PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.     

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.     

Hepatitis C virus activates the mTOR/S6K1 signaling pathway in inhibiting IRS-1 function for insulin resistance

Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.     

Effects of streptozotocin-induced type 1 maternal diabetes on PI3K/AKT signaling pathway in the hippocampus of rat neonates

Diabetes in pregnancy impairs hippocampus development in offspring, leading to behavioral problems and learning deficits. Phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway plays a pivotal role in the regulation of neuronal proliferation, survival and death. The present study was designed to examine the effects of maternal diabetes on PKB/Akt expression and phosphorylation in the developing rat hippocampus. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at first postnatal day (P1). The hippocampal expression and phosphorylation level of PKB/Akt, one of the key molecules in PI3K/AKT signaling pathway, was evaluated using real-time polymerase chain reaction (PCR) and western blot analysis. We found a significant bilateral downregulation of AKT1 gene expression in the hippocampus of pups born to diabetic mothers (p?<?0.05). Interestingly, our results revealed a marked upregulation of Akt1 gene in insulin-treated group compared with other groups (p?<?0.05). The western blot analysis also showed the reduction of phosphorylation level of all AKT isoforms in both diabetic and insulin-treated groups compared with control (p?<?0.05). Moreover, the results showed a significant increase in phosphorylation level of AKT in insulin-treated group compared with the diabetic group. These results represent that diabetes during pregnancy strongly influences the regulation of PKB/AKT in the developing rat hippocampus. Furthermore, although the control of glycemia by insulin administration is not sufficient to prevent the alterations in PKB/Akt expression, it modulates the phosphorylation process, thus ultimately resulting in a situation comparable to that found in the normal condition.     

Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6-hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa-induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.     

The CB1 endocannabinoid system modulates adipocyte insulin sensitivity

Mounting evidence suggests that the endocannabinoid system regulates energy metabolism through direct effects on peripheral tissues as well as central effects that regulate appetite. Here we examined the effect of cannabinoid receptor 1 (CB1) signaling on insulin action in fat cells. We examined effects of the natural CB1 agonist, 2-Arachidonoylglycerol (2-AG), and the synthetic CB1 antagonist, SR141716, on insulin action in cultured adipocytes. We used translocation of glucose transporter GLUT4 to plasma membrane (PM) as a measure of insulin action. 2-AG activation of the CB1 receptor promoted insulin sensitivity whereas antagonism by SR141716 reduced insulin sensitivity. Neither drug affected GLUT4 translocation in the absence of insulin or with high doses of insulin. Consistent with these results we found that insulin-stimulated phosphorylation of the protein kinase Akt was increased by 2-AG, attenuated by SR141716, and unaffected in the absence of insulin or by addition of high-dose insulin. These data provide a functional and molecular link between the CB1 receptor and insulin sensitivity, because insulin-stimulated phosphorylation of Akt is required for GLUT4 translocation to the PM. The sensitizing effects of 2-AG were abrogated by SR141716 and Pertussis toxin, indicating that the effects are mediated by CB1 receptor. Importantly, neither 2-AG nor SR141716 alone or in combination with maximal dose of insulin had effects on GLUT4 translocation and Akt phosphorylation. These data are consistent with a model in which the endocannabinoid system sets the sensitivity of the insulin response in adipocytes rather than directly regulating the redistribution of GLUT4 or Akt phosphorylation.     

The Akt substrate Girdin is a regulator of insulin signaling in myoblast cells

Akt kinases are important mediators of the insulin signal, and some Akt substrates are directly involved in glucose homeostasis. Recently, Girdin has been described as an Akt substrate that is expressed ubiquitously in mammals. Cells overexpressing Girdin show an enhanced Akt activity. However, not much is known about Girdin's role in insulin signaling. We therefore analyzed the role of Girdin in primary human myotubes and found a correlation between Girdin expression and insulin sensitivity of the muscle biopsy donors, as measured by a hyperinsulinemic–euglycemic clamp. To understand this finding on a cellular level, we then investigated the function of Girdin in C2C12 mouse myoblasts. Girdin knock-down reduced Akt and insulin receptor substrate-1 phosphorylation. In contrast, stable overexpression of Girdin in C2C12 cells strikingly increased insulin sensitivity through a massive upregulation of the insulin receptor and enhanced tyrosine phosphorylation of insulin receptor substrate-1. Furthermore, Akt and c-Abl kinases were constitutively activated. To investigate medium-term insulin responses we measured glucose incorporation into glycogen. The Girdin overexpressing cells showed a high basal glycogen synthesis that peaked already at 1 nM insulin. Taken together, we characterized Girdin as a new and major regulator of the insulin signal in myoblasts and skeletal muscle.     

Physiological and pathological changes in glucose regulate brain Akt and glycogen synthase kinase-3

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.     

Regulation of pancreatic duct cell differentiation by phosphatidylinositol-3 kinase

We have previously demonstrated that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling is essential for pancreatic regeneration after partial pancreatectomy in mice. In the present study, we examined a role of PI3K/Akt signaling for pancreatic duct cell differentiation into insulin-producing cells. Epithelial-like cells were isolated from mouse pancreas and confirmed to be positive for a duct cell marker cytokeratin-20 (CK-20) but negative for insulin. Incubation of these cells with epidermal growth factor, exhibited a gradual increase in Akt phosphorylation and expression of pancreatic duodenal homeobox-1 (PDX-1), a regulator of β-cell differentiation. Three weeks later, these CK-20-positive cells were noted to express insulin as determined by immunofluorescent double-staining. Akt phosphorylation, PDX-1 expression, and insulin production were effectively reduced by blocking the PI3K/Akt pathway using siRNA to the p85α regulatory subunit of PI3K. Our results demonstrate that PI3K/Akt activation has a critical role for pancreatic duct cell differentiation into insulin-producing cells.     

ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression

In addition to well-documented vascular growth-promoting effects, ANG II exerts proapoptotic effects that are poorly understood. IGF-1 is a potent survival factor for human vascular smooth muscle cells (hVSMC), and its antiapoptotic effects are mediated via the IGF-1 receptor (IGF-1R) through a signaling pathway involving phosphatidylinositol 3-kinase and Akt. We hypothesized that there would be cross talk between ANG II proapoptotic effects and IGF-1 survival effects in hVSMC. To investigate ANG II-induced apoptosis and the potential involvement of IGF-1, we exposed quiescent and nonquiescent hVSMC to ANG II. ANG II induced apoptosis only in nonquiescent cells but stimulated hypertrophy in quiescent cells. ANG II-induced apoptosis was characterized by marked inhibition of Akt phosphorylation and stimulation of membrane Fas ligand (FasL) expression, caspase-8 activation, and a reduction in soluble FasL expression. Adenovirally mediated overexpression of Akt rescued hVSMC from ANG II-induced apoptosis. IGF-1R activation increased Akt phosphorylation and soluble FasL expression, and these effects were completely blocked by coincubating hVSMC with ANG II. In conclusion, ANG II-induced apoptosis of hVSMC is characterized by marked inhibition of Akt phosphorylation and stimulation of an extrinsic cell death signaling pathway via upregulation of membrane FasL expression, caspase-8 activation, and a reduction in soluble FasL expression. Furthermore, ANG II antagonizes the antiapoptotic effect of IGF-1 by blocking its ability to increase Akt phosphorylation and soluble FasL. These findings provide novel insights into ANG II-induced apoptotic signaling and have significant implication for understanding ANG II-induced remodeling in hypertension and atherosclerosis.     

The effects of intracellular calcium depletion on insulin signaling in 3T3-L1 adipocytes

We have examined the requirement for intracellular calcium (Ca(2+)) in insulin signal transduction in 3T3-L1 adipocytes. Using the Ca(2+) chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), we find both augmentation and inhibition of insulin signaling phenomena. Pretreatment of cells with 50 microM BAPTA-AM did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 or insulin receptor (IR)beta. The decreased mobility of IRS1 normally observed after chronic stimulation with insulin, due to serine phosphorylation, was completely eliminated by Ca(2+) chelation. Correlating with decreased insulin-induced serine phosphorylation of IRS1, phosphotyrosine-mediated protein-protein interactions involving p85, IRS1, IRbeta, and phosphotyrosine-specific antibody were greatly enhanced by pretreatment of cells with BAPTA-AM. As a result, insulin-mediated, phosphotyrosine-associated PI3K activity was also enhanced. BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca(2+) depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca(2+) in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca(2+) is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca(2+) blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.     

Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3beta (GSK-3beta), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by insulin in controls. The protein expression of Akt/PKB, GSK-3beta, and p70 S6 kinase was unaltered by burn injury. However, insulin-stimulated activation of ERK, a signaling pathway parallel to Akt/PKB, was not affected by burn injury. These results demonstrate that burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle and suggest that attenuated Akt/PKB activation may be involved in deranged metabolism and muscle wasting observed after burn injury.     

Pioglitazone upregulates adiponectin receptor 2 in 3T3-L1 adipocytes

AimsPioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes.Main methodsGlucose uptake was evaluated by 2-[³H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT–PCR.Key findingsWe first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ.SignificanceOur results suggest that pioglitazone increases insulin sensitivity, at least partly, by PPARγ-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. In conclusion, the upregulation of AdipoR2 expression may be one of the mechanisms by which pioglitazone improves insulin resistance in 3T3-L1 adipocytes.     

Effects of erythropoietin on glucose metabolism

We purposed to determine the impact of erythropoietin on altering glucose metabolism in the settings of in vitro and in vivo experiments. The acute effect of erythropoietin on lowering blood glucose levels was studied in animal experiments. In [3H]-deoxy-D-glucose isotope studies we measured glucose uptake with insulin and erythropoietin using 3T3-L1 cells cultured under normal or high glucose conditions. Altered activation of Akt and ERK pathways was evaluated in immunoblot analyses. Immunocytochemistry was conducted to determine the glucose transporter 4 translocation to the plasma membrane. Addition of erythropoietin significantly lowered blood glucose levels in vivo in rats. The glucose uptake was markedly increased by erythropoietin treatment (at concentrations 0.15, 0.3, and 0.625 ng/ml) in adipocytes grown in high glucose medium (p<0.05), but it remained unaltered in cells under normal glucose conditions. Significant increase of phosphorylation of ERK and Akt was detected due to erythropoietin (p<0.05). Co-administration of erythropoietin and insulin resulted in higher phosphorylation of Akt and [3H]-deoxy-D-glucose uptake in adipocytes than insulin treatment alone. We found that erythropoietin induced the trafficking of glucose transporter 4 to the plasma membrane. Our data showed that erythropoietin significantly decreased blood glucose levels both in vivo and in vitro, in part, by increasing glucose uptake via the activation of Akt pathway. Preliminary data revealed that adipocytes most likely exhibit a specific receptor for erythropoietin.     

Insulin signaling in vascular endothelial cells: a key role for heterotrimeric G proteins revealed by siRNA-mediated Gbeta1 knockdown

Activation of insulin receptors stimulates the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in vascular endothelial cells. Heterotrimeric G proteins appear to modulate some of the cellular responses that are initiated by receptor tyrosine kinases, but the roles of specific G protein subunits in signaling are less clearly defined. We found that insulin treatment of cultured bovine aortic endothelial cells (BAEC) activates the alpha isoform of PI3-K (PI3-Kalpha) and discovered that purified G protein Gbeta1gamma2 inhibits PI3-Kalpha enzyme activity. Transfection of BAEC with a duplex siRNA targeting bovine Gbeta1 leads to a 90% knockdown in Gbeta1 protein levels, with no effect on expression of other G protein subunits. siRNA-mediated Gbeta1 knockdown markedly and specifically potentiates insulin-dependent activation of kinase Akt, likely reflecting the removal of the inhibitory effect of Gbetagamma on PI3-Kalpha activity. Insulin-induced tyrosine phosphorylation of insulin receptors is unaffected by Gbeta1 siRNA. By contrast, Gbeta1 knockdown leads to a significant decrease in the level of serine phosphorylation of the insulin receptor substrate IRS-1. We explored the effects of siRNA on several serine/threonine protein kinases that have been implicated in insulin signaling. Gbeta1 siRNA significantly attenuates phosphorylation of the 70 kDa ribosomal protein S6 kinase (p70S6K) in the basal state and following insulin treatment. We also found that IGF-1-initiated activation of Akt is significantly enhanced after siRNA-mediated Gbeta1 knockdown, while IGF-1-induced p70S6K activation is markedly suppressed following transfection of Gbeta1 siRNA. We propose that Gbeta1 participates in the activation of p70S6K, which in turn promotes the serine phosphorylation and inhibition of IRS-1. Taken together, these studies suggest that Gbeta1 plays an important role in insulin and IGF-1 signaling in endothelial cells, both by inhibiting the activity of PI3-Kalpha and by stimulating pathways that lead to activation of protein kinase p70S6K and to the serine phosphorylation of IRS-1.     

Melatonin enhances leptin expression by rat adipocytes in the presence of insulin

Leptin and melatonin play an important role in the regulation of body mass and energy balance. Both hormones show a circadian rhythm, with increasing values at night. In addition, melatonin receptors were recently described in adipocytes, where leptin is synthesized. Here, we investigated the influence of melatonin and its interaction with insulin and dexamethasone on leptin expression. Isolated rat adipocytes were incubated with melatonin (1 nM) alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) for 6 h. Melatonin or insulin alone did not affect leptin expression, but together they increased it by 120%. Dexamethasone increased leptin mRNA content (105%), and this effect was not enhanced by melatonin. Simultaneous treatment with the three hormones provoked a further increase in leptin release (250%) and leptin mRNA (100%). Melatonin prevented the forskolin-induced inhibition (95%) of leptin expression. In addition, melatonin's ability to stimulate leptin release (in the presence of insulin) was completely blocked by pertussis toxin and luzindole. To gain further insight into the molecular basis of melatonin and insulin synergism, the insulin-signaling pathway was investigated. Melatonin increased the insulin-induced insulin receptor-beta tyrosine phosphorylation, which led to an increased serine phosphorylation of the downstream convergent protein Akt. We concluded that melatonin interacts with insulin and upregulates insulin-stimulated leptin expression. These effects are caused by melatonin binding to the pertussis toxin-sensitive G(i) protein-coupled membrane receptor (MT1 subtype) and the cross talk with insulin, since insulin receptor and its convergent target Akt are coactivated by melatonin.     

Ceramide inhibits insulin-stimulated Akt phosphorylation through activation of Rheb/mTORC1/S6K signaling in skeletal muscle

Ceramide is a negative regulator of insulin activity. At the molecular level, it causes a decrease in insulin-stimulated Akt Ser473 phosphorylation in C2C12 myotubes. Interestingly, we found that the phosphorylation of S6K at Thr389 was increased under the same conditions. Utilizing both rapamycin to inhibit mTORC1 activity and shRNA to knock down Rheb, we demonstrated that the decrease in Akt Ser473 phosphorylation stimulated by insulin after C2-ceramide incubation can be prevented. The mechanism by which C2-ceramide impairs signaling would seem to involve a negative feedback of activated S6K via phosphorylation of insulin receptor substrate-1 at Ser636/639, since S6K inhibitor can block this phenomenon. Finally, rapamycin treatment was found not to affect C2-ceramide-induced PKCζ activation, suggesting that the pathway revealed in this study is parallel to the one involving PKCζ activation. We proposed a novel pathway/mechanism involving Rheb/mTORC1/S6K signaling to explain how C2-ceramide impairs insulin signaling via Akt phosphorylation. The existence of multiple pathways involved in insulin signaling impairment by C2-ceramide treatment implies that different strategies might be needed to ameliorate insulin resistance caused by C2-ceramide.     

Akt activation and augmented fibronectin production in hyperhexosemia

Dysmetabolic state in diabetes may lead to augmented synthesis of extracellular matrix (ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin (FN) production and that of its splice variant, EDB(+)FN, is regulated by protein kinase B (PKB, also known as Akt). In this study, we investigated the role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKBalpha knockout mice and wild-type control littermates. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 wk starting at 6 wk of age. We investigated FN mRNA, EDB(+)FN mRNA, and transforming growth factor (TGF)-beta mRNA expression, Akt phosphorylation, Akt kinase activity, and NF-kappaB and AP-1 activation in the retina, heart, and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB(+)FN, and TGF-beta in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which was prevented in the Akt knockout mice. NF-kappaB and AP-1 activation was pronounced in galactose-fed wild-type mice and prevented in the galactose-fed Akt1/PKBalpha-deficient group. In the retina and kidney, Ser473 was the predominant site for Akt phosphorylation, whereas in the heart it was Thr308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation may be an important mechanism leading to NF-kappaB and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.