OBJECTIVES: Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. METHODS: The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. RESULTS: Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). CONCLUSIONS p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.
Animals ; Humans ; Mice ; Carboxymethylcellulose Sodium/pharmacology* ; Diabetes Mellitus, Type 2/metabolism* ; Endothelium, Vascular/metabolism* ; Glucose/metabolism* ; Human Umbilical Vein Endothelial Cells ; Mice, Inbred C57BL ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Phosphorylation ; Pyruvate Kinase/metabolism* ; Vasodilation

A series of experiments was performed to determine the lactic dehydrogenase activity of various parasitic helminths. The enzyme activity was determined by the modified method of Wroblewshi and LaDue (1955) using tissue homogenate of 16 kinds of worm parasites. The worms were mostly collected alive from local abattoir and removed from the organ or tissues of the naturally infected animal host and some materials were also obtained from the human host. They were thoroughly washed and homogenized in chilled glass tissue grinder, and then centrifuged. The supernatants were designated as enzyme preparations, and their enzyme activity was measured by spectrophotometry at the wave length of 340 millimicron. In order to know the effects of temperature and substrate concentration on the enzyme activity, the extinction of reduced Coenzyme I(NADH) was measured at the various conditions of incubation temperature and substrate concentration. The results of this experiments were as follows: The lactic dehydrogenase activity occurred over all kinds of parasites used in this study. Most worms of nematodes and trematodes displayed their maximum activity in the range of pH 2.7-3.5, and cestodes revealed their maximum activity in the ranges of both pH 2.7-3.5 and pH 7.4. In nematodes and trematodes, the lactic dehydrogenase activity increased slowly as incubation temperature increases except in the case of Eurytrema pancreaticum, while the activity in cestodes decreased inversely. The lactic dehydrogenase activity increased in proportion to the increase of substrate concentration in most of worm parasites.
parasitology-nematode-trematoda-helminth ; lacticdehydrogenase ; nicotinamide dinucloetide ; sodium pyruvate ; Ascaris lumbricoides ; Ascaridia galli ; Dirofilaria immitis ; Fasciola hepatica ; Eurytrema pancreaticum ; Paramphistomum sp. ; Clonorchis sinensis ; Paragonimus westermani ; Taenia saginata ; Taenia solium ; Taenia pisiformis ; Dipylidium caninum ; Diphyllobothrium mansoni ; sparganum, Cysticercus cellulosae ; Cysticercus fasciolaris ; biochemistry- enzyme