Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 β (GSK-3β) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.     

Essential role of p38 MAPK for activation of skeletal muscle glucose transport by lithium

Lithium increases glucose transport and glycogen synthesis in insulin-sensitive cell lines and rat skeletal muscle, and has been used as a non-selective inhibitor of glycogen synthase kinase-3 (GSK-3). However, the molecular mechanisms underlying lithium action on glucose transport in mammalian skeletal muscle are unknown. Therefore, we examined the effects of lithium on glucose transport activity, glycogen synthesis, insulin signaling elements (insulin receptor (IR), Akt, and GSK-3beta), and the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) in the absence or presence of insulin in isolated soleus muscle from lean Zucker rats. Lithium (10 mM LiCl) enhanced basal glucose transport by 62% (p < 0.05) and augmented net glycogen synthesis by 112% (p < 0.05). Whereas lithium did not affect basal IR tyrosine phosphorylation or Akt ser(473) phosphorylation, it did enhance (41%, p < 0.05) basal GSK-3beta ser(9) phosphorylation. Lithium further enhanced (p < 0.05) the stimulatory effects of insulin on glucose transport (43%), glycogen synthesis (44%), and GSK-3beta ser(9) phosphorylation (13%). Lithium increased (p < 0.05) p38 MAPK phosphorylation both in the absence (37%) and presence (41%) of insulin. Importantly, selective inhibition of p38 MAPK (using 10 microM A304000) completely prevented the basal activation of glucose transport by lithium, and also significantly reduced (52%, p < 0.05) the lithium-induced enhancement of insulin-stimulated glucose transport. Theses results demonstrate that lithium enhances basal and insulin-stimulated glucose transport activity and glycogen synthesis in insulin-sensitive rat skeletal muscle, and that these effects are associated with a significant enhancement of GSK-3beta phosphorylation. Importantly, we have documented an essential role of p38 MAPK phosphorylation in the action lithium on the glucose transport system in isolated mammalian skeletal muscle.     

Unlike insulin, amino acids stimulate p70S6K but not GSK-3 or glycogen synthase in human skeletal muscle

Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating PKB (or Akt), which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal 70-kDa protein S6 kinase (p70(S6K)), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70(S6K), and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70(S6K). To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70(S6K), and GSK-3 phosphorylation, and on the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a mixed amino acid solution (1.26 micromol.kg(-1).min(-1) x 6 h, n = 9), a physiological dose of insulin (1 mU.kg(-1).min(-1) euglycemic hyperinsulinemic clamp x 2 h, n = 6), or a pharmacological dose of insulin (20 mU.kg(-1).min(-1) euglycemic hyperinsulinemic clamp x 2 h, n = 9). Whole body glucose disposal rates were assessed by calculating the steady-state glucose infusion rates, and vastus lateralis muscle was biopsied before and at the end of the infusion. Both amino acid infusion and physiological hyperinsulinemia enhanced p70(S6K) phosphorylation without affecting GSK-3 phosphorylation, but only physiological hyperinsulinemia also increased whole body glucose disposal and GS activity. In contrast, a pharmacological dose of insulin significantly increased whole body glucose disposal, p70(S6K), GSK-3 phosphorylation, and GS activity. We conclude that amino acids at physiological concentrations mediate p70(S6K) but, unlike insulin, do not regulate GSK-3 and GS phosphorylation/activity in human skeletal muscle.     

In vivo and in vitro application of black soybean peptides in the amelioration of endoplasmic reticulum stress and improvement of insulin resistance

AimsHepatic endoplasmic reticulum (ER) stress plays a key role in the development of obesity-induced insulin resistance. This study evaluated the effects of peptides from black soybean (BSP) on ER stress and insulin signaling in vitro and in vivo.Main methodsUsing C2C12 myotubes or HepG2 cells, we evaluated the effects of BSP on the expression of proteins involved in insulin signaling and in the ER stress response in insulin-sensitive or insulin-resistant cells. BSP was given orally to db/db mice for 5 weeks to investigate its antidiabetic effects in vivo and the underlying mechanisms.Key findingsBSP increased GLUT4 translocation and glucose transport in myotubes and stimulated Akt-mediated glycogen synthase kinase-3β (GSK-3β) and Foxo1 phosphorylation in HepG2 cells. BSP significantly restored the suppression of insulin-mediated Akt phosphorylation in insulin-resistant cells. BSP significantly inhibited the activation of ER stress-responsive proteins by thapsigargin. BSP also significantly reduced blood glucose and improved glucose tolerance in db/db mice. The serum lipid profile (triglyceride and high-density lipoprotein concentrations) improved concomitantly with the BSP-induced downregulation of hepatic fatty acid synthase expression in db/db mice. Consistent with the results observed in HepG2 cells, BSP downregulated the elevated hepatic ER stress response in diabetic mice concomitantly with an increased expression of phospho-Foxo1.SignificanceA peptide mixture, BSP, showed beneficial effects through multiple mechanisms involving the suppression of hepatic ER stress and restoration of insulin resistance, suggesting that it has potential as an antidiabetic agent.     

Famotidine inhibits glycogen synthase kinase-3β: An investigation by docking simulation and experimental validation

Famotidine was investigated as an inhibitor of glycogen synthase kinase-3β (GSK-3β) in an attempt to explain the molecular mechanism of its hypoglycemic side effects. The investigation included simulated docking experiments, in vitro enzyme inhibition assay, glycogen sparing studies using animal models and single dose oral glucose tolerance test (OGTT). Docking studies showed how famotidine is optimally fit within the binding pocket of GSK-3β via numerous attractive interactions with some specific amino acids. Experimentally, famotidine could inhibit GSK-3β (IC₅₀ = 1.44 μM) and increased significantly liver glycogen spares in fasting animal models. Moreover, a single oral dose of famotidine was shown to decrease the glycemic response curve after 75 g OGTT     

Molecular properties and antioxidant activities of polysaccharide–protein complexes from selected mushrooms by ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) was evaluated for isolation of polysaccharide–protein (PSP) complexes from three important medicinal mushrooms (Grifola frondosa, Coriolus versicolor and Lentinus edodes). Compared with those of conventional hot-water extraction (HWE), the PSP yield of UAE was similar with G. frondosa, notably higher with L. edodes but lower with C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE had higher protein but lower carbohydrate contents than those by HWE, and their molecular weight (MW) profiles exhibited an overall shift to lower MW and a major low-MW peak near 1.0 kDa. All PSPs from UAE but none from HWE exhibited 3–4 distinct protein bands between 10 and 130 kDa. The antioxidant activities of PSPs extracted by UAE were generally higher than those by HWE.     

Role of the NO synthase system in response to abiotic stress factors for basidiomycetes <Emphasis Type="Italic">Lentinula edodes</Emphasis> and <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Effect of stressors (unfavorable pH and temperature or carbon and nitrogen limitation) on the synthesis of the components of the NO synthase signaling system was studied in submerged cultures of xylotrophic basidiomycetes Lentinula edodes and Grifola frondosa. Marker compounds of the NO synthase signaling system were found in both cultures. A simultaneous increase of the concentrations of NO and citrulline in the culture liquid of the basidiomycetes grown at superoptimal pH and in nitrogen-limited medium indicates the activation of the NO synthase signal system under such stress conditions.     

Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats

A role for elevated glycogen synthase kinase-3 (GSK-3) activity in the multifactorial etiology of insulin resistance is now emerging. However, the utility of specific GSK-3 inhibition in modulating insulin resistance of skeletal muscle glucose transport is not yet fully understood. Therefore, we assessed the effects of novel, selective organic inhibitors of GSK-3 (CT-98014 and CT-98023) on glucose transport in insulin-resistant muscles of Zucker diabetic fatty (ZDF) rats. Incubation of type IIb epitrochlearis and type I soleus muscles from ZDF rats with CT-98014 increased glycogen synthase activity (49 and 50%, respectively, P < 0.05) but did not alter basal glucose transport (2-deoxyglucose uptake). In contrast, CT-98014 significantly increased the stimulatory effects of both submaximal and maximal insulin concentrations in epitrochlearis (37 and 24%) and soleus (43 and 26%), and these effects were associated with increased cell-surface GLUT4 protein. Lithium enhanced glycogen synthase activity and both basal and insulin-stimulated glucose transport in muscles from ZDF rats. Acute oral administration (2 x 30 mg/kg) of CT-98023 to ZDF rats caused elevations in GSK-3 inhibitor concentrations in plasma and muscle. The glucose and insulin responses during a subsequent oral glucose tolerance test were reduced by 26 and 34%, respectively, in the GSK-3 inhibitor-treated animals. Thirty minutes after the final GSK-3 inhibitor treatment, insulin-stimulated glucose transport was significantly enhanced in epitrochlearis (57%) and soleus (43%). Two hours after the final treatment, insulin-mediated glucose transport was still significantly elevated (26%) only in the soleus. These results indicate that specific inhibition of GSK-3 enhances insulin action on glucose transport in skeletal muscle of the insulin-resistant ZDF rat. This unique approach may hold promise as a pharmacological treatment against insulin resistance of skeletal muscle glucose disposal.     

Glycogen synthase: the existence of a single demonstrable from in bovine adipose tissue.

Glycogen synthase from bovine adipose tissue has been kinetically characterized. Glucose 6-phosphate increased enzyme activity 50-fold with an activation constant (A_0.5) of 2.6 mm. Mg²⁺ reversibly decreased this A_0.5 to 0.75 mm without changing the amount of stimulation by glucose 6-phosphate. Mg²⁺ did not alter the apparent K_m for UDP-glucose (0.13 mm). The pH optimum was broad and centered at pH 7.6. The glucose 6-phosphate activation of the enzyme was reversible and competitively inhibited by ATP (K_i = 0.6 mm) and P_i(K_i = 2.0 mm). The use of exogenous sources of glycogen synthase and glycogen synthase phosphatase suggests that (i) adipose tissue glycogen synthase phosphatase activity in fed mature steers is low or undetectable, and (ii) endogenous bovine adipose tissue glycogen synthase can be activated to other glucose 6-phosphate-dependent forms by addition of adipose tissue extracts from fasted steers or fed rats.     

Oxidative stress-induced insulin resistance in rat skeletal muscle: role of glycogen synthase kinase-3

Oxidative stress can contribute to the multifactorial etiology of whole body and skeletal muscle insulin resistance. No investigation has directly assessed the effect of an in vitro oxidant stress on insulin action in intact mammalian skeletal muscle. Therefore, the purpose of the present study was to characterize the molecular actions of a low-grade oxidant stress (H(2)O(2)) on insulin signaling and glucose transport in isolated skeletal muscle of lean Zucker rats. Soleus strips were incubated in 8 mM glucose for 2 h in the absence or presence of 100 mU/ml glucose oxidase, which produces H(2)O(2) at approximately 90 microM. By itself, H(2)O(2) significantly (P < 0.05) activated basal glucose transport activity, net glycogen synthesis, and glycogen synthase activity and increased phosphorylation of insulin receptor (Tyr), Akt (Ser(473)), and GSK-3beta (Ser(9)). In contrast, this oxidant stress significantly inhibited the expected insulin-mediated enhancements in glucose transport, glycogen synthesis, and these signaling factors and allowed GSK-3beta to retain a more active form. In the presence of CT-98014, a selective GSK-3 inhibitor, the ability of insulin to stimulate glucose transport and glycogen synthesis during exposure to this oxidant stress was enhanced by 20% and 39% (P < 0.05), respectively, and insulin stimulation of the phosphorylation of insulin receptor, Akt, and GSK-3 was significantly increased by 36-58% (P < 0.05). These results indicate that an oxidant stress can directly and rapidly induce substantial insulin resistance of skeletal muscle insulin signaling, glucose transport, and glycogen synthesis. Moreover, a small, but significant, portion of this oxidative stress-induced insulin resistance is associated with a reduced insulin-mediated suppression of the active form of GSK-3beta.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Contraction activates glucose uptake and glycogen synthase normally in muscles from dexamethasone-treated rats

Glucocorticoids cause insulin resistance in skeletal muscle. The aims of the present study were to investigate the effects of contraction on glucose uptake, insulin signaling, and regulation of glycogen synthesis in skeletal muscles from rats treated with the glucocorticoid analog dexamethasone (1 mg x kg(-1) x day(-1) ip for 12 days). Insulin resistance in dexamethasone-treated rats was confirmed by reduced insulin-stimulated glucose uptake (approximately 35%), glycogen synthesis (approximately 70%), glycogen synthase activation (approximately 80%), and PKB Ser(473) phosphorylation (approximately 40%). Chronic dexamethasone treatment did not impair glucose uptake during contraction in soleus or epitrochlearis muscles. In epitrochlearis (but not in soleus), the presence of insulin during contraction enhanced glucose uptake to similar levels in control and dexamethasone-treated rats. Contraction also increased glycogen synthase fractional activity and dephosphorylated glycogen synthase at Ser(645), Ser(649), Ser(653), and Ser(657) normally in muscles from dexamethasone-treated rats. After contraction, insulin-stimulated glycogen synthesis was completely restored in epitrochlearis and improved in soleus from dexamethasone-treated rats. Contraction did not increase insulin-stimulated PKB Ser(473) or glycogen synthase kinase-3 (GSK-3) phosphorylation. Instead, contraction increased GSK-3beta Ser(9) phosphorylation in epitrochlearis (but not in soleus) in muscles from control and dexamethasone-treated rats. In conclusion, contraction stimulates glucose uptake normally in dexamethasone-induced insulin resistant muscles. After contraction, insulin's ability to stimulate glycogen synthesis was completely restored in epitrochlearis and improved in soleus from dexamethasone-treated rats.     

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.     

Tissue-specific role of glycogen synthase kinase 3beta in glucose homeostasis and insulin action

Dysregulation of the protein kinase glycogen synthase kinase 3 (GSK-3) has been implicated in the development of type 2 diabetes mellitus. GSK-3 protein expression and kinase activity are elevated in diabetes, while selective GSK-3 inhibitors have shown promise as modulators of glucose metabolism and insulin sensitivity. There are two GSK-3 isoforms in mammals, GSK-3α and GSK-3β. Mice engineered to lack GSK-3β die in late embryogenesis from liver apoptosis, whereas mice engineered to lack GSK-3α are viable and exhibit improved insulin sensitivity and hepatic glucose homeostasis. To assess the potential role of GSK-3β in insulin function, a conditional gene-targeting approach whereby mice in which expression of GSK-3β was specifically ablated within insulin-sensitive tissues were generated was undertaken. Liver-specific GSK-3β knockout mice are viable and glucose and insulin tolerant and display “normal” metabolic characteristics and insulin signaling. Mice lacking expression of GSK-3β in skeletal muscle are also viable but, in contrast to the liver-deleted animals, display improved glucose tolerance that is coupled with enhanced insulin-stimulated glycogen synthase regulation and glycogen deposition. These data indicate that there are not only distinct roles for GSK-3α and GSK-3β within the adult but also tissue-specific phenotypes associated with each of these isoforms.     

Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.     

Effects of lithium and insulin on glycogen synthesis in L6 myocytes: additive effects on inactivation of glycogen synthase kinase-3

In insulin-sensitive L6 myocytes, insulin stimulated glycogen synthesis in a dose-dependent manner and lithium further stimulated glycogen synthesis at all insulin concentrations. Lithium alone at 20 mM stimulated glycogen synthesis to the degree similar to the maximal insulin response. Effects of lithium and insulin were fully additive for both glycogen synthesis and glycogen synthase activity. In L6 myocytes, insulin increased phosphorylation of Akt1 and glycogen synthase kinase-3 alpha and beta (GSK-3 alpha and beta), resulting in its activation and inactivation, respectively. Unlike insulin, lithium directly inhibited GSK-3 (both alpha and beta) without affecting phosphorylation of GSK-3. Moreover, lithium in vitro could further inhibit enzyme activity of GSK-3 (both alpha and beta) that was isolated from insulin-stimulated cells (thus already phosphorylated and inactivated by insulin). In summary, insulin increases glycogen synthesis by the Akt1/GSK-3/glycogen synthase pathway, but lithium increases glycogen synthesis by direct inhibition of GSK-3 in L6 myocytes. Inhibitory effects of lithium and insulin on GSK-3 (both alpha and beta) were additive, which may account, at least in part, for their additive effects on glycogen synthase activity and glycogen synthesis in L6 myocytes.     

6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: Exploitation of GSK-3 for treating leishmaniasis

Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3′-oxime (6-BIO), 6-Br-indirubin-3′acetoxime and 6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC₅₀) values ?1.2 μM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3β, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3β and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals.     

GSK-3beta regulation in skeletal muscles by adrenaline and insulin: evidence that PKA and PKB regulate different pools of GSK-3

We have recently shown that while adrenaline alone has no effect on the activation of Protein Kinase B (PKB) in rat soleus muscle, it greatly potentiates the effects of insulin (Brennesvik et al., Cellular Signalling 17: 1551-1559, 2005). In the current study we went on to investigate whether this was paralleled by a similar effect on GSK-3, which is a major PKB target. Surprisingly adrenaline alone increased phosphorylation of GSK-3beta Ser9 and GSK-3alpha Ser21 and adrenaline's effects were additive with those of insulin but did not synergistically potentiate insulin action. Dibutyryl-cAMP (5 mM) and the PKA specific cAMP analogue N6-Benzoyl-cAMP (2 mM) increased GSK-3beta Ser9 phosphorylation, whereas the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (1 mM) did not. Wortmannin (PI 3-kinase inhibitor; 1 microM) blocked insulin-stimulated GSK-3 phosphorylation completely, but adrenaline increased GSK-3beta Ser9 phosphorylation in the presence of wortmannin. The PKA inhibitor H89 (50 microM) reduced adrenaline-stimulated GSK-3beta Ser9 phosphorylation but did not influence the effects of insulin. Insulin-stimulated GSK-3 Ser9 phosphorylation was paralleled by decreased glycogen synthase phosphorylation at the sites phosphorylated by GSK-3 as expected. However, adrenaline-stimulated GSK-3 Ser9 phosphorylation was paralleled by increased glycogen synthase phosphorylation indicating this pool of GSK-3 may not be directly involved in phosphorylation of glycogen synthase. Our results indicate the existence of at least two distinct pools of GSK-3beta in soleus muscle, one phosphorylated by PKA and another by PKB. Further, we hypothesise that each of these pools is involved in the control of different cellular processes.     

Palbinone and triterpenes from Moutan Cortex (Paeonia suffruticosa,Paeoniaceae) stimulate glucose uptake and glycogen synthesis via activation of AMPK in insulin-resistant human HepG2 Cells

Moutan Cortex is a well-known herb in traditional Korean, Chinese, and Japanese anti-diabetic formulae. In the current study, we investigated the metabolic effects of isolated triterpenes (1–7) in HepG2 cells under high glucose conditions. These compounds remakably stimulated AMP-activated protein kinase (AMPK), GSK-3β, and ACC phosphorylation. The compounds also increased glucose uptake and enhanced glycogen synthesis. Among these, compound 1 displayed the greatest potential anti-diabetic activity though the AMPK activation pathway. Compound 1 significantly increased the levels of phospho-AMPK, phospho-ACC, and phospho-GSK-3β and stimulated glucose uptake and glycogen synthesis in a dose-dependent manner. In conclusion, our results suggest that these compounds, especially compound 1, may have beneficial roles in glucose metabolism via the AMPK pathway.