禁食对蛋鸡肝脏腺苷-磷酸激活的蛋白激酶活性的影响

腺苷 -磷酸 (AMP)激活的蛋白激酶 (AMP- acti-vated protein kinase,AMPK)是丝氨酸激酶家族的一员 ,由 AMP和其上游激酶 AMPK激酶所活化 ,对细胞内 AMP/ATP的变化非常敏感 [1] ,被称为真核细胞的“代谢感受器”[2 ]。研究发现在跑步 [3 ,4 ]、电刺激肌肉 [5,6]或禁食应激后 [7] ,大鼠肝脏和肌肉中的AMPK活性升高数倍 ,乙酰辅酶 A羧化酶 (ACC)活性显著下降甚至丧失 ,丙二酸单酰辅酶 A产量降低甚至为零 ,脂肪酸合成受抑。同时 ,AMPK活化后 ,脂肪酸的氧化率显著提高 ,CO2 和酮体生成量明显增加[4 ,8] 。这些均表明 ,AMPK活化…     

二甲双胍通过AMPK/STAT3信号通路抑制星形胶质细胞的反应性

探讨二甲双胍对体外培养的大鼠脊髓星形胶质细胞反应性的作用及机制。方法 使用三因子（TNF-α、IL-1α、C1q）诱导产生反应性星形胶质细胞,RT-qPCR及Westernblot检测补体3（C3）的表达;使用5、10、20 mM浓度的二甲双胍处理,Westernblot检测星形胶质细胞中腺苷酸激活蛋白激酶（AMPK）与信号转导和转录激活因子3（STAT3）磷酸化水平;采用AMPK抑制剂Compound C处理,检测AMPK和STAT3磷酸化水平。结果 形态学及RT-qPCR结果显示：与对照组相比,三因子处理改变了星形胶质细胞形态并显著提高了C3的表达（P＜0.05）,表明成功建立反应性星形胶质细胞模型。RT-qPCR结果及Westernblot结果显示：与对照组相比,二甲双胍对C3表达有明显的抑制作用,呈现浓度依赖性（P＜0.05）。在诱导星形胶质细胞反应性过程中,与对照组相比,AMPK的磷酸化水平显著降低（P＜0.05）,使用不同浓度二甲双胍处理后,AMPK的磷酸化水平显著增加且呈浓度依赖性（P＜0.05）,同时信号传导及STAT3的磷酸化水平显著降低且呈浓度依赖性（P＜0.05）。AMPK抑制剂Compound C处理后显著抑制了二甲双胍导致的AMPK活性升高及STAT3活性降低,同时抑制了C3表达下调（P＜0.05）。结论 二甲双胍通过AMPK/STAT3信号通路抑制星形胶质细胞的反应性     

Vascular Effects of the Polyphenolic Nutraceutical Supplement Taurisolo®: Focus on the Protection of the Endothelial Function

Preservation of vascular endothelium integrity and functionality represents an unmet medical need. Indeed, endothelial dysfunction leads to decreased nitric oxide biosynthesis, which is prodromic of hypertension and hypercoagulability. In this panorama, the nutraceutical supplement Taurisolo^®, a polyphenolic extract from Aglianico cultivar grape, rich in catechin and procyanidins, was evaluated as a vasoprotective, vasorelaxing, anti-hypertensive and anti-coagulant agent in: cell lines, isolated vessels, in vivo models of chronic hypertension and hypercoagulability, and in clinical tests of endothelial reactivity. Taurisolo^® demonstrated to fully protect vascular cell viability from oxidative stimulus at 100 µg/mL and evoke vasorelaxing effects (Emax = 80.6% ± 1.9 and pEC50 = 1.19 ± 0.03) by activation of the Sirtuins-AMPK-pathway. Moreover, Taurisolo^®, chronically administered at 20 mg/Kg/die in in vivo experiments, inhibited the onset of cardiac hypertrophy (heart weight/rat weight = 3.96 ± 0.09 vs. 4.30 ± 0.03), hypercoagulability (decrease of fibrinogen vs. control: p < 0.01) and hypertension (mean of Psys: 200 ± 2 vs. control 234 ± 2 mmHg) and improved endothelial function (Emax = 88.9% ± 1.5 vs. control 59.6% ± 3.6; flow-mediated dilation in healthy volunteers after 400 mg twice daily for 8 weeks vs. baseline: p = 0.019). In conclusion, Taurisolo^® preserves the vascular function against ox-inflamm-ageing process and the consequent cardiovascular accidents.     

TWIST2通过调控FGF21介导的AMPK/mTOR通路诱导卵巢癌细胞氧化应激和凋亡

目的探究TWIST2对卵巢癌细胞氧化应激和凋亡的作用及相关机制。方法收集2018年6月至2019年10月在山东省妇幼保健院进行治疗的30例卵巢癌患者的癌组织和癌旁组织,将pcDNA3.1-TWIST2质粒(TWIST2组)和pcDNA3.1阴性对照质粒(阴性对照组)分别转染卵巢癌细胞(SK-OV-3和HO-8910),未转染质粒组为空白对照组。通过实时荧光定量PCR(qRT-PCR)和免疫印迹(Western blot)实验检测组织和细胞中TWIST2 mRNA和蛋白质的表达量;细胞计数试剂盒CCK-8检测TWIST2组、阴性对照组和空白对照组的细胞活力;Western blot实验检测3组的凋亡相关蛋白caspase-3、Bax和Bcl-2,AMPK/mTOR通路相关蛋白FGF21、p-AMPK和p-mTOR的蛋白表达水平;试剂盒检测3组活性氧(ROS)基因、ATP的水平和超氧化物歧化酶(SOD)的酶活性。通过检测不同处理组的细胞凋亡率和ROS水平,研究TWIST2与FGF21的相互作用。结果TWIST2在卵巢癌组织和卵巢癌细胞中的表达量显著低于癌旁组织和正常卵巢上皮细胞的表达量(P<0.05)。与空白对照组和阴性对照组相比,TWIST2组的细胞活力显著降低,细胞凋亡率显著提高(P<0.05);ROS的水平显著升高,SOD酶活力、ATP水平、NAD~+/NADH水平显著降低(P<0.05)。TWIST2可以抑制FGF21、p-AMPK、p-mTOR蛋白的表达(P<0.05);在TWIST2组中加入FGF21或AMPK激动剂时,p-AMPK、p-mTOR蛋白表达水平显著升高,卵巢癌细胞的凋亡率和ROS水平显著降低(P<0.05),TWIST2与FGF21的表达量呈负相关(r=-0.6254,P<0.01)。结论TWIST2能够促进卵巢癌细胞氧化应激和凋亡,其作用机制是通过调控FGF21介导的AMPK/mTOR通路来实现的。     

Buformin suppresses proliferation and invasion via AMPK/S6 pathway in cervical cancer and synergizes with paclitaxel

Buformin is an old anti-diabetic agent and manifests potent anti-tumor activities in several malignancies. In the present study, we aimed to explore the functions of buformin in human cervical cancer. As our data shown, buformin exhibited significant anti-proliferative effects in a dose-dependent manner in 4 cervical cancer cell lines. Compared to the control, buformin notably suppressed colony formation and increased ROS production in C33A, Hcc94 and SiHa cells. Flow cytometric analysis showed that buformin induced marked cell cycle arrest but only resulted in mild apoptosis. The invasion of C33A and SiHa cells sharply declined with buformin treatment. Consistently, western blotting showed that buformin activated AMPK and suppressed S6, cyclin D1, CDK4, and MMP9. Moreover, we found that buformin enhanced glucose uptake and LDH activity, increased lactate level, while decreased ATP production in cervical cancer cells. In addition, low doses of buformin synergized with routine chemotherapeutic drugs (such as paclitaxel, cisplatin, and 5-FU) to achieve more significant anti-tumor effects. In vivo, a single use of buformin exerted moderate anti-tumor effects, and the combination with buformin and paclitaxel exhibited even greater suppressive effects. Buformin also consistently showed synergistic effects with paclitaxel in treating primary cultures of cervical cancer cells. Take together, we are the first to demonstrate that buformin suppresses cellular proliferation and invasion through the AMPK/S6 signaling pathway, which arrests cell cycle and inhibits cellular invasion. Buformin also could synergize with routine chemotherapies, producing much more powerful anti-tumor effects. With these findings, we strongly support buformin as a potent choice for treating cervical cancer, especially in combination with routine chemotherapy.     

Dissecting the role of AMP-activated protein kinase in human diseases

AMP-activated protein kinase (AMPK), known as a sensor and a master of cellular energy balance, integrates various regulatory signals including anabolic and catabolic metabolic processes. Accompanying the application of genetic methods and a plethora of AMPK agonists, rapid progress has identified AMPK as an attractive therapeutic target for several human diseases, such as cancer, type 2 diabetes, atherosclerosis, myocardial ischemia/reperfusion injury and neurodegenerative disease. The role of AMPK in metabolic and energetic modulation both at the intracellular and whole body levels has been reviewed elsewhere. In the present review, we summarize and update the paradoxical role of AMPK implicated in the diseases mentioned above and put forward the challenge encountered. Thus it will be expected to provide important clues for exploring rational methods of intervention in human diseases.     

负荷渐增式训练对老年小鼠骨骼肌卫星细胞AMPK磷酸化的影响

目的 探讨负荷渐增式训练对老年小鼠骨骼肌卫星细胞腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）磷酸化的影响。方法 实验小鼠分为 3 组：青年对照组（YC组，n=12）、老年对照组（OC组，n=12）与老年运动组（OT组，n=12）。OT组进行负荷渐增式训练，流式细胞分选技术分离CD45-/CD31-/Sca1-/VCAM(CD106)+细胞群体，分选细胞通过desmin、Myod肌原性染色以及成肌分化诱导培养进行肌卫星细胞鉴定，免疫组化结合Western blotting方法检测肌卫星细胞p-AMPK水平。结果 YC组骨骼肌卫星细胞AMPK及p-AMPK表达水平显著高于OC组（P<0.05）；OT组与OC组AMPK表达无明显变化（Ｐ>0.05），而OT组p-AMPK表达水平显著高于OC组（P<0.05）。结论 负荷渐增式训练可促进老年小鼠骨骼肌卫星细胞AMPK磷酸化，改善老年小鼠骨骼肌能量代谢。     

High expressions of LDHA and AMPK as prognostic biomarkers for breast cancer

ObjectivesThe purpose of this study was to investigate the potential correlation between lactate dehydrogenase A (LDHA) and AMP-activated protein kinase (AMPK) and their clinicopathologic significance in breast cancer.Materials and MethodsWestern blot and qRT-PCR were used to detect the expression levels of LDHA and AMPK in eight breast cancer lines and eight breast cancer tissues. In addition, LDHA and AMPK were detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs) of 112 patients. The association between LDHA and AMPK expression levels was statistically analyzed. So were the prognostic roles and clinicopathologic significances in breast cancer.ResultsThe expression levels of LDHA and AMPK were relatively higher in triple-negative breast cancer (TNBC) cell lines than in non-triple-negative breast cancer (NTNBC) cell lines. LDHA and AMPK were also further up-regulated in TNBC tissues than in NTNBC tissues. Correlation analysis showed a positive correlation between LDHA and AMPK expression levels. Expression of LDHA and AMPK were significantly correlated with TNM stage, distant metastasis, Ki67 status and survival outcomes of patients. Patients with both positive expression of LDHA and AMPK showed shorter overall survival (OS) and disease-free survival (DFS).ConclusionsThese findings improve our understanding of the expression pattern of LDHA and AMPK in breast cancer and clarify the role of LDHA and AMPK as promising prognostic biomarkers for breast cancer.     

17-β estradiol attenuates ovariectomy-induced changes in cardiomyocyte contractile function via activation of AMP-activated protein kinase

Menopause increases the risk of cardiometabolic diseases in women. This circumstance is usually attributed to a deficiency in circulating estrogen levels although the underlying mechanism remains elusive. Given the pivotal role of AMP-activated protein kinase (AMPK) in the regulation of energy metabolism and cardiac function, this study was designed to examine the role of AMPK in estrogen deficiency and replacement-exerted cardiomyocyte responses. Adult female WT and AMPK kinase dead (KD) mice were subjected to bilateral ovariectomy (OVX) or sham operation. A cohort of ovariectomized mice received 17β-estradiol (E2) (40 μg/kg/day, i.p.) for 6 weeks. Mechanical and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90%-relengthening (TR₉₀), and maximal velocity of shortening/relengthening (±dL/dt). Levels of AMPK, Akt JNK, ACC, SERCA, membrane Glut4, AS160 and PGC-1α were assessed using Western blot. OVX significantly decreased PS, ±dL/dt and intracellular Ca²⁺ rise in responsible to electric stimulus, prolonged TR₉₀ and intracellular Ca²⁺ decay without affecting TPS and resting intracellular Ca²⁺, the effects of which were reconciled by E2 replacement. Western blot analysis depicted that OVX suppressed phosphorylation of Akt AMPK and ACC although it promoted JNK phosphorylation, the effects of which were mitigated or significantly attenuated by E2 treatment in WT but not KD mice. Moreover, OVX procedure downregulated SERCA2a and membrane Glut4 while inhibiting AS160 phosphorylation without affecting PGC-1α levels. In vitro study revealed that E2 corrected cardiomyocyte contractile dysfunction elicited by OVX in cardiomyocytes from WT but not the AMPK kinase dead mice. Taken together, these data suggest that E2 treatment ameliorates estrogen deficiency-induced changes in cardiac contractile function possibly through an AMPK-dependent mechanism.