Let-7b对人黑色素瘤细胞增殖及有氧糖酵解的影响

目的研究let-7b对人黑色素瘤细胞增殖及有氧糖酵解的影响。方法将人工合成的microRNA单核苷酸let-7bmimics及inhibitor通过脂质体转染人黑色素瘤细胞株A375,上调或抑制let-7b在细胞内的表达水平,不同时相点检测细胞葡萄糖消耗和乳酸产量,MTT法检测细胞增殖。运用单因素方差分析探讨let-7b表达水平与黑色素瘤细胞有氧糖酵解及增殖的关系。结果let-7bmimics组24、48h时相点检测MTr吸光度值分别为0．396±0．030、0．525±0．037,低于其他组（P〈0．05）,提示高表达let-7b抑制细胞增殖;各组黑色素瘤细胞24、48h葡萄糖消耗及乳酸产量差异无统计学意义（P〉0．05）,乳酸产量与葡萄糖消耗比值各组间差异无统计学意义（P〉0．05）,其中空白组在24h和48h内葡萄糖转化为乳酸效率约为57％和43％。结论黑色素瘤A375细胞具有显著的有氧糖酵解表型,过表达let-7b可抑制人恶性黑色素瘤细胞A375增殖,但对于细胞的有氧糖酵解效率,尚未发现let-7b对其有显著影响。     

A role of PKC in the improvement of energy metabolism in preconditioned heart

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function. Received: 29 January 1999, Returned for 1. revision: 26 February 1999, 1. Revision received: 27 April 1999, Returned for 2. revision: 18 May 1999, 2. Revision received: 12 July 1999, Returned for 3. revision: 26 July 1999, 3. Revision received: 25 October 1999, Accepted: 3 November 1999     

Glycolysis revisited

Summary Glycolysis is usually considered as a paradigm metabolic pathway, due to the fact that it is present in most organisms, and also because it is the pathway by which an important nutrient, glucose, is consumed. Far from being completely understood, the regulation of this pathway witnessed several important progresses during the last few years. One of these is the discovery of fructose 2,6-bisphosphate, a potent stimulator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase. Originally found in the liver during the course of a study on the mechanism by which glucagon acts on gluconeogenesis, this compound is now recognized as a major element in the control of glycolysis and/or gluconeogenesis in many cell types and in various organisms. The other finding is that of a regulatory protein that modulates the activity of glucokinase, the enzyme that phosphorylates glucose in the liver and in the beta cells of pancreatic islets.Presented as the Minkowski Lecture, EASD Meeting Prague, Czechoslovakia 1992     

治疗前18F-FDG PET/CT代谢参数对原发宫颈癌预后的预测价值

目的：评估不同百分比SUVmax阈值分段代谢参数预测经同步放化疗宫颈癌预后的价值。方法：45名宫颈癌患者经治疗前18F-FDG PET/CT检查,获得代谢参数SUVmax、MTV、TLG。利用不同百分比SUVmax阈值分割法分割MTVs、TLGs(SUVmax的阈值范围为10%-85%,以5%的递增量递增)。通过ROC曲线分析找出最佳cut-off值,Kaplan-Meier曲线进行生存分析。结果:本研究患者的中位随访时间为18.5月(3.0-39.0)。进展组与非进展组SUVmax、MTV10%、MTV75%-MTV85%、TLG75%-TLG85%差异无统计学意义(P＞0.05),MTV15%-MTV70%、TLG10%-MTV70%在进展组和非进展组间差异有统计学意义(P＜0.05)。对宫颈癌预后预测作用最强的SUVmax阈值为40%。ROC曲线分析,MTV40%的cut-off值为21.09,此时AUC最大(AUC=0.823),敏感性为78.60%,特异性为87.10%；TLG40%的cut-off值为136.71,此时AUC最大(AUC=0.788),敏感性为78.60% ,特异性为77.40% 。结论：治疗前18F-FDG PET/CT代谢参数MTV40%、TLG40%可用于预测经同步放化疗治疗的原发宫颈癌预后。     

Heterogeneity of glycolysis in cancers and therapeutic opportunities

Upregulated glycolysis, both in normoxic and hypoxic environments, is a nearly universal trait of cancer cells. The enormous difference in glucose metabolism offers a target for therapeutic intervention with a potentially low toxicity profile. The past decade has seen a steep rise in the development and clinical assessment of small molecules that target glycolysis. The enzymes in glycolysis have a highly heterogeneous nature that allows for the different bioenergetic, biosynthetic, and signaling demands needed for various tissue functions. In cancers, these properties enable them to respond to the variable requirements of cell survival, proliferation and adaptation to nutrient availability. Heterogeneity in glycolysis occurs through the expression of different isoforms, posttranslational modifications that affect the kinetic and regulatory properties of the enzyme. In this review, we will explore this vast heterogeneity of glycolysis and discuss how this information might be exploited to better target glucose metabolism and offer possibilities for biomarker development.     

Loss of ATP13A2 impairs glycolytic function in Kufor-Rakeb syndrome patient-derived cell models

BackgroundKufor-Rakeb syndrome (KRS) is an autosomal recessive, juvenile-onset Parkinson's disease (PD) caused by loss-of-function mutations in ATP13A2 (PARK9). Impaired energy metabolism is considered a pathogenic mechanism in PD and mitochondrial dysfunction resulting from Zn²⁺ dyshomeostasis has been found in KRS patient-derived cells. In addition to mitochondrial energy production, glycolysis plays an important role in cellular energy metabolism and glucose hypometabolism has been reported in PD. However, glycolytic status in KRS remains undetermined despite its potential importance.MethodsWe assessed glycolytic function in ATP13A2-deficient KRS patient-derived human olfactory neurosphere cells and fibroblasts and determined the effect of pyruvate supplementation on improving cellular energy production.ResultsWe found impaired extracellular acidification, reduction in pyruvate production and a decrease in the NAD⁺/NADH ratio, indicative of glycolytic dysfunction. In addition, gene expression analysis revealed an altered expression profile for several glycolytic enzymes. Glycolytic dysfunction was aggravated when the intracellular Zn²⁺ concentration was increased, while ATP13A2 overexpression and pyruvate supplementation blocked the observed Zn²⁺-mediated toxicity. Moreover, supplementation with pyruvate significantly increased basal mitochondrial ATP production and abolished Zn²⁺-induced cell death.ConclusionsThese findings indicate that glycolytic dysfunction contributes to pathogenesis and pyruvate supplementation improves overall cellular bioenergetics in our KRS patient-derived cell model, highlighting a therapeutic potential.     

Lidocaine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxemia in mice by suppressing HIF1α-induced glycolysis

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infections. Previous studies have indicated that lidocaine, an amide local anesthetic, has anti-inflammatory properties; however, the underlying mechanism remains unclear. In this study, we have shown that lidocaine dose-dependently inhibits lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in macrophages and that lidocaine protects mice from LPS-induced inflammation. Moreover, we have demonstrated that lidocaine reduces the release of TNF-α and IL-6 through the reduction of the expression of GLUT1 and HK2 to further suppress HIF1α-induced aggravation of inflammatory cascades. Lidocaine can inhibit the enhanced glycolysis and glycolytic capacity induced by LPS in the macrophages. As an inhibitor of PHDs (prolyl hydroxylases), Dimethyloxalylglycine (DMOG) can reduce the anti-inflammatory effects of lidocaine. In conclusion, the present study indicates that lidocaine can be used as a potential therapeutic agent for sepsis.     

The mTOR-glycolytic pathway promotes T-cell immunobiology in oral lichen planus

Oral lichen planus (OLP) is a T-cell-mediated inflammatory mucosal disease. T cells require rapid metabolic reprogramming for their effector functions after activation by immunologic stimuli. The mammalian target of rapamycin (mTOR) is a central player in the metabolic reprogramming and immune responses of T cells. The present study investigated the role of mTOR in the immunometabolism of OLP. mTOR and its direct target eukaryotic initiation factor 4E binding protein 1 (4E-BP1) were highly phosphorylated in peripheral T cells of OLP patients. Rapamycin-mediated blockage of mTOR activation restrained both T-cell proliferation and DNA synthesis, promoted apoptosis, and decreased Th1/Th2 and Th17/Treg ratios. Dual blockage of mTOR and phosphatidylinositol 3-kinase (PI3K) exerted stronger inhibition on T-cell immunobiology than selective repression of PI3K alone. Rapamycin also blocked the glycolytic pathway in T cells. Moreover, glucose-induced activation of mTOR-glycolytic pathway increased T-cell proliferation, DNA synthesis, and the Th17/Treg ratio, and decreased T-cell apoptosis. In contrast, inhibition of glycolysis by 2-Deoxy-d-glucose (2-DG) yielded the opposite effects on T-cell immunobiology by blocking the mTOR pathway. In conclusion, enhanced activation of the mTOR-glycolytic pathway promoted T-cell immunobiology, suggesting that dysregulation of immunometabolism might be associated with T-cell dysfunction in OLP.     

Pyruvate restores contractile function and antioxidant defenses of hydrogen peroxide-challenged myocardium

PURPOSE: Pyruvate, a natural energy-yielding fuel in myocardium, neutralizes peroxides by a direct decarboxylation reaction, and indirectly augments the glutathione (GSH) antioxidant system by generating NADPH reducing power via citrate formation. The possibility that pyruvate's antioxidant actions could mediate its enhancement of contractile performance in prooxidant-challenged myocardium was investigated in isolated working guinea-pig hearts reversibly injured by hydrogen peroxide. METHODS: Hearts were challenged by 10 min perfusion with 100 microM H(2)O(2), followed by 90 min H(2)O(2)-free perfusion. Metabolic and antioxidant treatments (each 5m M) were administered at 30-90 min post-H(2)O(2). Phosphocreatine phosphorylation state, GSH/glutathione disulfide redox potential (GSH/GSSG) and key enzyme activities were measured in snap-frozen myocardium. RESULTS: H(2)O(2) exposure depleted myocardial energy and antioxidant reserves and produced marked contractile impairment that persisted throughout the H(2)O(2) washout period. Relative to untreated post-H(2)O(2) myocardium, pyruvate restored contractile performance, increased GSH/GSSG 52% and maintained phosphocreatine phosphorylation state; in contrast, lactate lowered cardiac performance and phosphorylation state. Neither the pharmacological antioxidant N -acetylcysteine (NAC) nor the pyruvate analog alpha-ketobutyrate increased cardiac function; both treatments increased GSH/GSSG but lowered phosphocreatine potential. H(2)O(2) partially inactivated aconitase, creatine kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), but all three enzymes spontaneously recovered during H(2)O(2) washout. Pyruvate did not further activate these enzymes and unexpectedly inhibited GAPDH by 60-70%. CONCLUSION: Pyruvate promoted robust contractile recovery of H(2)O(2)-challenged myocardium by the combination of citrate-mediated antioxidant mechanisms and maintenance of myocardial energy reserves.