage. 4. Evidence is presented showing that the rate of this synthesis of glycogen is controlled by factors other than the intracellular concentration of glycogen synthetase. 5. Our results are discussed in the context of the theory that the rates of glycogen synthesis and degradation act as a control mechanism for cell differentiation. 6. Criteria are discussed for deciding whether a biochemical event is causally or secondarily related to morphogenesis.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHIC STUDY OF GLYCOGEN SYNTHESIS IN THE RABBIT RETINA

Glycogen is present in the rabbit retina in monoparticulate form. Beta particles (~ 229 A) are abundant in Müller cell cytoplasm, particularly in its inner portion, decreasing in number outwards along the cell. They are slightly larger (~ 250 A) and much scarcer in neurons, though regularly present in the juxtanuclear Golgi region of ganglion cells. When the retina was incubated in a glucose-free medium, it was rapidly depleted of native glycogen. On further incubation in medium containing glucose-³H plus unlabeled glucose, glycogen reappeared in the form of beta particles of the same size and distribution as native ones, while radioautography revealed the appearance of amylase-labile radioactivity in the same locations. This newly formed glycogen was not associated with any particular organelle. The rate of synthesis, as judged from the amount of radioactivity, was high in the inner portion of Müller cells and declined uniformly toward the cell outer end, following a logarithmic gradient. The rate of synthesis was low in ganglion cells, at best approaching values in the outer portion of Müller cells. The concentration of glycogen in the inner portion of Müller cells is consistent with the view that it may be the source of glucose for the anaerobic glycolysis prevailing in the inner retina.     

Interactions between insulin and alpha 1-adrenergic agents in the regulation of glycogen metabolism in isolated hepatocytes

Addition of insulin to liver cells from fed rats incubated in the absence of other hormones resulted in a 2-fold increase in glycogen synthase activity. This direct effect of insulin has been characterized and compared with the antagonism by insulin of alpha 1-adrenergic effects on glycogen metabolism. The activation of glycogen synthase by insulin developed slowly (20-25 min) and was most effective when the enzyme was partially preactivated by glucose. With glucose concentrations above 15 mM the effects of insulin and glucose were additive. In contrast to glucose, which caused inverse changes in phosphorylase and glycogen synthase activity, insulin activated glycogen synthase without affecting phosphorylase a. Treatment of hepatocytes with phenylephrine led to an activation of phosphorylase and inactivation of glycogen synthase, which could be partially blocked by insulin. This antagonistic effect of insulin was rapid (complete within 5 min of insulin addition) and showed an identical time course for both enzymes. The activation of glycogen synthase by insulin and inactivation by phenylephrine both resulted principally from alterations in the Vmax. Insulin added alone did not alter the basal cytosolic free Ca2+ concentration, which was 160 nM as measured with Quin 2 as an intracellular Ca2+ indicator. Both the magnitude and the initial rate of cytosolic free Ca2+ increase induced by phenylephrine were reduced by about 50% in cells pretreated with insulin. It is concluded that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase, whereas insulin antagonizes the effects of alpha 1-agonists by interfering with their ability to elevate cytosolic free Ca2+.     

The content of glycogen and its fractions in human hepatocytes in traumatic disease

A cytofluorometric study was made of the total glycogen and its of fractions in liver cells of patients with hard mechanic trauma with or without intoxication. For studying glycogen dynamics in the course of traumatic illness, the aspiration biopsy material was obtained (30 patients) using repeated liver biopsy of one and the same patient. The total glycogen was found to change insignificantly in liver cells of patients with traumatic illness, both under favourable conditions and with intoxication, and at the normal level. The labile glycogen fraction in liver cells of patients with traumatic illness without intoxication is contained almost at the normal level (80-95%) of the total glycogen and is not changed for a long time. At that time the relative content of the labile glycogen fraction decreases appreciably in some cases to 45-50% due to intoxication development. A relative content of the labile glycogen fraction in hepatocytes with hard mechanical intoxication correlates well with the degree of intoxication. This makes hepatocyte glycogen microfluorometry a diagnostic tool in measuring the functional state of liver in the course of intoxication.     

Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.     

Ultrastructural localization of glycogen in erythrocytes and developing erythrocytic cells in normal human bone marrow

Summary The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) reaction was employed for the ultrastructural cytochemical localization of saliva-labile glycogen in the erythrocytic cells in normal human blood and bone marrow. Particulate glycogen was demonstrated in the cytoplasm of all developmental forms of erythrocytic cells from the proerythroblast through the reticulocyte; a few particles of glycogen also were present in mature erythrocytes even in the peripheral blood. Statistical evaluation of the number of glycogen particles in mid-plane cell sections at each morphological stage of development indicated a significant and stepwise decrease during cellular maturation. This change in glycogen content may reflect both cellular utilization and mitosis during the maturational sequence.Supported by Grant No. SR01AM 12084-15 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson, Barbara Speakmon and Marjorie Griffith for their technical assistance and to Dr. Gerald King for performing the bone marrow aspirations.     

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.     

Glycogen metabolism in novikoff ascites-hepatoma cells

A study of the enzymes of the glycogen pathway in Novikoff ascites hepatoma shows that glycogen synthetase has the lowest activity and that the tumour contains no high-K(m) soluble glucokinase. However, incubation of tumour cells with metabolizable sugars in vitro, or intraperitoneal administration of glucose into the tumour-bearing rat, results in glycogen accumulation by the tumour cells. Glycogen synthesis in the tumour is supported by aerobically produced ATP but is decreased anaerobically and by uncouplers of oxidative phosphorylation. Absence of P(i) from the incubation medium increases glycogen synthesis and decreases glycolysis. The optimum temperature for glycogen synthesis is 37 degrees . The capacity of the intact tumour cell to degrade deposited glycogen is low, but is accelerated by 2,4-dinitrophenol. Tumour homogenates prepared after osmotic shock do not incorporate [(14)C]glucose into glycogen. The glucose moiety of glucose 1-phosphate and of UDP-glucose is incorporated into glycogen by the homogenates and the incorporation of glucose 1-phosphate is greatly enhanced by AMP. Glucose 6-phosphate is a poor precursor of glycogen in the homogenate system, probably because it inhibits activation of phosphorylase b by AMP.     

Glycogen synthase association with the striated muscle glycogen-targeting subunit of protein phosphatase-1. Synthase activation involves scaffolding regulated by beta-adrenergic signaling

Glycogen-binding subunits for protein phosphatase-1 (PP1) target the PP1 catalytic subunit (PP1C) to glycogen particles, where the enzymes glycogen synthase and glycogen phosphorylase are concentrated. Here we identify sites within the striated muscle glycogen-binding subunit (G(M)) that mediate direct binding to glycogen synthase. Both PP1C and glycogen synthase were coimmunoprecipitated with a full-length FLAG-tagged G(M) transiently expressed in COS7 cells or C2C12 myotubes. Deletion and mutational analysis of a glutathione S-transferase (GST) fusion of the N-terminal domain of G(M) (residues 1-240) identified two putative sites for binding to glycogen synthase, one of which is the WXNXGXNYX(I/L) motif that is conserved among the family of PP1 glycogen-binding subunits. Either deletion of this motif or Ala substitution of Asn-228 in this motif disrupted the binding of glycogen synthase. Expression of full-length FLAG-G(M) in cells increased the activity of endogenous glycogen synthase, but protein disabled in either PP1 binding or glycogen synthase binding did not produce synthase activation. The results show that efficient activation of glycogen synthase requires a scaffold function of G(M) that involves simultaneous binding of both PP1C and glycogen synthase. Isoproterenol and forskolin treatment of cells decreased glycogen synthase binding to FLAG-G(M), thereby limiting synthase activation by PP1. This response was insensitive to inhibition by H-89, therefore probably not involving cAMP-dependent protein kinase, but did require inclusion of microcystin-LR during cell lysis, implying that phosphorylation was modulating binding of glycogen synthase. Phosphorylation control of binding to a scaffold site on the G(M) subunit of PP1 offers a new mechanism for regulation of muscle glycogen synthase in response to beta-adrenergic signals.     

An attempt to describe the ultrastructure and ultrahistochemistry of ciliary processes in mammals

The aim of the present paper was to reexamine fine structural characteristics and glycogen topochemistry of ciliary processes in small laboratory mammals (hamsters, guinea-pigs and mice). A two-layered epithelium continuously covered all ciliary processes. The epithelium consisted of inner nonpigmented and outer pigmented cells whose apices faced each other. They were linked by desmosomes and tight junctions. Basal cell aspects showed extensively interdigitating processes adjacent to the inner (rarely also outer) basal lamina. The ciliary process core was made up of reticular fibers, few fibrocytes, and capillaries with or without fenestrations. No glycogen particles were found in the ciliary epithelium using the PA-TSC-SP procedure.     

Non-hormonal and hormonal control of glycogen metabolism in isolated sheep liver cells

1. Control of glycogen metabolism by various substrates and hormones was studied in ruminant liver using isolated hepatocytes from fed sheep. 2. In these cells glucose appeared uneffective to stimulate glycogen synthesis whereas fructose and propionate activated glycogen synthase owing to (i) a decrease in phosphorylase a activity and (ii) changes in the intracellular concentrations of glucose 6-phosphate and adenine nucleotides. 3. The activation of hepatic glycogenolysis by glucagon and alpha 1-adrenergic agents was associated with increased phosphorylase a and decreased glycogen synthase activities. 4. The simultaneous changes in these two enzyme activities suggest that in sheep liver, activation of phosphorylase a is not a prerequisite step for synthase inactivation. 5. In sheep hepatocytes, in the presence of propionate and after a lag period, insulin activated glycogen synthase without affecting phosphorylase a. 6. This latter result suggests that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase. Insulin also antagonized glucagon effect on glycogen synthesis by counteracting the rise of cAMP.     

Regulation of glycogen metabolism in astrocytoma and neuroblastoma cells in culture.

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in aprallel with the medium glucose. The critical glucose concentration is 2.5 mM for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations. When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E1 is effective with the neuroblastoma cells.     

Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.