Hormonal regulation of hepatic glycogen synthase phosphatase

Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.     

Expression and regulation of glycogen synthase kinase 3 in human neutrophils

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase involved in the regulation of cellular processes ranging from glycogen metabolism to cell cycle regulation. Its two known isoforms, α and β, are differentially expressed in tissues throughout the body and exert distinct but often overlapping functions. GSK-3 is typically active in resting cells, inhibition by phosphorylation of Ser21 (GSK-3α) or Ser9 (GSK-3β) being the most common regulatory mechanism. GSK-3 activity has been linked recently with immune system function, yet little is known about the role of this enzyme in neutrophils, the most abundant leukocyte type. In the present study, we examined GSK-3 expression and regulation in human neutrophils. GSK-3α was found to be the predominant isoform, it was constitutively expressed and cell stimulation with different agonists did not alter its expression. Stimulation by fMLP, LPS, GM-CSF, Fcγ receptor engagement, or adenosine A_2A receptor engagement all resulted in phosphorylation of Ser21. The use of metabolic inhibitors revealed that combinations of Src kinase, PKC, PI3K/AKT, ERK/RSK and PKA signaling pathways could mediate phosphorylation, depending on the agonist. Neither PLC nor p38 were involved. We conclude that GSK-3α is the main isoform expressed in neutrophils and that many different pathways can converge to inhibit GSK-3α activity via Ser21-phosphorylation. GSK-3α thus might be a hub of cellular regulation.     

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.     

A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and carbohydrate metabolism

Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.     

Differential regulation of glycogen synthase kinase 3beta by insulin and Wnt signaling

Glycogen synthase kinase 3beta (GSK3beta) is a key component in many biological processes including insulin and Wnt signaling. Since the activation of each signaling pathway results in a decrease in GSK3beta activity, we examined the specificity of their downstream effects in the same cell type. Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of beta-catenin. In contrast, Wnt increases the cytosolic pool of beta-catenin but not glycogen synthase activity. We found that, unlike insulin, neither the phosphorylation status of the serine9 residue of GSK3beta nor the activity of protein kinase B is regulated by Wnt. Although the decrease in GSK3beta activity is required, GSK3beta may not be the limiting component for Wnt signaling in the cells that we examined. Our results suggest that the axin-conductin complexed GSK3beta may be dedicated to Wnt rather than insulin signaling. Insulin and Wnt pathways regulate GSK3beta through different mechanisms, and therefore lead to distinct downstream events.     

多功能的蛋白:糖原合成酶激酶-3

糖原合成酶激酶-3(GSK-3)是一个多功能的丝氨酸／苏氨酸类激酶,在真核生物中普遍存在。在哺乳动物中包括两个亚型,即GSK-3a和GSK-3β。GSK-3至少在三条细胞通路上有作用：Wnt/wingless,P13-kinase以及Hedgehog信号通路,该酶的作用主要包括调节糖原的合成代谢,参与细胞的分化与增殖等。研究发现,GSK-3在某些疾病,如阿尔茨海默病和非胰岛素依赖型糖尿病(NIDDM)中,其活性会异常升高。现已发现了几种针对该酶的抑制剂,如aloisine,paullones和马来酰胺类化合物等。这些抑制剂的确在分子水平特异性地抑制GSK-3的活性,而对其他激酶几乎没有作用。关于这些抑制剂的研究工作也已经在细胞水平和动物模型上开展起来,为开发以GSK-3为靶点的新的治疗药物创造了良好的基础。     

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).     

Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.     

Glycogen synthase kinase 3: an emerging therapeutic target

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that has recently emerged as a key target in drug discovery. It has been implicated in multiple cellular processes and linked with the pathogenesis of several diseases. GSK-3 inhibitors might prove useful as therapeutic compounds in the treatment of conditions associated with elevated levels of enzyme activity, such as type 2 diabetes and Alzheimer's disease. The pro-apoptotic feature of GSK-3 activity suggests a potential role for its inhibitors in protection against neuronal cell death, and in the treatment of traumatic head injury and stroke. Finally, selective inhibitors of GSK-3 could mimic the action of mood stabilizers such as lithium and valproic acid and be used in the treatment of bipolar mood disorders.     

Calmodulin-dependent glycogen synthase kinase

A cAMP-independent glycogen synthase kinase has been purified from rabbit liver. This kinase is completely dependent on the presence of calmodulin and Ca2+ for activity. Half-maximal activation required about 0.1 microM calmodulin. Complete inhibition was obtained in the presence of ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid or trifluoperazine. This calmodulin-dependent synthase kinase does not phosphorylate phosphorylase, myosin light chain, casein, or histone. It rapidly incorporates 0.4 to 0.5 mol of 32P/mol of synthase subunit into the NH2-terminal domain, resulting in partial inactivation of glycogen synthase. These results indicate the existence of a calmodulin-dependent kinase which may be specific for glycogen synthase.     

Glycogen synthase kinase 3 in the world of cell migration

Glycogen synthase kinase 3 (GSK3) is one of the few master switch kinases that regulate many aspects of cell functions. Recent studies on cell polarization and migration have shown that GSK3 is also essential for proper regulation of these processes. GSK3 influences cell migration as one of the regulators of the spatiotemporally controlled dynamics of the actin cytoskeleton, microtubules, and cell‐to‐matrix adhesions. In this mini‐review, the effects of GSK3 on these three aspects of cell migration will be discussed.     

miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting

Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.