Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

Background: Glycogen synthase kinase-3 (GSK-8) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3.

Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats

The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was their infused with glucagon (1 nM), isoproterenot (2 mu M), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase. Copyright (c) 2008 John Wiley & Sons, Ltd.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A Genome-wide Screen for Neurospora crassa Transcription Factors Regulating Glycogen Metabolism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Muscle glycogen metabolism changes in rats fed early postnatal a fructose-rich diet after maternal protein malnutrition: effects of acute physical exercise at the maximal lactate steady-state intensity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Abnormal Glycogen Storage by Retinal Neurons in Diabetes

PURPOSE: It is widely held that neurons of the central nervous system do not store glycogen and that accumulation of the polysaccharide may cause neurodegeneration. Since primary neural injury occurs in diabetic retinopathy, we examined neuronal glycogen status in the retina of streptozotocin-induced diabetic and control rats.

METHODS: Glycogen was localized in eyes of streptozotocin-induced diabetic and control rats using light microscopic histochemistry and electron microscopy, and correlated with immunohistochemical staining for glycogen phosphorylase and phosphorylated glycogen synthase (pGS).

RESULTS: Electron microscopy of 2-month-old diabetic rats (n = 6) showed massive accumulations of glycogen in the perinuclear cytoplasm of many amacrine neurons. In 4-month-old diabetic rats (n = 11), quantification of glycogen-engorged amacrine cells showed a mean of 26 cells/mm of central retina (SD ± 5), compared to 0.5 (SD ± 0.2) in controls (n = 8). Immunohistochemical staining for glycogen phosphorylase revealed strong expression in amacrine and ganglion cells of control retina, and increased staining in cell processes of the inner plexiform layer in diabetic retina. In control retina, the inactive pGS was consistently sequestered within the cell nuclei of all retinal neurons and the retinal pigment epithelium (RPE), but in diabetics nuclear pGS was reduced or lost in all classes of retinal cell except the ganglion cells and cone photoreceptors.

CONCLUSIONS: The present study identifies a large population of retinal neurons that normally utilize glycogen metabolism but show pathologic storage of the polysaccharide during uncontrolled diabetes.

Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis

Glycogen synthase kinase 3 (GSK3, of which there are two isoforms, GSK3alpha and GSK3beta) was originally characterized in the context of regulation of glycogen metabolism, though it is now known to regulate many other cellular processes. Phosphorylation of GSK3alpha(Ser21) and GSK3beta(Ser9) inhibits their activity. In the heart, emphasis has been placed particularly on GSK3beta, rather than GSK3alpha. Importantly, catalytically-active GSK3 generally restrains gene expression and, in the heart, catalytically-active GSK3 has been implicated in anti-hypertrophic signalling. Inhibition of GSK3 results in changes in the activities of transcription and translation factors in the heart and promotes hypertrophic responses, and it is generally assumed that signal transduction from hypertrophic stimuli to GSK3 passes primarily through protein kinase B/Akt (PKB/Akt). However, recent data suggest that the situation is far more complex. We review evidence pertaining to the role of GSK3 in the myocardium and discuss effects of genetic manipulation of GSK3 activity in vivo. We also discuss the signalling pathways potentially regulating GSK3 activity and propose that, depending on the stimulus, phosphorylation of GSK3 is independent of PKB/Akt. Potential GSK3 substrates studied in relation to myocardial hypertrophy include nuclear factors of activated T cells, beta-catenin, GATA4, myocardin, CREB, and eukaryotic initiation factor 2Bvarepsilon. These and other transcription factor substrates putatively important in the heart are considered. We discuss whether cardiac pathologies could be treated by therapeutic intervention at the GSK3 level but conclude that any intervention would be premature without greater understanding of the precise role of GSK3 in cardiac processes.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Evaluation of placental glycogen storage in mild diabetic rats

Purpose: To evaluate the placental glycogen storage and fetal development in the pregnancy of neonatally streptozocin-induced diabetic rats and to establish relation with glycemia and insulin levels. Methods: At the birth day, 147 female rats were randomly distributed in two experimental groups: 1) Non-diabetic Group (Control, n=45) - received the vehicle; 2) Diabetic Group (STZ, n=102) received 100 mg streptozocin/kg in neonatal period. At day 0 of pregnancy, adult female rats were included in the control group when presented glycemia below 120 mg/dL and, in the group STZ with glycemia between 120 and 300 mg/dL. At day 21 of pregnancy, blood samples were collected for glycemia and insulin determination, and placentas withdrawn for placental glycogen determination. The newborns (NB) were classified in small (SGA), appropriate (AGA) and large (LGA) for gestational age. Results: Rats STZ presented higher glycemia at days 0 and 14 of pregnancy. At end of pregnancy, rats STZ showed higher proportion of NB SGA and LGA; reduced rate of NB AGA and unaltered glycemia, insulin and placental glycogen determinations. Conclusion: Mild diabetes altered the maternal glycemia in the early pregnancy, impairing future fetal development, but it caused no alteration on insulin and placental glycogen determination, confirming that this glycemic intensity was insufficient to change glycogen metabolism.

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 31 kDa, 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. (C) 2004 Elsevier B.V. All rights reserved.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

External assessment of myocardial glucose metabolism with $\sp{18}$F-2 -deoxy-2 -fluoro-D-glucose

Despite the popularity of the positron emitting glucose analog, ($\sp{18}$F) -2-deoxy-2-fluoro-D-glucose (2FDG), for the noninvasive "metabolic imaging" of organs with positron emission tomography (PET), the physiological basis for the tracer has not been tested, and the potential of 2FDG for the rapid kinetic analysis of altered glucose metabolism in the intact heart has not been fully exploited. We, therefore, developed a quantitative method to characterize metabolic changes of myocardial glucose metabolism noninvasively and with high temporal resolution.^ The first objective of the work was to provide direct evidence that the initial steps in the metabolism of 2FDG are the same as for glucose and that 2FDG is retained by the tissue in proportion to the rate of glucose utilization. The second objective was to characterize the kinetic changes in myocardial glucose transport and phosphorylation in response to changes in work load, competing substrates, acute ischemia and reperfusion, and the addition of insulin. To assess changes in myocardial glucose metabolism isolated working rat hearts were perfused with glucose and 2FDG. Tissue uptake of 2FDG and the input function were measured on-line by external detection. The steady state rate of 2FDG phosphorylation was determined by graphical analysis of 2FDG time-activity curves.^ The rate of 2FDG uptake was linear with time and the tracer was retained in its phosphorylated form. Tissue accumulation of 2FDG decreased within seconds with a reduction in work load, in the presence of competing substrates, and during reperfusion after global ischemia. Thus, most interventions known to alter glucose metabolism induced rapid parallel changes in 2FDG uptake. By contrast, insulin caused a significant increase in 2FDG accumulation only in hearts from fasted animals when perfused at a sub-physiological work load. The mechanism for this phenomenon is not known but may be related to the existence of two different glucose transporter systems and/or glycogen metabolism in the myocardial cell.^ It is concluded that (1) 2FDG traces glucose uptake and phosphorylation in the isolated working rat heart; and (2) early and transient kinetic changes in glucose metabolism can be monitored with high temporal resolution with 2FDG and a simple positron coincidence counting system. The new method has revealed transients of myocardial glucose metabolism, which would have remained unnoticed with conventional methods. These transients are not only important for the interpretation of glucose metabolic PET scans, but also provide insights into mechanisms of glucose transport and phosphorylation in heart muscle. ^

Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^