有氧运动对小鼠骨骼肌结节性硬化复合物蛋白2基因表达及胰岛素受体底物1丝氨酸磷酸化的影响

背景：研究表明,胰岛素受体底物蛋白1(insulin receptor substrate 1,IRS1)的丝氨酸磷酸化和结节性硬化复合物蛋白2的表达及哺乳动物雷帕霉素靶蛋白/核糖体S6激酶1信号通路的异常可诱导机体产生胰岛素抵抗。 目的：观察有氧运动对小鼠骨骼肌结节性硬化复合物蛋白2基因表达及IRS1丝氨酸磷酸化的影响。 方法：将C57BL/6小鼠随机分为安静组和运动组,运动组进行6周的75%VO2max强度的有氧跑台运动,安静组不做任何干预。采用RT-PCR,Western blot和免疫荧光组织化学染色等方法检测各组大鼠骨骼肌结节性硬化复合物蛋白2,IRS1,pIRS1-Ser307和pIRS1-Ser636/639的表达和定位。 结果与结论：运动组结节性硬化复合物蛋白2 mRNA及蛋白表达高于安静组(P < 0.05),两组间胰岛素受体底物蛋白1 mRNA及蛋白表达差异无显著性意义。运动组胰岛素受体底物蛋白1的丝氨酸307和丝氨酸636/639位点的磷酸化明显低于安静组(P< 0.05)。结果提示,有氧运动可增强骨骼肌组织结节性硬化复合物蛋白2基因表达,进而抑制胰岛素受体底物蛋白1的丝氨酸磷酸化。     

观察炎症状态和高脂状态对mTOR/S6K/IRS1-Ser和IRS1-Tyr信号通路表达的影响

目的观察在体内外炎症状态下哺乳动物雷帕霉素靶蛋白(mTOR)-70S核糖体蛋白S6激酶(S6K)-胰岛素受体底物1(IRS1)信号传导通路异常磷酸化表达对胰岛素抵抗的影响。方法体外实验,将HepG2分为4组:对照组、高脂组[给予100 mg/L低密度脂蛋白(LDL)处理]、炎症介入组[给予20μg/L肿瘤坏死因子-α(TNF-α)处理]、联合干预组(给予20μg/L TNF-α+100 mg/L LDL处理)。采用实时定量PCR和Western blotting方法检测mTOR、S6K和IRS1 mRNA及蛋白表达水平;体内实验,8周龄c57BL/6J小鼠随机分为4组(正常饮食组、正常饮食加炎症组、高脂饮食组和高脂饮食加炎症组),分别给予隔日皮下注射生理盐水和酪蛋白建立炎症模型,8周后将实验小鼠于深度麻醉下处死并检测其肝脏组织中mTOR、S6K和IRS1 mRNA及蛋白表达水平。结果体内外实验均显示与对照组相比,炎症组与高脂组mTOR、S6K和IRS1-Ser mRNA及蛋白表达增加,而联合干预组表达明显增高,IRS1-Tyr mRNA及蛋白表达下降,联合干预组表达明显降低。结论炎症及高脂状态激活mTOR/s6k/IRS1-Ser信号通路,而抑制IRS1-Tyr信号通路,从而加重胰岛素抵抗。     

Frequent [corrected] hyperphosphorylation of ribosomal protein S6 [corrected] in lymphangioleiomyomatosis-associated angiomyolipomas.

Lymphangioleiomyomatosis is a progressive lung disease characterized by a diffuse proliferation of pulmonary smooth muscle cells and cystic degeneration. Lymphangioleiomyomatosis can occur either independently of other disease or in association with tuberous sclerosis complex, a tumor-suppressor gene syndrome caused by mutations that inactivate either TSC1 or TSC2. TSC2 mutations and loss of heterozygosity have been identified in sporadic lymphangioleiomyomatosis-associated angiomyolipomas, thus implicating the TSC/Ras homolog-enriched in brain (Rheb)/mammalian target of Rapamycin (mTOR)/p70 S6 kinase signaling pathway in their pathogenesis. This study was undertaken to determine whether the mTOR/p70 S6 kinase signaling pathway is activated in lymphangioleiomyomatosis-associated angiomyolipomas lacking TSC1/TSC2 loss of heterozygosity. Phospho-ribosomal protein S6 (Ser235/236) immunohistochemistry was performed on five lymphangioleiomyomatosis-associated angiomyolipomas, two matched lymphangioleiomyomatosis pulmonary samples, and three sporadic angiomyolipomas. TSC1/TSC2 loss of heterozygosity was previously excluded in these angiomyolipomas. Moderate or strong phospho-ribosomal protein S6 immunoreactivity was found in all lymphangioleiomyomatosis-associated and sporadic angiomyolipomas, suggesting a high incidence of mTOR/p70 S6 kinase signaling pathway activation despite a lack of TSC1/TSC2 loss of heterozygosity. Focally positive phospho-S6 staining was also evident in both lymphangioleiomyomatosis pulmonary samples. We hypothesized that this S6 hyperphosphorylation could reflect mutational activation of Rheb or Rheb-like protein (RhebL1), Ras family members which directly activate mTOR. Mutational analysis performed on DNA from these eight angiomyolipomas plus five additional sporadic angiomyolipomas did not reveal mutations in exons 3 and 4 (homologous sites of Ras activating mutations) of either Rheb or RhebL1. These data suggest that activation of the Rheb/mTOR/p70 S6 kinase pathway is related to the pathogenesis of lymphangioleiomyomatosis-associated and sporadic angiomyolipomas lacking TSC1/TSC2 loss of heterozygosity. This high incidence of mTOR signaling pathway activation suggests that treatment with mTOR inhibitors, such as Rapamycin, may benefit patients with angiomyolipomas independent of the detection of TSC1/TSC2 loss of heterozygosity.     

Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. Here we report that TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth.     

Renin angiotensin system modulates mTOR pathway through AT2R in HIVAN

Mammalian target of rapamycin (mTOR) has been reported to contribute to the development of HIV-associated nephropathy (HIVAN). We hypothesized that HIV may be activating renal tissue mTOR pathway through renin angiotensin system (RAS) via Angiotensin Receptor Type II receptor (AT2R). Renal tissues of Vpr transgenic and Tg26 (HIVAN) mice displayed enhanced phosphorylation of mTOR and p70S6K. Aliskiren, a renin inhibitor attenuated phosphorylation of both mTOR and p70S6K in renal tissues of HIVAN mice. Interestingly, Angiotensin Receptor Type I (AT1R) blockade did not modulate renal tissue phosphorylation of mTOR in HIVAN mice; on the other hand, AT2R blockade attenuated renal tissue phosphorylation of mTOR in HIVAN mice. In vitro studies, both renin and Ang II displayed enhanced mouse tubular cell (MTC) phosphorylation of p70S6K in a dose dependent manner. HIV/MTC also displayed enhanced phosphorylation of both mTOR and p70S6K; interestingly this effect of HIV was further enhanced by losartan (an AT1R blocker). On the other hand, AT2R blockade attenuated HIV-induced tubular cell phosphorylation of mTOR and p70S6K, whereas, AT2R agonist enhanced phosphorylation of mTOR and p70S6K. These findings indicate that HIV stimulates mTOR pathway in HIVAN through the activation of renin angiotensin system via AT2R.     

Activation of the mTOR pathway in sporadic angiomyolipomas and other perivascular epithelioid cell neoplasms

Angiomyolipoma (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-tuberous sclerosis complex (TSC), non-lymphangioleiomyomatosis-associated AMLs, and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14 of 15 cases of extrarenal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.     

Targeting the Phosphatidylinositol 3-Kinase Pathway in Airway Smooth Muscle

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in regulating cell growth, proliferation, survival, and motility. Structural alterations, e.g. airway remodeling, in asthma and chronic obstructive pulmonary disease (COPD) are associated with increased airway smooth muscle (ASM) cell growth and proliferation due to the frequent stimulation of ASM by inflammatory mediators, contractile agonists, and growth factors. The critical role of the PI3K signaling pathway in regulating ASM cell growth and proliferation is well established. However, recent discovery of the tumor suppressor proteins tuberous sclerosis complex 1 (TSC1) and TSC2, also known as hamartin and tuberin, as downstream effectors of PI3K and upstream regulators of the mammalian target of rapamycin (mTOR) and S6 kinase 1(S6K1) shed a new light on the PI3K signaling cascade in regulating cell growth and proliferation. The activity of TSC1/TSC2 is regulated by growth factors, nutrients, and energy; thus, TSC1/TSC2 serves as a signaling module for protein translational regulation, cell cycle progression, and cell size, which are key events controlling cell growth and proliferation. This article highlights the potential contribution of the PI3K-TSC1/TSC2-mTOR/S6K1 pathway in smooth muscle remodeling. Pharmacologic manipulation of this signaling pathway could have a major impact on treatment of asthma and COPD.     

Over-expression of NYGGF4 (PID1) inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway

IntroductionDefects in insulin-stimulated glucose uptake in muscle are the important early events in the pathogenesis of insulin resistance. NYGGF4 (also named PID1) is a recently discovered gene which is suggested to be associated with obesity-associated insulin resistance. In this study, we aimed to investigate the effects of NYGGF4 on glucose uptake and insulin signaling in rat skeletal muscle cells.MethodsRat L6 myoblasts were transfected with either an empty vector or an NYGGF4-expressing vector and induced to differentiate into mature L6 skeletal myotubes. Glucose uptake was determined by measuring uptake of 2-deoxy-d-[³H] glucose. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting was also used to measure phosphorylation and total protein levels of the insulin signaling proteins including insulin receptor (IR), insulin receptor substrate 1 (IRS1), Akt, extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun-N-terminal kinase (JNK).ResultsNYGGF4 over-expression in L6 skeletal myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, or JNK.ConclusionsOver-expression of NYGGF4 inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway. These observations highlight the potential role of NYGGF4 in glucose homeostasis and the development of insulin resistance in obesity.     

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.     

Resistance exercise, but not endurance exercise, induces IKKβ phosphorylation in human skeletal muscle of training-accustomed individuals

The mammalian target of rapamycin complex 1 (mTORC1) is considered an important role in the muscular adaptations to exercise. It has been proposed that exercise-induced signaling to mTORC1 do not require classic growth factor PI3K/Akt signaling. Activation of IKKβ and the mitogen-activated protein kinases (MAPKs) Erk1/2 and p38 has been suggested to link inflammation and cellular stress to activation of mTORC1 through the tuberous sclerosis 1 (TSC1)/tuberous sclerosis 2 (TSC2) complex. Consequently, activation of these proteins constitutes potential alternative mechanisms of mTORC1 activation following exercise. Previously, we demonstrated that mTOR is preferentially activated in response to resistance exercise compared to endurance exercise in trained individuals without concomitant activation of Akt. In the present study, we extended this investigation by examining IκB kinase complex (IKK), TSC1, MAPK, and upstream Akt activators, along with gene expression of selected cytokines, in skeletal muscles from these subjects. Biopsies were sampled prior to, immediately after, and in the recovery period following resistance exercise, endurance exercise, and control interventions. The major finding was that IKKβ phosphorylation increased exclusively after resistance exercise. No changes in TSC1, Erk1/2, insulin receptor, or insulin receptor substrate 1 phosphorylation were observed in any of the groups, while p38 phosphorylation was higher in the resistance exercise group compared to both other groups immediately after the intervention. Resistance and endurance exercise increased IL6, IL8, and TNFα gene expression immediately after exercise. The non-exercise control group demonstrated that cytokine gene expression is also sensitive to repeated biopsy sampling, whereas no effect of repeated biopsy sampling on protein expression and phosphorylation was observed. In conclusion, resistance exercise, but not endurance exercise, increases IKKβ phosphorylation in trained human subjects, which support the idea that IKKβ can influence the activation of mTORC1 in human skeletal muscle.     

Exercise improves skeletal muscle insulin resistance without reduced basal mTOR/S6K1 signaling in rats fed a high-fat diet

Exercise improves high-fat diet (HFD)-induced skeletal muscle insulin resistance, but the mechanism is unresolved. This study aims to explore whether the improvement in response to exercise is associated with mTOR/S6K1 signaling and whether the signaling changes are muscle-specific. Male SD rats (150–180 g) were used for this study. After the experimental period, 6 weeks of exercise improved HFD-impaired intraperitoneal glucose tolerance and insulin-stimulated 2-deoxyglucose uptake in soleus (SOL) and extensor digitorum longus (EDL) muscles. Furthermore, 6 weeks of the HFD resulted in a reduced type I fiber ratio of SOL, an increased type I ratio of EDL, and a reduced fiber size of EDL, whereas exercise increased type I fiber ratio of SOL as well as type I fiber cross-sectional areas of EDL. However, the HFD had a main effect on basal cytosolic phosphorylation of S6K1 on Thr³⁸⁹ content in SOL, which was also influenced by a significant interaction between the diet and exercise in EDL. Exercise had no direct effect on the basal phosphorylation of Akt on Ser⁴⁷³, mTOR on Ser²⁴⁴⁸, S6K1 on Thr³⁸⁹ content in SOL. On the contrary, exercise prevented HFD-induced decrease in basal phosphorylation of S6K1 on Thr³⁸⁹ content in EDL. These results indicate that 6 weeks of HFD and exercise lead to alterations in fiber type shift, fiber size, and basal phosphorylation of S6K1 on Thr³⁸⁹ content in a muscle-specific pattern. Exercise prevents HFD-induced skeletal muscle insulin resistance, which is not associated with a reduced basal phosphorylation of mTOR/S6K1 alteration in the muscles.     

Differential expression of mTOR signaling pathway proteins in lichen planopilaris and frontal fibrosing alopecia

Dysregulation of the mammalian target of rapamycin (mTOR) signaling pathway has a variety of effects on the immune system and stem cell proliferation. Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are inflammatory scalp conditions resulting in permanent alopecia, which are thought to be related to stem cell damage. Here we investigate the expression of mTOR signaling pathway proteins in human hair follicles of LPP and FFA patients. The expression of mTOR pathway proteins in biopsy specimens from lesional and non-lesional scalp areas of eight LPP and five FFA patients were compared to control scalp biopsies from patients undergoing surgical excisions of sebaceous cysts. We performed immunohistochemical evaluation using a panel of antibodies including mTOR, phospho-mTOR (Ser2448), phospho-p70S6K (Thr389), phospho-4EBP1 (Thr37146), and phospho-tuberin (T1462), as well as Western blot analysis for phospho-p70S6K (Thr389) expression. All evaluated mTOR pathway proteins were similarly expressed in the control and patient non-lesional scalps. While mTOR expression did not show significant alterations between the groups, p-mTOR, p-p70S6K, p-4EBP1, and p-tuberin expressions decreased in the interfollicular epidermis in the lesional scalps of patients. p-p70S6K and p-4EBP1 expression decreased in the outer root sheath (ORS) and inner root sheath (IRS) of the bulge of hair follicles in the lesional scalps of patients. p-mTOR and p-p70S6K expression increased in the lower follicle ORS and bulb of the hair follicles, and p-4EBP1 expression decreased in the bulb of the hair follicles in the lesional scalps of patients. Phospho-tuberin expression increased in the IRS of the bulge and lower follicle ORS of the hair follicles in the lesional scalps of patients, whereas its expression decreased in the bulb. Our results indicate that the mTOR signaling pathway proteins are localized throughout normal hair follicles and that expression of mTOR signaling pathway proteins is altered in the hair follicles of LPP and FFA patients. Further research is required to understand the mechanism by which mTOR operates in the pathogenesis of these diseases.     

Constant allelic alteration on chromosome 16p (TSC2 gene) in perivascular epithelioid cell tumour (PEComa): genetic evidence for the relationship of PEComa with angiomyolipoma

Perivascular epithelioid cell tumours (PEComas) are a family of tumours including classic angiomyolipoma, lymphangioleiomyomatosis, and clear epithelioid cell tumours reported under a variety of names such as epithelioid angiomyolipoma, pulmonary and extrapulmonary clear cell sugar tumour, and PEComa. Our previous comparative genomic hybridization study of PEComas demonstrated recurrent chromosomal aberrations including deletions on chromosome 16p, where the TSC2 gene is located. In this study, we focused on the alteration of chromosome 16p, including TSC2. We collected ten sporadic and two tuberous sclerosis complex-associated PEComas, as well as 14 sporadic classic hepatic and renal angiomyolipomas (AMLs) as controls. We used 16 microsatellite markers distributed along chromosome 16p to test for allelic imbalances on chromosome 16p and at TSC2, and two markers for TSC1. Furthermore, we carried out immunohistochemical staining for phospho-p706K, phospho-AKT, and phospho-S6 to evaluate the effect of TSC2 alterations on the mTOR signalling pathway. Loss of heterozygosity (LOH) was found in 11 PEComas and involved the region of the TSC2 locus in seven. Six classic angiomyolipomas had allelic changes at chromosome 16p. Microsatellite instability was detected in two PEComas. The incidence of genetic aberrations was significantly higher in the PEComa group. Only one PEComa showed LOH at the TSC1 locus. Eleven PEComas and 13 AMLs revealed elevated phospho-p70S6K accompanied by reduced phospho-AKT. Five PEComas and eight classic angiomyolipomas were positive for phospho-S6. The phosphorylation profile indicates functional activation of the mTOR pathway through a disrupted TSC1/2 complex. Our observations of frequent deletion of TSC2 and the mTOR signalling pathway provide evidence that the oncogenetic lineage of PEComa, as a distinct TSC2-linked neoplasm, is similar to that of angiomyolipoma.     

Cortical neurons develop insulin resistance and blunted Akt signaling: a potential mechanism contributing to enhanced ischemic injury in diabetes

Patients with diabetes are at higher risk of stroke and experience increased morbidity and mortality after stroke. We hypothesized that cortical neurons develop insulin resistance, which decreases neuroprotection via circulating insulin and insulin-like growth factor-I (IGF-I). Acute insulin treatment of primary embryonic cortical neurons activated insulin signaling including phosphorylation of the insulin receptor, extracellular signal-regulated kinase (ERK), Akt, p70S6K, and glycogen synthase kinase-3β (GSK-3β). To mimic insulin resistance, cortical neurons were chronically treated with 25?mM glucose, 0.2?mM palmitic acid (PA), or 20?nM insulin before acute exposure to 20?nM insulin. Cortical neurons pretreated with insulin, but not glucose or PA, exhibited blunted phosphorylation of Akt, p70S6K, and GSK-3β with no change detected in ERK. Inhibition of the phosphatidylinositol 3-kinase (PI3-K) pathway during insulin pretreatment restored acute insulin-mediated Akt phosphorylation. Cortical neurons in adult BKS-db/db mice exhibited higher basal Akt phosphorylation than BKS-db(+) mice and did not respond to insulin. Our results indicate that prolonged hyperinsulinemia leads to insulin resistance in cortical neurons. Decreased sensitivity to neuroprotective ligands may explain the increased neuronal damage reported in both experimental models of diabetes and diabetic patients after ischemia-reperfusion injury.     

Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serine-threonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1

We have previously found that both mitogen-activated protein kinase (MAPK)- and Rho kinase (ROCK)-related signaling pathways are necessary for the induction of pulmonary artery smooth muscle cell (SMC) proliferation by serotonin (5-hydroxytryptamine [5-HT]). In the present study, we investigated the possible additional participation of a phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K1) pathway in this growth response. We found transient activation of Akt (Ser473) and more prolonged activation of S6K1 by 5-HT. Inhibition of PI3K with Wortmannin and LY294002 completely blocked these activations, but not that of MAPK or the ROCK substrate myosin phosphatase targeting subunit. Similarly, inhibition of MAPK and ROCK failed to block the Akt activation. Inhibition of Akt with NL-71-101 and downregulation of Akt expression with Akt small interfering RNA blocked 5-HT-induced S6K1 phosphorylation. Wortmannin, LY294002, and NL-71-101 dose-dependently inhibited 5-HT-induced SMC proliferation. 5-HT stimulated mTOR phosphorylation and the mTOR inhibitor, rapamycin, blocked activations of S6K1 and S6 ribosomal protein, and inhibited 5-HT-induced SMC proliferation. Akt phosphorylation and cell proliferation were also blocked by the antioxidants, N-acetyl-l-cysteine, Ginko biloba 501, and tiron, the reduced nicotinamide adenine dinucleotide phosphate oxidase inhibitor, diphenyleneiodonium, and the 5-HT2 receptor antagonists ketanserin and mianserin, but not by the 5-HT serotonin transporter or 5-HT 1B/1D receptor antagonists. We conclude from these studies that a parallel PI3K- and reactive oxygen species-dependent Akt/mTOR/S6K1 pathway participates independently from MAPK and Rho/ROCK in the mitogenic effect of 5-HT on pulmonary artery SMCs. From these and other studies, we postulate that independent signaling pathways leading to 5-HT-induced SMC proliferation are initiated through multiple 5-HT receptors and serotonin transporter at the cell surface.     

Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome

Tuberous sclerosis complex (TSC) and Peutz-Jeghers syndrome (PJS) are dominantly inherited benign tumor syndromes that share striking histopathological similarities. Here we show that LKB1, the gene mutated in PJS, acts as a tumor suppressor by activating TSC2, the gene mutated in TSC. Like TSC2, LKB1 inhibits the phosphorylation of the key translational regulators S6K and 4EBP1. Furthermore, we show that LKB1 activates TSC2 through the AMP-dependent protein kinase (AMPK), indicating that LKB1 plays a role in cell growth regulation in response to cellular energy levels. Our results suggest that PJS and other benign tumor syndromes could be caused by dysregulation of the TSC2/mTOR pathway.     

mTOR、S6K1在双胍类降糖药物改善2型糖尿病中的作用研究

目的 探讨mTOR、S6K1在双胍类降糖药物改善2型糖尿病中的作用和意义.方法 选取本院2015年3月至2017年3月收治的100例糖尿病患者,随机分为观察一组和观察二组,观察一组患者给予常规胰岛素治疗,观察二组患者在此基础上接受二甲双胍治疗3个月.选取同期健康体检者50例为对照组.治疗前后分别检测受试者血清中mTOR、S6K1信号通路的水平变化.结果 与对照组相比,观察一组和观察二组患者体内mTOR和S6K1水平显著升高,IL-6、TNF-α和IL-Iβ水平显著上升,差异具有统计学意义(P＜0.05).观察一组和观察二组患者与治疗前相比,治疗后mTOR、S6K1、IL-6、TNF-α和IL-Iβ水平显著降低,差异具有统计学意义(P＜0.05).与观察一组患者治疗后相比,观察二组患者二甲双胍治疗后,血清mTOR、S6K1、IL-6、TNF-α和IL-Iβ水平显著降低,差异具有统计学意义(P＜0.05).患者血液mTOR和S6K1与其使用二甲双胍治疗的疗效存在显著的负相关关系,Person相关系数为负值,P＜0.01.结论 双胍类降糖药物可以明显改善糖尿病患者的临床症状;mTOR、S6K1信号通路在糖尿病患者的疾病发展和临床治疗中发挥重要的作用.     

The molecular basis for oxidative stress-induced insulin resistance

Reactive oxygen and nitrogen molecules have been typically viewed as the toxic by-products of metabolism. However, accumulating evidence has revealed that reactive species, including hydrogen peroxide, serve as signaling molecules that are involved in the regulation of cellular function. The chronic and/or increased production of these reactive molecules or a reduced capacity for their elimination, termed oxidative stress, can lead to abnormal changes in intracellular signaling and result in chronic inflammation and insulin resistance. Inflammation and oxidative stress have been linked to insulin resistance in vivo. Recent studies have found that this association is not restricted to insulin resistance in type 2 diabetes, but is also evident in obese, nondiabetic individuals, and in those patients with the metabolic syndrome. An increased concentration of reactive molecules triggers the activation of serine/threonine kinase cascades such as c-Jun N-terminal kinase, nuclear factor-kappaB, and others that in turn phosphorylate multiple targets, including the insulin receptor and the insulin receptor substrate (IRS) proteins. Increased serine phosphorylation of IRS reduces its ability to undergo tyrosine phosphorylation and may accelerate the degradation of IRS-1, offering an attractive explanation for the molecular basis of oxidative stress-induced insulin resistance. Consistent with this idea, studies with antioxidants such as vitamin E, alpha-lipoic acid, and N-acetylcysteine indicate a beneficial impact on insulin sensitivity, and offer the possibility for new treatment approaches for insulin resistance.     

高脂饲养致小鼠胰岛素抵抗对脂肪肝形成的影响

目的:探讨高脂饲养致小鼠脂肪肝形成的机制。方法:随机将8周雄性C57BL/6J小鼠分成高脂饲养组(给予含60%卡路里的高饱和脂肪酸饲养)和正常对照组,饲养12周。监测体重、肝重、血甘油三酯、血总胆固醇、血糖和血胰岛素水平,通过高胰岛素正葡萄糖钳夹实验反映胰岛素敏感性,HE染色、苏丹IV染色及肝脂含量反映肝组织脂质沉积情况,确定高脂饲养致小鼠脂肪肝的形成。通过Western blot法检测磷酸化胰岛素受体底物1(IRS1)和蛋白激酶B(Akt)水平反映胰岛素信号通路激活情况,检测固醇调节元件结合蛋白1(SREBP-1)和脂肪酸合成酶(FAS)蛋白水平反映肝内脂质合成的情况。结果:高脂饲养组小鼠体重及肝重较正常对照组小鼠明显增加。与正常对照组相比,高脂组血和肝组织内甘油三酯和总胆固醇含量显著升高,血清胰岛素水平升高,葡萄糖输注率减少,磷酸化IRS1和Akt水平降低。肝组织HE染色可见高脂组肝细胞胞浆内充满大量脂肪空泡,苏丹IV染色可见肝细胞内存在大量大小不一的红色脂滴;SREBP-1和FAS蛋白水平明显升高。给予外源性油酸干预原代正常肝细胞48 h,磷酸化IRS1和Akt水平呈浓度依赖性减低,而SREBP-1和FAS蛋白表达明显升高。结论:高脂饲养导致小鼠肝脏发生胰岛素抵抗,并通过激活SREBP-FAS脂肪合成途径,促进肝脏脂质沉积,从而诱发脂肪肝。     

Dominant Splice Site Mutations in <Emphasis Type="Italic">PIK3R1</Emphasis> Cause Hyper IgM Syndrome,Lymphadenopathy and Short Stature

The purpose of this research was to use next generation sequencing to identify mutations in patients with primary immunodeficiency diseases whose pathogenic gene mutations had not been identified. Remarkably, four unrelated patients were found by next generation sequencing to have the same heterozygous mutation in an essential donor splice site of PIK3R1 (NM_181523.2:c.1425 + 1G > A) found in three prior reports. All four had the Hyper IgM syndrome, lymphadenopathy and short stature, and one also had SHORT syndrome. They were investigated with in vitro immune studies, RT-PCR, and immunoblotting studies of the mutation’s effect on mTOR pathway signaling. All patients had very low percentages of memory B cells and class-switched memory B cells and reduced numbers of naïve CD4+ and CD8+ T cells. RT-PCR confirmed the presence of both an abnormal 273 base-pair (bp) size and a normal 399 bp size band in the patient and only the normal band was present in the parents. Following anti-CD40 stimulation, patient’s EBV-B cells displayed higher levels of S6 phosphorylation (mTOR complex 1 dependent event), Akt phosphorylation at serine 473 (mTOR complex 2 dependent event), and Akt phosphorylation at threonine 308 (PI3K/PDK1 dependent event) than controls, suggesting elevated mTOR signaling downstream of CD40. These observations suggest that amino acids 435–474 in PIK3R1 are important for its stability and also its ability to restrain PI3K activity. Deletion of Exon 11 leads to constitutive activation of PI3K signaling. This is the first report of this mutation and immunologic abnormalities in SHORT syndrome.