Cell signalling of the GLP-1 action in rat liver

GLP-1, incretin with insulin-independent antidiabetic properties, is insulinomimetic upon glucose metabolism in extrapancreatic tissues, acting through specific receptors not associated to adenylate cyclase activation. We investigated the role of enzymes mediating insulin actions, in the GLP-1-induced glycogen synthase a activation in rat hepatocytes. GLP-1, like insulin, activates PI3K/PKB, p70s6k, p44 and p42 MAP-kinase. Wortmannin (PI3K/PKB inhibitor) blocked the stimulatory action of insulin on glycogen synthase a and reduced that of GLP-1; rapamycin (p70s6k inhibitor) was ineffective and PD98059 (MEK/MAPK inhibitor) decreased only the insulin effect; okadaic acid (PP-2A inhibitor) was ineffective, while TNFalpha (PP-1 inhibitor) blocked the action of insulin and reduced that of GLP-1; H-7 or Ro 31-8220 (PKC inhibitors) decreased the GLP-1 effect, while only H-7 reduced that of insulin. The activation of PI3K/PKB, PKC and PP-1, but not PP-2A, seems to mediate the GLP-1 stimulatory action on glycogen synthase a in rat hepatocytes, while MAPKs and p70s6k could participate in other GLP-1 effects.     

Effects of glucagon-like peptide-1 and exendins on kinase activity, glucose transport and lipid metabolism in adipocytes from normal and type-2 diabetic rats

Several kinases have been implicated in the metabolic response of human and rat myocytes to glucagon-like peptide-1 (GLP-1), exendin-4 (Ex-4) and exendin-9 (Ex-9). We have investigated, in isolated rat adipocytes, the changes caused by GLP-1, Ex-4 and Ex-9 compared with those provoked by insulin or glucagon, upon the activity of phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB), p42/44 MAP kinases (MAPKs) and p70s6 kinase (p70s6k), and the participation of these kinases and protein kinase C (PKC) in their action upon 2-deoxy-d-glucose uptake, lipolysis and lipogenesis. The study was conducted in normal rats, and extended to a streptozotocin-induced type-2 diabetic model (STZ-rats). The participation of distinct kinases was estimated by using potential kinase inhibitors, including wortmannin, PD98059, rapamycin, H-7 and RO31-8220. In normal rat adipocytes, GLP-1 and both exendins share with insulin an increasing action upon the activity of all kinases studied (except PKB), PI3K, p44 and p42 MAPKs and possibly PKC, all being required for their stimulating effect upon glucose uptake. Ex-4 and Ex-9, like GLP-1 and insulin, have lipogenic action, while only Ex-4 shares with GLP-1 its lipolytic effect which is antagonized by Ex-9. MAP kinases and PKC seem to have an essential role in the GLP-1 and Ex-4 lipolytic action, as does PI3K in that of Ex-4. An increase in PI3K and MAPKs activity for the lipogenic effect of Ex-4, Ex-9 and GLP-1 are required, and in the case of Ex-4 and Ex-9, a stimulation of p70s6k activity is also needed. In cells from STZ-rats the magnitude of the above parameters was, in general, comparable to that in normal animals, with some exceptions: basal PI3K activity and lipogenesis were higher, GLP-1, Ex-4 and Ex-9 failed to modify basal lipogenesis but increased PKB activity, insulin failed to affect the activity of MAPKs and the insulin-induced glucose uptake was impaired. The impaired insulin effects upon some of the variables in the STZ-rat, distinct from those of GLP-1 and exendins, adds knowledge to the mechanism of the beneficial action of GLP-1 and Ex-4 in diabetic states.     

Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor

Aims/hypothesis Treatment with glucocorticoids, especially at high doses, induces insulin resistance. The aims of the present study were to identify the potential defects in insulin signalling that contribute to dexamethasone-induced insulin resistance in skeletal muscles, and to investigate whether the glycogen synthase-3 (GSK-3) inhibitor CHIR-637 could restore insulin-stimulated glucose metabolism.Materials and methods Skeletal muscles were made insulin-resistant by treating male Wistar rats with dexamethasone, a glucocorticoid analogue, for 12 days. Insulin-stimulated glucose uptake, glycogen synthesis and insulin signalling were studied in skeletal muscles in vitro.Results Dexamethasone treatment decreased the ability of insulin to stimulate glucose uptake, glycogen synthesis and glycogen synthase fractional activity. In addition, the dephosphorylation of glycogen synthase by insulin was blocked. These defects were paralleled by reduced insulin-stimulated protein kinase B (PKB) and GSK-3 phosphorylation. While expression of PKB, GSK-3 and glycogen synthase was not reduced by dexamethasone treatment, expression of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) was increased. Inhibition of GSK-3 by CHIR-637 increased glycogen synthase fractional activity in soleus muscle from normal and dexamethasone-treated rats, although the effect was more pronounced in control rats. CHIR-637 did not improve insulin-stimulated glucose uptake in muscles from dexamethasone-treated rats.Conclusions/interpretation We demonstrated that chronic dexamethasone treatment impairs insulin-stimulated PKB and GSK-3 phosphorylation, which may contribute to insulin resistance in skeletal muscles. Acute pharmacological inhibition of GSK-3 activated glycogen synthase in muscles from dexamethasone-treated rats, but GSK-3 inhibition did not restore insulin-stimulated glucose uptake.     

RO 31-8220 activates c-Jun N-terminal kinase and glycogen synthase in rat adipocytes and L6 myotubes. Comparison to actions of insulin

The activation of c-Jun N-terminal kinase (JNK) by insulin and anisomycin has been reported to result in increases in glycogen synthase (GS) activity in rat skeletal muscle (Moxham et al., J Biol Chem, 1996, 271:30765-30773). In addition, the protein kinase C (PKC) inhibitor, RO 31-8220, has been reported to activate JNK in rat-1 fibroblasts (Beltman et al., J Biol Chem, 1996, 271:27018-27024). Presently, we found that the RO 31-8220, as well as insulin, activated JNK and GS and stimulated glucose incorporation into glycogen in rat adipocytes and L6 myotubes. In contrast to activation of JNK, RO 31-8220 inhibited extracellular response kinases 1 and 2 (ERK1/2) and had no significant effects on protein kinase B (PKB). Stimulatory effects of RO 31-8220 on JNK and glycogen metabolism were not explained by PKC inhibition, as other PKC inhibitors were without effect on glucose incorporation into glycogen and have no effect on JNK (Beltman et al., J Biol Chem, 1996, 271:27018). Insulin, on the other hand, activated JNK, as well as PKB and ERK1/2. However, stimulatory effects of insulin on GS and glucose incorporation into glycogen appeared to be fully intact and additive to those of RO 31-8220, despite the fact that insulin did not provoke additive increases in JNK activity above those observed with RO 31-8220 alone. Our findings suggest that JNK serves to activate GS during the action of RO 31-8220 in rat adipocytes and L6 myotubes; insulin, on the other hand, appears to activate GS largely independently of JNK.     

Involvement of the serine/threonine p70S6 kinase in TGF-beta1-induced ADAM12 expression in cultured human hepatic stellate cells

BACKGROUND/AIMS: In chronic liver injury, quiescent hepatic stellate cells change into proliferative myofibroblast-like cells, which are a main source of fibrosis. We have recently reported that these cells synthesize ADAM12, a disintegrin and metalloprotease whose expression is up-regulated by TGF-beta1 in liver cancers. Here, we studied the role of the serine/threonine p70S6 kinase (p70S6K) in regulating TGF-beta1-induced ADAM12 expression. RESULTS: The phophatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the mitogen-activated protein kinase inhibitor, UO126, decreased the TGF-beta1-dependent ADAM12 expression and prevented the phosphorylation of p70S6K. In addition, TGF-beta1-induced ADAM12 up-regulation was blocked by the Frap/mTOR inhibitor rapamycin, which abrogated the phosphorylation of p70S6K. In untreated cells, LY294002 but not rapamycin diminished the basal ADAM12 expression related to inhibition of Akt and the glycogen synthase kinase-3 phosphorylation. CONCLUSIONS: The data suggest that TGF-beta1 induces ADAM12 gene expression through both the PI3K/Frap-mTOR/p70S6K and MEK/ERK pathways. In addition, activation of the PI3 pathway might be involved in the basal ADAM12 expression in cultured hepatic stellate cells. The involvement of PI3K in ADAM12 expression, similar to that previously observed for collagen I and fibronectin, suggests common pathways for gene up-regulation in hepatic stellate cells that occur during liver fibrogenesis and contribute to tumor progression.     

Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1

OBJECTIVE: Insulin-responsive adipogenic signaling molecules include insulin receptor substrates (IRS)-1 and -2, phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB; also known as Akt). Mammalian target of rapamycin (mTOR) is a PKB substrate, and regulates p70 S6 kinase (p70 S6K). Since p70 S6K is an insulin-responsive kinase downstream of PI3K and PKB, its potential role in adipogenic insulin signaling was investigated. DESIGN: We measured the effect of rapamycin, a specific inhibitor of mTOR, on insulin-induced 3T3-L1 adipogenesis and on insulin-stimulated p70 S6K activation. RESULTS: Rapamycin partially reduced differentiation, measured by Oil Red O staining, triacylglycerol accumulation (by up to 46%), and peroxisome proliferator-activated receptor gamma protein expression (by 50%). In contrast, rapamycin completely inhibited insulin-stimulated p70 S6K activation, assessed by phosphorylation of p70 S6K and its substrate, S6. Expression of a constitutively activated form of p70 S6K did not promote 3T3-L1 adipogenesis. The considerable residual differentiation in the presence of rapamycin, despite the complete blockade of p70 S6K activation, prompted us to measure the phosphorylation of another rapamycin-sensitive protein, eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Insulin-stimulated 4E-BP1 phosphorylation in 3T3-L1 preadipocytes was only partially affected by rapamycin, consistent with the differentiation data. Phosphorylation of eIF4E itself, an expected consequence of 4E-BP1 phosphorylation, was also only partially inhibited. CONCLUSION: Our data suggest that adipogenic mTOR signaling occurs via the 4E-BP1/eIF4E pathway, rather than through p70 S6K.     

Homocysteine thiolactone inhibits insulin-stimulated DNA and protein synthesis: possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation

Hyperhomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of homocysteine are related to the formation of homocysteine thiolactone and the consequent increase in oxidative stress. We have recently found that homocysteine thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress seemed to be the mechanism underlying these effects, since glutathione was able to restore the insulin signaling as well as the insulin-mediated glycogen synthesis. In the present work we have further investigated insulin receptor signaling studying mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. Again, homocysteine thiolactone (50 microM) prevented insulin-mediated MAPK, GSK-3 and p70 S6K phosphorylation and these effects were blocked by glutathione (250 microM). Since MAPK and PI3K pathways, including GSK3 and S6K, seem to mediate insulin-mediated growth and proliferation, we measured DNA and protein synthesis. We have found that homocysteine thiolactone (50 microM) inhibits insulin-mediated growth and proliferation, as previously shown for glycogen synthesis. Again, these effects seem to be mediated by oxidative stress, since 250 microM glutathione completely abolished the effects of homocysteine thiolactone on insulin-stimulated DNA and protein synthesis. In conclusion, these data suggest that homocysteine thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in the action of insulin on growth and proliferation.     

The role of protein kinase B/Akt in insulin-induced inactivation of phosphorylase in rat hepatocytes

Aims/hypothesis An insulin signalling pathway leading from activation of protein kinase B (PKB, also known as Akt) to phosphorylation (inactivation) of glycogen synthase kinase-3 (GSK-3) and activation of glycogen synthase is well characterised. However, in hepatocytes, inactivation of GSK-3 is not the main mechanism by which insulin stimulates glycogen synthesis. We therefore tested whether activation of PKB causes inactivation of glycogen phosphorylase. Materials and methods We used a conditionally active form of PKB, produced using recombinant adenovirus, to test the role of acute PKB activation in the control of glycogen phosphorylase and glycogen synthesis in hepatocytes. Results Conditional activation of PKB mimicked the inactivation of phosphorylase, the activation of glycogen synthase, and the stimulation of glycogen synthesis caused by insulin. In contrast, inhibition of GSK-3 caused activation of glycogen synthase but did not mimic the stimulation of glycogen synthesis by insulin. PKB activation and GSK-3 inhibition had additive effects on the activation of glycogen synthase, indicating convergent mechanisms downstream of PKB involving inactivation of either phosphorylase or GSK-3. Glycogen synthesis correlated inversely with the activity of phosphorylase-a, irrespective of whether this was modulated by insulin, by PKB activation or by a selective phosphorylase ligand, supporting an essential role for phosphorylase inactivation in the glycogenic action of insulin in hepatocytes. Conclusions/interpretation In hepatocytes, the acute activation of PKB, but not the inhibition of GSK-3, mimics the stimulation of glycogen synthesis by insulin. This is explained by a pathway downstream of PKB leading to inactivation of phosphorylase, activation of glycogen synthase, and stimulation of glycogen synthesis, independent of the GSK-3 pathway.     

Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism

Summary Isolated skeletal muscle from healthy individuals was used to evaluate the role of phosphoinositide 3-kinase (PI 3-kinase) in insulin signalling pathways regulating mitogen activated protein kinase (MAP-kinase) and protein kinase-B and to investigate whether MAP-kinase was involved in signalling pathways regulating glucose metabolism. Insulin stimulated glycogen synthase activity ( ≈ 1.7 fold), increased 3-o-methylglucose transport into human skeletal muscle strips ( ≈ 2 fold) and stimulated phosphorylation of the p42 ERK-2 isoform of MAP-kinase. This phosphorylation of p42 ERK2 was not blocked by the PI 3-kinase inhibitors LY294002 and wortmannin although it was blocked by the MAP-kinase kinase (MEK) inhibitor PD 98059. However, PD98059 (up to 20 μmol/l) did not block insulin activation of glycogen synthase or stimulation of 3-o-methylglucose transport. Wortmannin and LY294002 did block insulin stimulation of protein kinase-B (PKB) phosphorylation and stimulation of 3-o-methylglucose transport was inhibited by wortmannin (IC₅₀≈ 100 nmol/l). These results indicate that MAP-kinase is activated by insulin in human skeletal muscle by a PI 3-kinase independent pathway. Furthermore this activation is not necessary for insulin stimulation of glucose transport or activation of glycogen synthase in this tissue. [Diabetologia (1997) 40: 1172–1177]     

Delays in insulin signaling towards glucose disposal in human skeletal muscle

We explored whether the delay that occurs between a rise in plasma insulin and the increase of glucose disposal occurs before, at, or downstream of steps that are believed to be part of the insulin signaling cascade. Skeletal muscle biopsies were obtained from 16 nondiabetic subjects before, and 20 and 180 min after plasma insulin levels had been augmented in euglycemic hyperinsulinemic glucose clamps. Although plasma insulin had reached 98% of its final concentration within 10 min, insulin receptor kinase (IRK) activity, p85 associated with insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity, and Thr(308)-protein kinase B (PKB) phosphorylation in the muscle biopsies at 20 min had reached only 60, 48, 34 and 47% respectively of those at 180 min. This suggests a delay before the level of IRK and little or no delay between IRK and PKB activation. The observation that glycogen synthase activity and glucose disposal at 20 min had both only reached 25% of the respective values at 180 min suggests an additional delay downstream of the investigated signaling steps.     

Glucocorticoids differentially modulate insulin-mediated protein and glycogen synthetic signaling downstream of protein kinase B in rat myocardium

Insulin and protein kinase B (or Akt) play critical roles in cardiomyocytic growth and survival. High concentrations of glucocorticoids antagonize insulin's action. To examine whether endogenous glucocorticoids modulate insulin's effect on Akt signaling in the protein and glycogen synthetic pathways in myocardium, we studied three groups of rats (n = 12 each) 4 d after either a bilateral adrenalectomy (ADX), ADX with physiological stress dose dexamethasone treatment (ADX + DEX), or a sham operation. Rats received either a saline infusion or a 3 mU/kg.min euglycemic insulin clamp for 3 h. ADX had no effect on myocardial Akt or GSK-3 [glycogen synthase (GS) kinase 3] phosphorylation, but it decreased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (p70(S6K)) (P < 0.003 for both). Insulin enhanced the phosphorylation of Akt (P < 0.04), 4E-BP1 (P < 0.002), and p70(S6K) (P < 0.0001) in ADX, but not in sham rats. Dexamethasone restored the levels of 4E-BP1 and p70(S6K) phosphorylation and abrogated the insulin-stimulated Akt, 4E-BP1, and p70(S6K) phosphorylation. ADX rats had higher GS activity (P = 0.058) and lower glycogen content (P < 0.0001) than sham rats. GSK-3 phosphorylation after insulin infusion was greater in ADX rats. Insulin did not alter GS activity. Although insulin did not change the glycogen content in sham or ADX rats, it increased glycogen content by approximately 50% in ADX + DEX rats (P < 0.02). We conclude that endogenous glucocorticoids differentially modulate the regulation of Akt-4E-BP1/p70(S6K) and Akt-GSK-3-GS signaling pathways in heart by physiologic hyperinsulinemia over a range from deficiency to physiological stress concentrations.     

Insulin Signaling, GSK-3, Heat Shock Proteins and the Natural History of Type 2 Diabetes Mellitus: A Hypothesis

Metabolic syndrome and type 2 diabetes are progressive, indolent, multi-organ diseases. Understanding the abnormalities of heat shock proteins (HSPs) in these diseases is paramount to understanding their pathogenesis. In insulin resistant states and diabetes, heat shock factor 1(HSF-1) is low in insulin sensitive tissues, resulting in low Hsp 60, 70, and 90 levels. We propose that low Hsps levels are the result of decreased insulin action leading to less phosphorylation of PI3K, PKB, and glycogen synthase kinase-3 (GSK-3). Importantly, less GSK-3 phosphorylation (and thus more GSK-3 activity) will lower HSF-1. Low Hsps make organs vulnerable to injury, impair the stress response, accelerate systemic inflammation, raise islet amyloid polypeptide, and increase insulin resistance. Feeding this cycle is excess saturated fat and calorie consumption, hypertension, inactivity, aging, and genetic predisposition- all of which are a associated with high GSK-3 activity and low Hsps. Support for the proposed "vicious" cycle is based on the observation that GSK-3 inhibition and Hsp stimulation result in increased insulin sensitivity, reduced accumulation of degenerative proteins with in the cell, improved wound healing, decreased organ damage and improved recovery from vascular ischemia. Recognizing GSK-3 and Hsps in the pathogenesis of insulin resistance, the central common feature of the metabolic syndrome, and type 2 diabetes will expand our understanding of the disease, offering new therapeutic options.     

Impaired muscle glycogen synthase in type 2 diabetes is associated with diminished phosphatidylinositol 3-kinase activation

Insulin signaling pathways potentially involved in regulation of skeletal muscle glycogen synthase were compared in differentiated human muscle cell cultures from nondiabetic and type 2 diabetic patients. Insulin stimulation of glycogen synthase activity as well as phosphorylation of MAPK, p70 S6 kinase, and protein kinase B (Akt) were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin (50 nM) and LY294002 (10 microM). In contrast to lean and obese nondiabetic subjects, where there were minimal effects (15-20% inhibition), insulin stimulation of glycogen synthase in muscle cultures from diabetic subjects was greatly diminished ( approximately 75%) by low concentrations of wortmannin (25 nM) or LY294002 (2 microM). This increased sensitivity of diabetic muscle to impairment of insulin-stimulated glycogen synthase activity occurs together with diminished insulin-stimulation (by 40%) of IRS-1-associated phosphatidylinositol 3-kinase activity in the same cells. Protein expression of IRS-1, p85, p110, Akt, p70 S6 kinase, and MAPK were normal in diabetic cells, as was insulin-stimulated phosphorylation of Akt, p70 S6 kinase, and MAPK. These studies indicate that, despite prolonged growth and differentiation of diabetic muscle under normal metabolic culture conditions, defects of insulin-stimulated phosphatidylinositol 3-kinase and glycogen synthase activity in diabetic muscle persist, consistent with intrinsic (rather than acquired) defects of insulin action.     

软脂酸诱导HepG2细胞胰岛素抵抗及花生四烯酸防治作用的机制研究

目的 探讨高浓度软脂酸(PA)诱导HepG2细胞胰岛素抵抗(IR)的机制及花生四烯酸(AA)对IR的防治作用。方法 (1)用高浓度软脂酸(PA)或10^-7mol／L高胰岛素(HI)培养HepG2细胞建立具有IR的细胞模型，测定培养液中葡萄糖含量及细胞内糖原含量作为鉴定指标；(2)用Western blot检测胞内糖原合酶(GS)和蛋白激酶B(PKB)蛋白水平；(3)用磷脂酰肌醇3激酶(P13K)抑制剂Wortmannin(WT)探讨其对胰岛素信号通路的影响；(4)观察AA是否对PA引起的IR有防治作用。结果 (1)0．20mmol／L PA或川培养HepG2细胞36h后，培养液中葡萄糖含量极显著增高，细胞内糖原含量极显著减少；(2)高浓度PA使磷酸化的PKB(P-Ser473)蛋白水平显著减少，磷酸化的糖原合酶(P-Ser641 GS)蛋白水平极显著增加；(3)WT使对照组GS活性及胞内糖原含量极显著减少，HI组和PA组胞内糖原含量均无统计学差异，但各实验组PKB活性都极显著减少；(4)PA AA组培养液中葡萄糖含量显著低于PA组，GS和PKB活性及胞内糖原含量显著增加。结论 高浓度PA或HI培养HepG2细胞能够诱导IR，其机制可能是其引起胰岛素信号传递途径中自PKB下游到GS之间的信号通路受阻所致。AA能改善PA引起的IR。     

Signalling pathways involved in the stimulation of glycogen synthesis by insulin in rat hepatocytes

Summary In hepatocytes glycogen storage is stimulated by insulin and this effect of insulin is counteracted by epidermal growth factor (EGF). The mechanism by which insulin stimulates glycogen synthesis in liver is unknown. We investigated the involvement of candidate protein kinases in insulin signalling in hepatocytes. Both insulin and EGF activated extracellular regulated kinase 2 (ERK-2), p70^rsk and protein kinase B (PKB) and inactivated glycogen synthase kinase-3 (GSK-3). Whereas EGF caused a greater activation of ERK-2 than insulin, the converse was true for PKB. The stimulation by insulin of ERK-2 was blocked by a mitogen-activated protein (MEK) inhibitor (PD 98 059) and of p70^rsk by rapamycin. However, these inhibitors, separately or in combination, did not block the stimulation of glycogen synthesis by insulin, indicating that activation of these kinases is not essential for the stimulation of glycogen synthesis by insulin. Mono Q fractionation of hepatocyte extracts resolved a single myelin basic protein (MBP) kinase peak from extracts of EGF-treated cells (peak 1, eluting at 200 mmol/l NaCl) and two peaks from insulin-treated cells (peak 1 eluting at 200 mmol/l NaCl and peak 2 eluting at 400 mmol/l NaCl). In the combined presence of insulin and EGF, activation of peak 2 was abolished. In situ MBP kinase assays and immunoblotting established that peak 1 coincides with ERK-2 and peak 2 is not an activated form of ERK-1 or ERK-2. It is concluded that PKB, which is activated to a greater extent by insulin than EGF, and peak 2, which is activated by insulin and counteracted by EGF, are possible candidates in mediating the stimulation of glycogen synthesis by insulin. [Diabetologia (1998) 41: 16–25] Received: 24 April 1997 and in revised form: 26 August 1997     

Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway

The activation of the glucagon-like peptide-1 (GLP-1) receptor has been shown to have an important role in the functional activity of islet beta-cells and in the expansion of the islet cell mass. Constant remodeling of islet cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of GLP-1 when cells were challenged by a proapoptotic stimulus. We have shown that activation of the GLP-1 receptor inhibits H(2)O(2)-induced apoptosis in a cultured mouse insulinoma cell line, termed MIN6. GLP-1 reduced DNA fragmentation and improved cell survival. This was mediated by an increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. GLP-1 also prevented the H(2)O(2)-dependent cleavage of poly-(ADP-ribose)-polymerase. Inhibition of the GLP-1-dependent increase of cAMP by Rp-cAMP blocked the antiapoptotic action of GLP-1, as determined by DNA fragmentation and poly-(ADP-ribose)-polymerase assays and by detection of Bcl-2 and Bcl-xL protein levels. Investigation of the role of the protein kinases, PI-3 kinase (PI3K) and MAPK, by use of the inhibitors PD098059 and LY294002 demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H(2)O(2). The present study provides evidence that GLP-1 has an antiapoptotic action mediated by a cAMP- and PI3K-dependent signaling pathway.     

Bile acids enhance the activity of the insulin receptor and glycogen synthase in primary rodent hepatocytes

Previously, we demonstrated that deoxycholic acid (DCA)-induced ERK1/2 and AKT signaling in primary hepatocytes is a protective response. In the present study, we examined the regulation of the phosphatidylinositol 3 (PI3) kinase/AKT/glycogen synthase (kinase) 3 (GSK3)/glycogen synthase (GS) pathway by bile acids. In primary hepatocytes, DCA activated ERBB1 (the epidermal growth factor receptor), ERBB2, and the insulin receptor, but not the insulin-like growth factor 1 (IGF-1) receptor. DCA-induced activation of the insulin receptor correlated with enhanced phosphorylation of insulin receptor substrate 1, effects that were both blocked by the insulin receptor inhibitor AG1024 and by expression of the dominant negative IGF-1 receptor (K1003R), which inhibited in trans. Expression of the dominant negative IGF-1 receptor (K1003R) also abolished DCA-induced AKT activation. Bile acid-induced activation of AKT and phosphorylation of GSK3 were blunted by the ERBB1 inhibitor AG1478 and abolished by AG1024. Bile acids caused activation of GS to a similar level induced by insulin (50 nM); both were blocked by inhibition of insulin receptor function and the PI3 kinase/AKT/GSK3 pathway. In conclusion, these findings suggest that bile acids and insulin may cooperate to regulate glucose storage in hepatocytes.     

Normalizing action of exendin-4 and GLP-1 in the glucose metabolism of extrapancreatic tissues in insulin-resistant and type 2 diabetic states

Exendin-4 (Ex-4) mimics glucagon-like peptide-1 (GLP-1 or GCG as listed in the HUGO database), being anti-diabetic and anorectic, in stimulating glucose and lipid metabolism in extrapancreatic tissues. We studied the characteristics of Ex-4 and GLP-1 action, during prolonged treatment, on GLUTs expression (mRNA and protein), glycogen content (GC), glucose transport (GT), glycogen synthase a (GSa), and kinase (PI3K and MAPKs) activity, in liver, muscle, and fat of insulin-resistant (IR, by fructose) and type 2 diabetic (T2D, streptozotocin at birth) rats compared with normal rats. In both IR and T2D, the three tissues studied presented alterations in all measured parameters. In liver, GLP-1 and also Ex-4 normalized the lower than normal Glut2 (Slc2a2) expression and showed a trend to normalize the reduced GC in IR, and GLP-1, like Ex-4, also in T2D, effects mediated by PI3K and MAPKs. In skeletal muscle, neither GLP-1 nor Ex-4 modified Glut4 (Slc2a4) expression in either experimental model but showed normalization of reduced GT and GSa, in parallel with the normalization of reduced PI3K activity in T2D and MAPKs in both models. In adipose tissue, the altered GLUT4 expression in IR and T2D, along with reduced GT in IR and increased GT in T2D, and with hyperactivated PI3K in both, became normal after GLP-1 and Ex-4 treatment; yet, MAPKs, that were also higher, became normal only after Ex-4 treatment. The data shows that Ex-4, as well as GLP-1, exerts a normalizing effect on IR and T2D states through a distinct post-receptor mechanism, the liver being the main target for Ex-4 and GLP-1 to control glucose homeostasis.