Effects of betaine on ethanol-stimulated secretion of IGF-I and IGFBP-1 in rat primary hepatocytes： involvement of p42/44 MAPK activation

AIM:To evaluate the effects of betaine on the ethanol-induced secretion of IGF-I and IGFBP-1 using radioimmunoassay and Western blotting, respectively, in primary cultured rat hepatocytes. METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10(-5), 10(-4), and 10(-3) mol/L). We measured IGF-I and IGFBP-1 using radioimmunoassay and Western blotting, respectively. RESULTS:The ethanol-induced inhibition of IGF-I secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10(-3) mol/L, betaine significantly increased IGF-I secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10(-3) mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-I secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion. CONCLUSION: Betaine modulates the secretion of IGF-I and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.     

Effects of betaine on etnanol-stimulated secretion of IGF-I and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation

AIM: To evaluate the effects of betaine on the ethanol-induced secretion of IGF-I and IGFBP-1 using radioim-munoassay and Western blotting, respectively, in primary cultured rat hepatocytes. METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10-5, 10-4, and 10-3 mol/L). We measured IGF-I and IGFBP-1 using radioimmunoassay and Western blotting, respectively. RESULTS: The ethanol-induced inhibition of IGF-I secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10?mol/L, betaine significantly increased IGF-I secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10-3 mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-I secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion. CONCLUSION: Betaine modulates the secretion of IGF-I and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.     

Effects of betaine on ethanol-stimulated secretion of IGF-Ⅰ and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation

AIM: To evaluate the effects of betaine on the ethanolinduced secretion of IGF-Ⅰ and IGFBP-1 using radioimmunoassay and Western blotting, respectively, in primary cultured rat hepatocytes.METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10-5,10-4, and 10-3 mol/L). We measured IGF-Ⅰ and IGFBP-1 using radioimmunoassay and Western blotting, respectively.RESULTS: The ethanol-induced inhibition of IGF-Ⅰ secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10-3 mol/L, betaine significantly increased IGF-Ⅰ secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10-3 mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-Ⅰ secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion.CONCLUSION: Betaine modulates the secretion of IGF-Ⅰ and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.     

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells

Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen‐activated protein kinases (MAPKs) [extracellular signal‐regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3‐kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12‐myristate 13‐acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA‐treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbα expression, DNA endoreduplication, and formation of platelet‐like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down‐regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.     

Involvement of p38 MAPK,JNK, p42/p44 ERK and NF-kappaB in IL-1beta-induced chemokine release in human airway smooth muscle cells

Asthma is an inflammatory disease, in which eotaxin, MCP-1 and MCP-3 play a crucial role. These chemokines have been shown to be expressed and produced by IL-1beta-stimulated human airway smooth muscle cells (HASMC) in culture. In the present study we were interested to unravel the IL-1beta-induced signal transduction leading to chemokine production. Using Western blot, we observed an activation of p38 MAPK, JNK kinase and p42/p44 ERK when HASMC were stimulated with IL-1beta. We also observed a significant decrease in the expression and the release of eotaxin, MCP-1 and MCP-3 in the presence of SB203580, an inhibitor of p38 MAPK (71 +/- 6%, P < 0.05, n = 8 and 39 +/- 10% P < 0.01, n = 10 respectively), curcumin, an inhibitor of JNK kinase (83 +/- 4.9% and 88 +/- 3.4% respectively, P < 0.01, n = 4). U0126, an inhibitor of p42/p44 ERK, also produced a significant decrease in chemokine production (46.3 +/- 9%, P < 0.01 n = 10 and 67.8 +/- 12%, P < 0.01, n = 12). Pyrrolydine dithiocarbamate, an inhibitor of NF-kappaB was also able to reduce the eotaxin, MCP-1 and MCP-3 expression and production (50 +/- 13%, P < 0.05, n = 10 and 23 +/- 7%, P < 0.05, n = 12). We conclude that p38 MAPK, JNK kinase, ERK and NF-kappaB are involved in the IL-1beta-induced eotaxin, MCP-1, and MCP-3 expression and release in HASMC.     

Inhibitory mechanisms of resveratrol in platelet activation: pivotal roles of p38 MAPK and NO/cyclic GMP

Resveratrol has been reported to have antiplatelet activity; however, the detailed mechanisms have not yet been resolved. This study aimed to systematically examine the detailed mechanisms of resveratrol in the prevention of platelet activation in vitro and in vivo. Resveratrol (0.05-0.25 micromol/l) showed stronger inhibition of platelet aggregation stimulated by collagen (1 microg/ml) than other agonists. Resveratrol (0.15 and 0.25 micromol/l) inhibited collagen-induced platelet activation accompanied by [Ca(+2)]i mobilization, thromboxane A(2) (TxA(2)) formation, phosphoinositide breakdown, and protein kinase C (PKC) activation. Resveratrol markedly increased levels of NO/cyclic guanosine monophosphate (GMP), and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. Resveratrol markedly inhibited p38 mitogen-activated protein kinase (MAPK) but not Jun N-terminal kinase or extracellular signal-regulated kinase-2 phosphorylation in washed platelets. Resveratrol-reduced hydroxyl radical (OH(-)) formation in the electron spin resonance study. In an in vivo study, resveratrol (5 mg/kg) significantly prolonged platelet plug formation of mice. In conclusion, the main findings of this study suggest that the inhibitory effects of resveratrol possibly involve (i) inhibition of the p38 MAPK-cytosolic phospholipase A(2)-arachidonic acid-TxA(2)-[Ca(+2)]i cascade and (ii) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C and/or PKC activation. Resveratrol is likely to exert significant protective effects in thromboembolic-related disorders by inhibiting platelet aggregation.     

慢性低氧对肺动脉高压大鼠肺血管ERK 1/2、p38MAPK表达的影响

目的：通过建立慢性低氧性肺动脉高压大鼠模型,研究慢性低氧对大鼠肺血管细胞外信号调节蛋白激酶(ERK1/2)、p38MAPK蛋白表达的影响。方法建立慢性常压低氧肺动脉高压大鼠模型,将雄性SD大鼠随机分为正常对照组、低氧1d、3d、7d、14d和21d组,应用免疫组织化学技术检测肺动脉高压形成过程中大鼠肺血管 ERK1/2、p38MAPK 蛋白表达水平。结果①RVSP 和 RV/(LV+S)比值较正常对照组明显增加(P<0.05),低氧后3 d、7 d、14 d和21 d后大鼠肺血管明显增厚;②ERK1/2、p38MAPK蛋白广泛分布于肺血管内皮细胞、平滑肌细胞和成纤维细胞中,且随着低氧时间的延长,ERK1/2、p38MAPK蛋白表达量增加。结论 ERK1/2、p38MAPK 蛋白表达量的上调可能参与了慢性低氧诱导的大鼠肺动脉高压肺血管重塑的发生、发展过程。     

Involvement of phosphoinositide-3-kinase and p70 S6 kinase in regulation of proliferation of rat lactotrophs in culture

Phosphoinositide-3-kinase (PI-3K) and p70 S6 kinase (p70^56k) are suggested as important molecules for mediating mitogenic actions of growth factors and cytokines in variety of cell types. The purpose of the present study was to investigate whether these kinases were involved in mediation of the mitogenic actions of not only the growth factor insulin but also cyclic adenosine monophosphate (cAMP) and estrogen on rat cultured lactotrophs. Treatment with wortmannin or LY294002, a PI-3K inhibtor, or rapamycin, a p70^S6k inhibitor, decreased basal levels of 5-bromo-2-deoxyuridine (BrdU)-labeling indices of lactotrophs in a dose-dependent manner. These inhibitors were effective in blocking an increase in BrdU-labeling indices induced by insulin. LY294002 and rapamycin also suppressed an increase in BrdU-labeling indices induced by forskolin, an adenylate cyclase activator, or dibutyryl cAMP, a membrane-permeable cAMP analog, as well as that induced by estradiol, a physiologic extracellular activator of lactotroph proliferation. However, the dibutyryl cAMP-, but not insulin-induced proliferation, acquired a resistance to LY294002 and rapamycin by pretreatment with bromocriptine, a dopaminergic agonist that is able to suppress lactotroph proliferation. These results suggest that the mitogenic actions of cAMP and estradiol on rat lactotrophs are mediated by PI-3K and p70^S6k, and that dopaminergic inhibition modifies the PI-3K and p70^S6k dependence of the regulation of lactotroph proliferation.     

泡球蚴感染对BALB/c小鼠肝细胞ERK1/2和p38表达变化的初步研究

目的通过检测ERK1/2和p38在泡球蚴感染小鼠肝组织中的表达,探讨其在肝泡球蚴病发生中的作用。方法采用半定量逆转录聚合酶链反应（RT-PCR）及免疫组织化学的方法检测16例感染组小鼠和16例对照组小鼠肝组织中的ERK1/2和p38的表达水平。结果感染组ERK1/2表达水平较对照组有统计学差异（P〈0.05）,p38的表达在感染组与对照组无统计学差异。结论在泡球蚴感染早期,肝细胞增殖占主导。     

Interleukin-1β induces cyclo-oxygenase-2 expression in gastric cancer cells by the p38 and p44/42 mitogen-activated protein kinase signaling pathways

BACKGROUND AND AIMS: Cyclo-oxygenase-2 (COX-2) is the inducible enzyme in the gastric mucosa responsible for prostaglandin production during inflammation and ulcer healing. The regulation of COX-2 gene expression in gastric epithelial cells is not well understood. In this study, we investigated the effect of interleukin (IL)-1beta on COX-2 expression in the human gastric cancer cell, and explored the signaling pathways involved. METHODS: Gastric cancer cell line AGS was treated with IL-1beta or the inhibitors of mitogen-activated protein-Erk kinase (MEK) and p38 mitogen-activated protein (MAP) kinase prior to the addition of IL-1beta. The COX-2 mRNA or protein levels were measured by using RT-PCR or western blot analysis, respectively. Prostaglandin E2 (PGE2) production/secretion was determined by using the prostaglandin E2 EIA assay. The phosphorylation/activation of p44/42 and p38 MAP kinases were determined by using western blot analysis and using phospho-specific antibodies. RESULTS: Interleukin-1beta treatment dose- and time-dependently increased COX-2 mRNA and protein expression levels, and enhanced PGE2 production/secretion in AGS cells. In contrast, IL-1beta had no effect on the level of the constitutively expressed COX-1. In parallel to the increase of COX-2, we showed that p44/42 and p38 MAP kinase activities were also upregulated by IL-1beta treatment. To demonstrate the cause-effect relationship, we showed that inhibition of MEK and p38 MAP kinase with specific inhibitors suppressed IL-1beta-mediated increases in COX-2 mRNA and protein levels, and the PGE2 production. CONCLUSIONS: Our results demonstrated that in human gastric cancer cells, IL-1beta upregulates the COX-2 gene expression through the activation of MEK/p44/42 and p38 MAP kinases pathway.     

针对STAT3的siRNA抑制HepG2细胞增殖和p44/42MAPK蛋白的表达

目的研究STAT3信号传导通路对HepG2细胞p44/42MAPK蛋白表达和细胞生长的影响。方法将针对STAT3的siRNA转染入HepG2细胞以沉默STAT3基因的表达,采用MTT法检测细胞生长,采用Western blot法检测STAT3和p44/42MAPK蛋白的表达。结果对照组和lipofectamineTM2000处理组细胞生长无明显差异(q=0.97,P0.05),而siRNA处理组细胞生长被明显抑制(q=9.36,P0.05);SiRNA转染后24h、48h、72h和96h,细胞抑制率分别为33.2%、39.6%4、3.1%和33.9%s,iRNA在96h后抑制作用减弱,细胞开始重新繁殖;转染细胞72h和96h后,可见STAT3蛋白表达均被抑制(t=14.12,P0.05),p44/42MAPK蛋白表达未被抑制(F=3.99,P0.05),而p-p44/42MAPK蛋白表达增加(t=16.30,P0.05)。结论 STAT3信号传导通路可以影响细胞生长及p44/42MAPK蛋白质的磷酸化,而p-p44/42MAPK的表达增加可能代偿了沉默STAT3引起的细胞生长抑制,使细胞重新繁殖。     

Role of cyclooxygenase 2, p38 and p42/44 MAPK in the secretion of prostacyclin induced by epidermal growth factor,endothelin-1 and angiotensin II in rat ventricular cardiomyocytes

We studied the respective roles of cyclooxygenases (COX) isoforms as well as the p38 and p42/44 MAP kinase cascades in angiotensin II (AngII)-, endothelin-1 (ET-1)- and epidermal growth factor (EGF)-induced prostacyclin (PGI(2)) secretion in neonatal rat ventricular cardiomyocytes. Exposure of these cells for 1 h to 100 nM AngII, ET-1 or EGF resulted in an increase in prostacyclin formation which was abolished by the COX-2 specific inhibitor NS-398 (1 microM), while the COX-1 inhibitor valeryl salicylate (5 microM) had no effect. Agonist-induced prostacyclin secretion was also abolished in the presence of cycloheximide (10 microg/ml), indicating that newly synthesized proteins are necessary for this response. In this context, the COX-2 protein amount was significantly increased following 1 h incubation of cardiomyocytes, with AngII, ET-1 and EGF. These results indicate that in cardiomyocytes AngII, ET-1 and EGF induce both the synthesis and the activity of COX-2. Investigating the role of MAPK in the stimulation of prostacyclin induced by these three agonists, we found that both the p42/44 MAPK inhibitor PD 98059 (50 microM) and the p38 MAPK blocker SB 203580 (5 microM) prevented agonist-induced PGI(2) secretion without affecting COX-2 activity or synthesis. Our results show that p42/44 and p38 MAPK activation is at the basis of AngII-, ET-1- and EGF-induced prostacyclin secretion in cardiomyocytes. They further suggest that these MAPK act on a target(s) located upstream of COX-2.     

胰岛素对血管平滑肌细胞4E结合蛋白1和核糖体蛋白S6激酶磷酸化的刺激作用

目的研究胰岛素对血管平滑肌细胞(VSMC)蛋白质合成翻译过程的两个调节子,4E-结合蛋白1(4E-BP1)和核糖体蛋白S6激酶,磷酸化的调节作用及其生物学意义。方法培养大鼠胸主动脉VSMC。用3H-亮氨酸和3H-TdR掺入法分别测定蛋白质合成和DNA合成;免疫印迹法检测4E-BP1和核糖体蛋白S6激酶的磷酸化。结果与对照相比,100nmol/L胰岛素显著增加了VSMC的3H-亮氨酸和3H-TdR掺入。同样浓度的胰岛素作用于VSMC,可以诱导4E-BP1和核糖体蛋白S6激酶发生磷酸化。二者的磷酸化分别于胰岛素刺激后,30min和10min达高峰。结论胰岛素可以刺激VSMC的4E-BP1和核糖体蛋白S6激酶发生磷酸化,这可能是胰岛素发挥促进VSMC生长作用的机制。     

Lesion-targeted thrombopoietin potentiates vasculogenesis by enhancing motility and enlivenment of transplanted endothelial progenitor cells via activation of Akt/mTOR/p70S6kinase signaling pathway

Thrombopoietin (TPO), a physiological regulator of megakaryocyte and platelet development, is a multifunctional positive regulator in early hematopoiesis by hematopoietic stem cells. In this study, we investigated the effect of TPO on endothelial progenitor cells (EPCs) for therapeutic vasculogenesis in vitro and in vivo, and the intracellular signaling mechanism exerting the activity of EPCs. 7-day culture-expanded EPCs derived from human peripheral blood mononuclear cells were applied to each assay. Flow cytometry demonstrated the expression of c-Mpl, the receptor of TPO, in cultured EPCs. In vitro experiments revealed enhanced migration and survival of cultured EPCs by TPO. In vivo, TPO was intramuscularly administered into the foci of ischemic hindlimbs in athymic nude mice, immediately followed by intravenous injection of cultured EPCs, to assess the booster effect of TPO on vascular regeneration. At day 4 post-transplantation, transplanted EPCs were 1.7-fold higher in TPO-treated animals compared to control. At day 28, blood perfusion was recovered in the TPO-treated group, accompanied by an increase in microvascular density. The signaling transduction pathway underlying TPO-mediated activities of cultured EPCs was assessed by Western blotting. TPO induced sequential phosphorylations of Akt to p70S6kinase through mTOR. Inhibition of the PI3-kinase/Akt/mTOR/p70S6kinase signaling pathway negated the biological functions of cultured EPCs, either migration (by LY294002 for PI3-kinase and Rapamycin for mTOR) or survival and tubulogenesis (by Rapamycin). These findings provide evidence that TPO possesses booster potential for therapeutic vasculogenesis, by activating the PI3-kinase/Akt/mTOR/p70S6kinase pathway crucial to the biological activities of EPCs.     

Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf‐1–ERK1/2–p70S6K signalling pathway

Background: Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase‐activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation. Aims: In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain. Methods and results: On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ‐stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ‐stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector‐stable clone, suggesting that downregulation of RhoA activity in DLC2γ‐stable clones inhibited cell cycle progression. In the DLC2γ‐stable clone, the levels of Raf‐1 and extracellular signal‐regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ–GAP defective mutant‐stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2‐stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho‐p70S6K and phospho‐ERK1/2 were upregulated. Conclusion: Our data show that DLC2 inhibits the activity of Raf‐1–ERK1/2–p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.     

Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta

Summary Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total ⁸⁶Rb⁺ (K⁺) uptake from 0.8 ± 0.04 to 1.02 ± 0.05 nmol · mg^–1· protein^–1· min^–1 (p < 0.005). These changes in K⁺ flux, though small, can alter the membrane potential. Uptake occurred through both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na⁺/K⁺/2Cl^– cotransporter prior to insulin action, no increase in ⁸⁶Rb⁺ uptake via the Na⁺/K⁺-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50–200 nmol/l) and LY294 002 (50 μmol/l) attenuated both total insulin-stimulated ⁸⁶Rb⁺ uptake as well as uptake via the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter. Neither the inhibitor of p70 S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098 059 (50 μmol/l), had any effect on insulin's stimulation of K⁺ influx. A 10 μmol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 μmol/l it was ineffective, suggesting involvement of the atypical PKC-ζ isoform. We conclude that insulin-stimulated K⁺ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-ζ. [Diabetologia (1998) 41: 1199–1204] Received: 20 March 1998 and in revised form: 2 June 1998     

PI3-kinase/Akt/mTOR signaling: Impaired on/off switches in aging,cognitive decline and Alzheimer's disease

The normal on and off switching of the PI3-K (phosphoinositide 3-kinase)/Akt pathway, particularly by its major activators insulin and IGF-1 (insulin-like growth factor-1), is a powerful integrator of physiological responses rudimentary to successful aging. This is highlighted by extensive studies showing that reducing, but not obliterating, activation of the PI3-K/Akt/mTOR signal, at several levels, can extend healthy lifespan in organisms from yeast to mammals. Moreover, aberrant control of the PI3-K/Akt axis is emerging to be a primary causative node in all major diseases of aging: cancer, type 2 diabetes mellitus (T2DM), heart disease and neurodegeneration. Aging is the major risk factor for AD, the most common dementia disorder. The integrated coordination of neuronal responses through the PI3-K/Akt pathway has significant functional impact on key events that go awry in Alzheimer's disease (AD), including: synaptic plasticity, neuronal polarity, neurotransmission, proteostasis, use-dependent translation, metabolic control and stress responses including DNA repair. Investigation of the status of the PI3-K/Akt system in brains of individuals who have had AD shows aberrant and sustained activation of neuronal PI3-K/Akt/mTOR signaling to be an early feature of the disease. This is mechanistically linked to progressive desensitization of normal brain insulin and IGF-1 responses, aberrant proteostasis of Aβ and tau, synaptic loss and cognitive decline in the disease. Notably, concomitantly with feedback inhibition of insulin and IGF-1 responses, increased activation of the neuronal PI3-K/Akt/mTOR axis is a major candidate effector system for transmission of pathophysiological signals from Aβ to tau in the context of defects in synaptic transmission that lead to cognitive decline. Therapeutic approaches targeted at normalizing signaling through either the neuronal PI3-kinase/Akt/mTOR pathway or its activation by insulin and IGF-1 have been shown to be protective against the development of AD pathology and cognitive decline in animal models of AD and some of these therapies are entering clinical trials in patients with the disease.