p53-regulated autophagy is controlled by glycolysis and determines cell fate

The tumor suppressor p53 regulates downstream targets that determine cell fate. Canonical p53 functions include inducing apoptosis, growth arrest, and senescence. Non-canonical p53 functions include its ability to promote or inhibit autophagy and its ability to regulate metabolism. The extent to which autophagy and/or metabolic regulation determines cell fate by p53 is unclear. To address this, we compared cells resistant or sensitive to apoptosis by the p53 activator Nutlin-3a. In resistant cells, glycolysis was maintained upon Nutlin-3a treatment, and activated p53 promoted prosurvival autophagy. In contrast, in apoptosis sensitive cells activated p53 increased superoxide levels and inhibited glycolysis through repression of glycolytic pathway genes. Glycolysis inhibition and increased superoxide inhibited autophagy by repressing ATG genes essential for autophagic vesicle maturation. Inhibiting glycolysis increased superoxide and blocked autophagy in apoptosis-resistant cells, causing p62-dependent caspase-8 activation. Finally, treatment with 2-DG or the autophagy inhibitors chloroquine or bafilomycin A1 sensitized resistant cells to Nutlin-3a-induced apoptosis. Together, these findings reveal novel links between glycolysis and autophagy that determine apoptosis-sensitivity in response to p53. Specifically, the findings indicate 1) that glycolysis plays an essential role in autophagy by limiting superoxide levels and maintaining expression of ATG genes required for autophagic vesicle maturation, 2) that p53 can promote or inhibit autophagy depending on the status of glycolysis, and 3) that inhibiting protective autophagy can expand the breadth of cells susceptible to Nutlin-3a induced apoptosis.     

Application of mitochondrial pyruvate carrier blocker UK5099 creates metabolic reprogram and greater stem-like properties in LnCap prostate cancer cells in vitro

Aerobic glycolysis is one of the important hallmarks of cancer cells and eukaryotic cells. In this study, we have investigated the relationship between blocking mitochondrial pyruvate carrier (MPC) with UK5099 and the metabolic alteration as well as stemness phenotype of prostatic cancer cells. It was found that blocking pyruvate transportation into mitochondrial attenuated mitochondrial oxidative phosphorylation (OXPHOS) and increased glycolysis. The UK5099 treated cells showed significantly higher proportion of side population (SP) fraction and expressed higher levels of stemness markers Oct3/4 and Nanog. Chemosensitivity examinations revealed that the UK5099 treated cells became more resistant to chemotherapy compared to the non-treated cells. These results demonstrate probably an intimate connection between metabolic reprogram and stem-like phenotype of LnCap cells in vitro. We propose that MPC blocker (UK5099) application may be an ideal model for Warburg effect studies, since it attenuates mitochondrial OXPHOS and increases aerobic glycolysis, a phenomenon typically reflected in the Warburg effect. We conclude that impaired mitochondrial OXPHOS and upregulated glycolysis are related with stem-like phenotype shift in prostatic cancer cells.     

“膀胱癌湿热瘀毒蕴积病因病机与其有氧糖酵解代谢异常相关”假说的提出

传统中医认为,在肿瘤的发生发展过程中"虚、痰、瘀、毒"是其主要病因病机,而正气虚弱是疾病发生的基础,《灵枢·水胀》中也记载"恶气乃起,息肉乃生"。因此,"虚、痰、瘀、毒"和机体相互作用产生"恶气",从而导致肿瘤。作者前期对膀胱癌的研究中,发现肿瘤细胞有氧糖酵解代谢异常过程可提供其持续生长的物质(核酸、磷脂、氨基酸、乳酸等)和能量(ATP),并且和细胞生长环境中的H+、HCO3-、O2等密切作用。结合中医肿瘤学"虚、痰、瘀、毒"的辨证认识和膀胱的脏腑特性,本文提出"膀胱癌湿热瘀毒蕴积病因病机与其有氧糖酵解代谢异常相关"的假说。     

EDRF Does Not Mediate Coronary Vasodilation Secondary to Simulated Ischemia: A Study on KATP Channels and Nω-nitro-L-arginine on Coronary Perfusion Pressure in Isolated Langendorff-Perfused Guinea-Pig Hearts

Several authors have alluded to the possible involvement of EDRF (NO) in ischemia-induced coronary artery dilation. Alternatively, it has been suggested that opening of ATP-dependent K channels could play a key role in this context. We studied the effects of sulfonylureas and N^G-nitro-L-arginine (LNNA), a specific inhibitor of endothelial NO (EDRF) synthesis, on ischemia-induced coronary vasodilation in isolated Langendorff-perfused guinea pig hearts arrested with 15 mM KCl in normal Tyrode, and isolated pig coronary arteries precontracted with 43 mM KCl. In Isolated Langerdorff-perfused guinea pig heart, when hypoxia was simulated by switching 100% O₂ in the perfusate to 100% N₂, coronary perfusion pressure (CPP) fell from 90 cm H₂O by 45 ± 5 cm H₂O. In the presence of LNNA, a specific inhibitor of NO synthetase in endothelial cells, CPP dropped by 44 ± 6 cm H₂O (n = 6; ± SEM, no statistically significant). On biochemical simulation of ischemia (addition of iodoacetate [IAA]), CPP dropped 40 ± 6 cm H₂O, and in experiments performed under the same conditions but in the presence of LNNA, CPP dropped by 38 ± 5 cm H₂O (n = 6; ± SEM; not statistically significant). When ischemia was simulated metabolically by equimolar replacement of 10 mM glucose with 2-deoxyglucose (DOG), an inhibitor of glycolysis CPP decreased by 24 ± 1 cm H₂O (n = 6; ± SEM) after 15 minutes. This fall in CPP was almost prevented by 20 M glibenclamide, whereas in the presence of 20 M LNNA the DOG-induced decrease in CPP was not significantly inhibited, and CPP decreased by 22 ± 2.6 cm H₂O (n = 6; ± SEM). In isolated pig coronary artery rings, maximal tension, achieved by depolarizing the smooth muscle cells by 43 mM KCl, decreased by 37 ± 7% upon simulated hypoxia by replacing 100% O₂ with 100% N₂ in the perfusate (n = 6; ± SEM) in arteries with intact endothelium. In arteries without endothelium, maximal tension also dropped by 35 ± 6% (not statistically significant). In the same experiments the decrease in tension could be largerly inhibited in the presence of 50 M glibenclamide. Our results clearly show that in isolated perfused guinea pig hearts, as well as in isolated pig coronary arteries, EDRF does not play a decisive role in the coronary dilatory response to hypoxia and ischemia.     

Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility

Although glycolysis is highly conserved, it is remarkable that several unique isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like its human ortholog (GAPD2), is the sole GAPDH isozyme in sperm. It is tightly bound to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. We disrupted Gapds expression by gene targeting to selectively block sperm glycolysis and assess its relative importance for in vivo sperm function. Gapds(-/-) males were infertile and had profound defects in sperm motility, exhibiting sluggish movement without forward progression. Although mitochondrial oxygen consumption was unchanged, sperm from Gapds(-/-) mice had ATP levels that were only 10.4% of those in sperm from WT mice. These results imply that most of the energy required for sperm motility is generated by glycolysis rather than oxidative phosphorylation. Furthermore, the critical role of glycolysis in sperm and its dependence on this sperm-specific enzyme suggest that GAPDS is a potential contraceptive target, and that mutations or environmental agents that disrupt its activity could lead to male infertility.     

The onset of liver glycogen synthesis in fasted-refed lean and genetically obese (fa/fa) rats

Summary Lean and genetically obese (fa/fa) rats were fed ad libitum, or fasted for 17 h and then meal-fed for varying time intervals. During refeeding, glucose-6-phosphatase activity of lean rats declined to the low value that was present in livers of fasted obese rats and which remained unchanged in the obese group during the meal. Refeeding also resulted in increases in hepatic concentrations of glucose-6-phosphate and fractose-6-phosphate, fructose 1,6-bisphosphate, fractose-2,6-bisphosphate, -glycerophosphate, pyruvate and lactate in lean and obese rats, absolute values being higher in the fasted obese than in the fasted lean group. Obese animals had higher postprandial portal blood insulin, glucose and lactate concentrations than lean animals. In spite of this, the rate of hepatic glycogen deposition was the same in both groups and was accompanied by similar glycogen synthase a levels. Following refeeding, phosphorylase was transiently inactivated in livers of lean but not of obese animals, while glycogen synthase was inactivated in both groups. The data suggest that (1) in lean animals refeeding was associated with a stimulation of liver glycolysis, presumably by insulin; (2) in fasted obese rats hepatic glycolysis was already in a stimulated state and was only slightly enhanced further after the meal, in keeping with their unaltered hyperinsulinaemia; (3) there was an increased turnover of liver glycogen or a resistance to insulin stimulation of glycogen synthesis in fa/fa rats during refeeding.     

Chromium-containing traditional Chinese medicine,Tianmai Xiaoke Tablet improves blood glucose through activating insulin-signaling pathway and inhibiting PTPIB and PCK2 in diabetic rats

OBJECTIVE： Chromium is an essential mineral that is thought to be necessary for normal glucose homeostasis. Numerous studies give evidence that chromium picolinate can modulate blood glucose and insulin resistance. The main ingredient of-13anmai Xiaoke （TMXK） Tablet is chromium picolinate. In China, TMXK Tablet is used to treat type 2 diabetes. This study investigated the effect of TMXK on glucose metabolism in diabetic rats to explore possible underlying molecular mechanisms for its action. METHODS： Diabetes was induced in rats by feeding a high-fat diet and subcutaneously injection with a single dose of streptozotocin （50 mg/kg, tail vein）. One week after streptozotocin-injection, model rats were divided into diabetic group, low dose of TMXK group and high dose of TMXK group. Eight normal rats were used as normal control. After 8 weeks of treatment, skeletal muscle was obtained and was analyzed using Roche NimbleGen mRNA array and quantitative polymerase chain reaction （qPCR）. Fasting blood glucose, oral glucose tolerance test and homeostasis model assessment of insulin resistance （HOMA-IR） index were also measured. RESULTS： The authors found that the administration of TMXK Tablet can reduce the fasting blood glucose and fasting insulin level and HOMA-IR index. The authors also found that 2 223 genes from skeletal muscle of the high-dose TMXK group had significant changes in expression （1 752 increased, 471 decreased）. Based on Kyoto encyclopedia of genes and genomes pathway analysis, the most three significant pathways were ＂insulin signaling pathway＂, ＂glycolysis/ gluconeogenesis＂ and ＂citrate cycle （-ICA）＂. qPCR showed that relative levels of forkhead box 03 （Fox03）, phosphoenolpyruvate carboxykinase 2 （Pck2）, and protein tyrosine phosphatase 1B （Ptplb） were significantly decreased in the high-dose TMXK group, while v-akt murine thymoma viral oncogene homolog 1 （Aktl） and insulin receptor substrate 2 （Its2） were increased. CONCLUSION： Our data show that TMXK Tablet reduces fasting glucose level and improves insulin resistance in diabetic rats. The mechanism may be linked to the inactivation of PTP1B and PCK enzymes, or through intracellular pathways, such as the insulin signaling pathway.     

MATERNAL LEUKOCYTE ENZYMES IN HUMAN FETAL MALNUTRITION

The impaired activity of maternal leukocyte pyruvate kinase and its response to allosteric modulators may be a cause and an indicator of fetal malnutrition.     

Effects of inhibition of glycolysis on calcium homeostasis and functional recovery of stunned myocardium

ＥｆｆｅｃｔｓｏｆｉｎｈｉｂｉｔｉｏｎｏｆｇｌｙｃｏｌｙｓｉｓｏｎｃａｌｃｉｕｍｈｏｍｅｏｓｔａｓｉｓａｎｄｆｕｎｃｔｉｏｎａｌｒｅｃｏｖｅｒｙｏｆｓｔｕｎｎｅｄｍｙｏｃａｒｄｉｕｍＧｕｏＷｅｎｙｉ（郭文怡）；ＯｕＪｉｎｘｉ（区晋禧）；ＪｉａＧｕｏｌ...     

Thiamine corrects delayed replication and decreases production of lactate and advanced glycation end-products in bovine retinal and human umbilical vein endothelial cells cultured under high glucose conditions

Summary This study aimed at verifying whether thiamine, a co-enzyme which decreases intracellular glycolysis metabolites by allowing pyruvate and glyceraldheyde 3-phosphate to enter the Krebs cycle and the pentose-phosphate shunt, respectively, corrects delayed replication caused by high glucose concentrations in cultured human umbilical vein (HUVEC) and bovine retinal endothelial cells (BREC). After incubation in physiological (5.6 mmol/l) or high (28.0 mmol/l) glucose with or without 150 μmol/l thiamine, cells were counted and proliferation assessed by mitochondrial dehydrogenase activity. Lactate was measured in both cell types as an index of glycolytic activity and fluorescent advanced glycosylation end-products (AGE) concentration was determined in the HUVEC lysate. Both cell counts and proliferation assays in either of the cell types confirmed the impairment to cell replication induced by high glucose. When thiamine was added to cells kept under high glucose conditions, the number of surviving cells was significantly increased and the reduced cell proliferation appeared to be corrected. Lactate assays confirmed the increased production of this metabolite by BREC and HUVEC in high glucose, which was reduced by thiamine. Fluorescent AGE determination showed that thiamine may prevent non-enzymatic glycation in HUVEC. Thiamine restores cell replication, decreases the glycolytic flux and prevents fluorescent AGE formation in endothelial cells cultured in high glucose, suggesting that abnormal levels of glycolytic metabolite(s) may damage cells. [Diabetologia (1996) 39: 1263–1268] Received: 18 March 1996 and in revised form: 12 June 1996