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.     

The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3alpha and Ser9 of GSK3beta. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3beta, but not GSK3alpha. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels.     

Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

Protein kinase B (also known as Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase 3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of three daily injections of 15 mg/kg cocaine or 1 mL/kg saline spaced 1 h apart for 1, 3, or 14 days. Amygdala, nucleus accumbens, caudate putamen, and hippocampus tissues were dissected 30 min following the last injection and analyzed for phosphorylated and total Akt and GSK3(alpha and beta) protein levels using western blot analysis. Phosphorylation of Akt on the threonine-308 (Thr308) residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 was significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure.     

Transient and rapid activation of Akt/GSK‐3β and mTORC1 signaling by D3 dopamine receptor stimulation in dorsal striatum and nucleus accumbens

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time‐course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK‐3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein‐S6 (Ser240/244), and eukaryotic initiation factor‐4E binding protein‐1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock‐out mice lacking D3R. In drd1a‐EGFP transgenic mice, quinelorane activated Akt/GSK‐3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK‐3β pathway in the two populations of medium‐size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.     

Hydroxysafflor Yellow A Protects Against Cerebral Ischemia–Reperfusion Injury by Anti-apoptotic Effect Through PI3K/Akt/GSK3β Pathway in Rat

Hydroxysafflor yellow A (HSYA) is the major active chemical component of the flower of the safflower plant, Carthamus tinctorius L. Previously, its neuroprotection against cerebral ischemia–reperfusion (I/R) injury was reported by anti-oxidant action and suppression of thrombin generation. Here, we investigate the role of HSYA in cerebral I/R-mediated apoptosis and possible signaling pathways. Male Wistar rats were subjected to transient middle cerebral artery occlusion for 2 h, followed by 24 h reperfusion. HSYA was administered via tail-vein injection just 15 min after occlusion. The number of apoptotic cells was measured by TUNEL assay, apoptosis-related proteins Bcl-2, Bax and the phosphorylation levels of Akt and GSK3β in ischemic penumbra were assayed by western blot. The results showed that administration of HSYA at the doses of 4 and 8 mg/kg significantly inhibited the apoptosis by decreasing the number of apoptotic cells and increasing the Bcl-2/Bax ratio in rats subjected to I/R injury. Simultaneously, HSYA treatment markedly increased the phosphorylations of Akt and GSK3β. Blockade of PI3K activity by wortmannin dramatically abolished its anti-apoptotic effect and lowered both Akt and GSK3β phosphorylation levels. Taken together, these results suggest that HSYA protects against cerebral I/R injury partly by reducing apoptosis via PI3K/Akt/GSK3β signaling pathway.     

Neuroprotection by urate on 6‐OHDA‐lesioned rat model of Parkinson's disease: linking to Akt/GSK3β signaling pathway

Higher plasma urate level is reported to be associated with a reduced risk and slower progression of Parkinson's disease (PD). In this study, we explored the effects of urate on dopaminergic neurons in nigrostriatal pathway in the 6‐hydroxydopamine (6‐OHDA) unilaterally lesioned rats. Uric acid (UA), when given twice daily at 200 mg/kg intraperitoneally for 10 consecutive days, elevated urate (the anionic form of UA) in plasma and striatum by 55% and 36.8%, respectively, as compared with vehicle group. This regimen of UA was found to ameliorate the behavioral deficits, dopaminergic neuron loss as well as dopamine depletion in the nigrostriatal system. Moreover, UA administration was capable of increasing glutathione level and superoxide dismutase activity while decreasing malondialdehyde accumulation in striatum. In addition, the phosphorylation of both protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) in the lesioned striata of 6‐OHDA‐lesioned rats was dramatically reduced as compared with sham‐operated rats. This reduction was attenuated in the Parkinsonian rats receiving UA treatment. Similarly, in vitro findings showed that UA alleviated the decrease in Akt activation and the increase in GSK3β activity caused by 6‐OHDA. Furthermore, neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9 was found to be abolished in the presence of PI3K inhibitor. Therefore, our findings demonstrated that urate was able to protect dopaminergic neurons in rat nigrostriatal pathway against the neurotoxicity of 6‐OHDA, and showed that its beneficial effects may be related to its regulation on Akt/GSK3β signaling.     

Astrocytic Expressions of Phosphorylated Akt,GSK3β and CREB Following an Excitotoxic Lesion in the Mouse Hippocampus

Glycogen synthase kinase 3β (GSK3β) is believed to play important roles in the regulation of synaptic plasticity, cell survival and circadian rhythms in the mature CNS. However, although several studies have been focused on the GSK3β, little is known about GSK3β changes in glial cells under neuropathological conditions. In this study, we evaluated the expressions of molecules associated with the GSK3β signaling pathway, following the induction of an excitotoxic lesion in mouse brain by kainic acid (KA) injection, which caused pyramidal cell degeneration in the hippocampal CA3 region. In injured hippocampi, Ser47-Akt (protein kinase B, PKB) phosphorylation increased from 4 h until 1 day post-injection (PI). Ser9-GSK3β and Ser133-cAMP responsive element-binding protein (CREB) phosphorylations showed similar spatiotemporal patterns in hippocampi at 1 day until 3 days PI. Double immunohistochemistry also showed that these phosphorylated forms of Akt, GSK3β and CREB were expressed in astrocytes. For the first time, our data demonstrate the injury-induced astrocytic changes in the levels of phosphorylation of Akt, -GSK3β and -CREB in vivo, which may reflect mechanisms of glial cells protection or adaptive response to damage. DW Kim and JH Lee contributed equally to this work.     

Differences in the effects of amphetamine and methylphenidate on brain dopamine turnover and serum prolactin concentration in reserpine-treated rats.

In the present study we compared the ability of amphetamine and methylphenidate to antagonize the elevation of serum prolactin produced by reserpine because of the differences in the actions of amphetamine and methylphenidate on brain dopamine turnover. Groups of male rats were treated with either methylphenidate (10 mg/kg) or amphetamine (5 mg/kg) alone or in combination with reserpine (5 mg/kg). The reserpine treatment was given 4 hours before methylphenidate or amphetamine, and the rats were killed 5 hours after reserpine. Neither amphetamine nor methylphenidate alone was able to suppress serum prolactin. Amphetamine but not methylphenidate was able to block the increase of serum prolactin in response to reserpine. Amphetamine lowered brain DOPAC in control and reserpine-treated rats, but methylphenidate elevated brain DOPAC in control rats and had no effect in reserpine-treated rats. These results indicate that the methylphenidate group of CNS stimulants can be differentiated on the basis of their neuroendocrine effects from the amphetamine group of stimulants.     

Kainate induces AKT, ERK and cdk5/GSK3beta pathway deregulation, phosphorylates tau protein in mouse hippocampus

Acute treatment with kainate 30 mg/kg (KA) produced behavioral alterations and reactive gliosis. However, it did not produce major death of mouse hippocampal neurons, indicating that concentrations were not cytotoxic. KA caused rapid and temporal Erk phosphorylation (at 6h) and Akt dephosphorylation (1-3 days). Concomitantly, the activation of GSK3beta was increased 1-3 days after KA. After 7 days, a reduction in GSK3beta activation was observed. Caspase-3 activity increased, but to a lesser extent than calpain activation (measured by fluorimetry and calpain-cleaved alpha-spectrin). As calpain is involved in cdk5 activation, and cdk5 is related to GSK3beta, the cdk5/p25 pathway was examined. Results showed that the p25/p35 ratio in KA-injected mice for 3 days was 73.6% higher than control levels. However, no changes in cdk5 expression were detected. Both Western blot and immunohistochemistry against p-Tau(Thr(231)) indicated an increase at this phosphorylated site of tau protein. Indeed an increase in p-Tau(Ser(199)) and p-Tau(Ser(396)) was observed by Western blot. Our results demonstrate that tau hyperphosphorylation, induced by KA, is due to an increase in GSK3beta/cdk5 activity in combination with an inactivation of Akt. This indicates that the calpain/cdk5 pathway for tau phosphorylation has a potential role in delayed apoptotic death evoked by excitotoxicity. Moreover, the subsequent activation of caspase and calpain proteases leads to dephosphorylation of tau, thus increasing microtubular destructuration. Taken together, our results provide new insights in the activation of several kinase-pathways implicated in cytoskeletal alterations that are a common feature of neurodegenerative diseases.     

Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser⁴⁷³ phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.     

Intracellular signaling pathways regulating net protein balance following diaphragm muscle denervation

Unilateral denervation (DNV) of rat diaphragm muscle increases protein synthesis at 3 days after DNV (DNV-3D) and degradation at DNV-5D, such that net protein breakdown is evident by DNV-5D. On the basis of existing models of protein balance, we examined DNV-induced changes in Akt, AMP-activated protein kinase (AMPK), and ERK½ activation, which can lead to increased protein synthesis via mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K), glycogen synthase kinase-3β (GSK3β), or eukaryotic initiation factor 4E (eIF4E), and increased protein degradation via forkhead box protein O (FoxO). Protein phosphorylation was measured using Western analyses through DNV-5D. Akt phosphorylation decreased at 1 h and 6 h after DNV compared with sham despite decreased AMPK phosphorylation. Both Akt and AMPK phosphorylation returned to sham levels by DNV-1D. Phosphorylation of their downstream effector mTOR (Ser2481) did not change at any time point after DNV, and phosphorylated p70S6K and eIF4E-binding protein 1 (4EBP1) increased only by DNV-5D. In contrast, ERK½ phosphorylation and its downstream effector eIF4E increased 1.7-fold at DNV-1D and phosphorylated GSK3β increased 1.5-fold at DNV-3D (P < 0.05 for both comparisons). Thus, following DNV there are differential effects on protein synthetic pathways with preferential activation of GSK3β and eIF4E over p70S6K. FoxO1 nuclear translocation occurred by DNV-1D, consistent with its role in increasing expression of atrogenes necessary for subsequent ubiquitin-proteasome activation evident by DNV-5D. On the basis of our results, increased protein synthesis following DNV is associated with changes in ERK½-dependent pathways, but protein degradation results from downregulation of Akt and nuclear translocation of FoxO1. No single trigger is responsible for protein balance following DNV. Protein balance in skeletal muscle depends on multiple synthetic/degradation pathways that should be studied in concert.     

Changes in Antioxidant Defense Enzymes after d-amphetamine Exposure: Implications as an Animal Model of Mania

Studies have demonstrated that oxidative stress is associated with amphetamine-induced neurotoxicity, but little is known about the adaptations of antioxidant enzymes in the brain after amphetamine exposure. We studied the effects of acute and chronic amphetamine administration on superoxide dismutase (SOD) and catalase (CAT) activity, in a rodent model of mania. Male Wistar rats received either a single IP injection of d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle (acute treatment). In the chronic treatment rats received a daily IP injection of either d-amphetamine (1 mg/kg, 2 mg/kg, or 4 mg/kg) or vehicle for 7 days. Locomotor behavior was assessed using the open field test. SOD and CAT activities were measured in the prefrontal cortex, hippocampus, and striatum. Acute and to a greater extent chronic amphetamine treatment increased locomotor behavior and affected SOD and CAT activities in the prefrontal cortex, hippocampus and striatum. Our findings suggest that amphetamine exposure is associated with an imbalance between SOD and CAT activity in the prefrontal cortex, hippocampus and striatum.     

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.     

Methylphenidate alters NCS-1 expression in rat brain

Methylphenidate has been used as an effective treatment for attention deficit hyperactivity disorder (ADHD). Methylphenidate (MPH) blocks dopamine and norepinephrine transporters causing an increase in extracellular levels. The use of psychomotor stimulants continues to rise due to both the treatment of ADHD and illicit abuse. Methylphenidate sensitization mechanism has still poor knowledge. Neuronal calcium sensor 1 was identified as a dopaminergic receptor interacting protein. When expressed in mammalian cells, neuronal calcium sensor 1 attenuates dopamine-induced D2 receptor internalization by a mechanism that involves a reduction in D2 receptor phosphorylation. Neuronal calcium sensor 1 appears to play a pivotal role in regulating D2 receptor function, it will be important to determine if there are alterations in neuronal calcium sensor 1 in neuropathologies associated with deregulation in dopaminergic signaling. Then, we investigated if methylphenidate could alter neuronal calcium sensor 1 expression in five brain regions (striatum, hippocampus, prefrontal cortex, cortex and cerebellum) in young and adult rats. These regions were chosen because some are located in brain circuits related with attention deficit hyperactivity disorder. Our results showed changes in neuronal calcium sensor 1 expression in hippocampus, prefrontal cortex and cerebellum mainly in adult rats. The demonstration that methylphenidate induces changes in neuronal calcium sensor 1 levels in rat brain may help to understand sensitization mechanisms as well as methylphenidate therapeutic effects to improve attention deficit hyperactivity disorder symptoms.     

Altered Regulation of Akt Signaling with Murine Cerebral Malaria,Effects on Long-Term Neuro-Cognitive Function,Restoration with Lithium Treatment

Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling likely underlies long-term neurological sequelae observed in ECM and may yield adjunctive therapeutic targets for the management of CM.     

Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro

Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling. The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt. We studied Akt-dependent phosphorylation of tau protein in vitro. We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau. Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1. The Akt-phosphorylated tau retained its initial electrophoretic mobility. Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100. Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3beta activity. On the other hand, GSK3beta selectively blocked Ser214 phosphorylation, which was prevented by lithium. The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases. Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.     

Glycogen synthase kinase 3beta is a novel regulator of high glucose- and high insulin-induced extracellular matrix protein synthesis in renal proximal tubular epithelial cells

High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.