Overexpression of Protein Phosphatase 2 Regulatory Subunit B"Alpha Promotes Glycolysis by Regulating Hexokinase 1 in Hepatocellular Carcinoma / 生物医学与环境科学（英文）

Objective@#To investigate the regulatory relationship of Protein Phosphatase 2 Regulatory Subunit B"Alpha ( PPP2R3A) and hexokinase 1 ( HK1) in glycolysis of hepatocellular carcinoma (HCC).@*Methods@#In HepG2 and Huh7 cells, PPP2R3A expression was silenced by small interfering RNA (siRNA) and overexpression by plasmid transfection. The PPP2R3A-related genes were searched by RNA sequencing. Glycolysis levels were measured by glucose uptake and lactate production. QRT-PCR, ELISA, western blot and immunofluorescence assay were performed to detect the changes of PPP2R3A and HK1. Cell proliferation, migration and invasion assay were used to study the roles of HK1 regulation by PPP2R3A.@*Results@#RNA sequencing data revealed that PPP2R3A siRNA significantly downregulated the expression of HK1. PPP2R3A gene overexpression promotes, while gene silencing suppresses, the level of HK1 and glycolysis in HCC cells. In HCC tissue samples, PPP2R3A and HK1 were colocalized in the cytoplasm, and their expression showed a positive correlation. HK1 inhibition abrogated the promotion of glycolysis, proliferation, migration and invasion by PPP2R3A overexpression in liver cancer cells.@*Conclusion@#Our findings showed the correlation of PPP2R3A and HK1 in the glycolysis of HCC, which reveals a new mechanism for the oncogenic roles of PPP2R3A in cancer.

Suppression of miR-181 a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

Pharmacological evaluation of hyperin for antihyperglycemic activity and effect on lipid profile in diabetic rats.

Antihyperglycemic potential of hyperin at 25 and 50 mg/kg doses for 30 days to streptozotocin induced diabetic rats has been reported. In oral glucose tolerance test, hyperin treated rats showed a significant reduction in blood glucose level after 120 min. It was found that hyperin exhibited dose dependent and significant antihyperglycemic activity in streptozotocin induced diabetic rats which were nearly similar with standard drug glybenclamide. Activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, glycogen phosphorylase, glycosylated haemoglobin and level of serum urea and creatinine were significantly decreased in hyperin supplemented diabetic rats, dose dependently. Activities of hexokinase and glycogen synthase were increased with augmentation in liver glycogen, insulin and haemoglobin content in hyperin treated diabetic rats. General hematological parameters did not show any significant change in hyperin treated diabetic rats hence it is safe at these doses. Histopathological studies showed significant morphological changes in pancreatic β-cells of streptozotocin induced diabetic rats. A decreased number of secretory granules of β- cells were observed in diabetic rats and these pathological abnormalities were normalized after treatment with hyperin and standard drug glybenclamide. Further, hyperin decreases significant in serum total cholesterol, triglyceride, low density lipoprotein, very low density lipoprotein levels coupled with elevation of high density lipoprotein in diabetic rats. These results suggest that hyperin has a pivotal role in blood glucose level in streptozotocin induced hyperglycemia by improving the function of pancreatic islets and increasing glycolysis and decreasing gluconeogenesis.

Trueness Assessment for Serum Glucose Measurement Using Commercial Systems through the Preparation of Commutable Reference Materials

BACKGROUND: Commutable reference materials (RMs) are suitable for end-users for evaluating the metrological traceability of values obtained using routine measurement systems. We assessed the performance of 6 routine measurement systems with validated secondary RMs. METHODS: We tested the homogeneity, stability, and commutability of 5 minimally processed human serum pools according to the standard guidelines. The serum pools were assigned values as per the reference procedure of the United States Centers for Disease Control and were used to evaluate the trueness of results from 6 commercial measurement systems based on enzymatic methods: 3 glucose oxidase (GOD) and 3 hexokinase (HK) methods. RESULTS: The prepared RMs were validated to be sufficiently homogenous, stable, and commutable with the patient samples. Method bias varied for different systems: GOD01, -0.17 to 2.88%; GOD02, 1.66 to 4.58%; GOD03, -0.17 to 3.14%; HK01, -3.48 to -0.85%; HK02, -3.83 to -0.11%, and HK03, -1.82 to -0.27%. CONCLUSIONS: We observed that the prepared serum glucose RMs were qualified for trueness assessment. Most of the measurement systems met the minimal quality specifications.

Measuring b-Galactosidase activity at pH 6 with a differential pH sensor

The b-Galactosidase activity at pH 6 is used as a cellular marker to identify senescent cell cultures. The classic method to identify this enzymatic activity is using cytochemical staining with X-Gal after 16 hrs. In this work, a differential pH sensor was used to measure b-Galactosidase activity at pH 6. The measurement is easy and only takes 3 min.

Tratamiento y post-tratamiento con lonidamina en la línea celular de carcinoma colónico humano HT-29 / Treatment and post-treatment with lonidamine in human colon carcinoma HT-29 cell line

Lonidamina (1-[ 2,4-diclorofenil metil]-1H indazol-3-ácido carboxílico), (lnd), es una droga antineoplásica cuyo mecanismo de acción se ejerce sobre el metabolismo intermedio de la glucosa. Los efectos de la lnd sobre el crecimiento celular y el metabolismo celular se investigaron en las células HT- 29, línea celular de carcinoma colónico humano, que requiere altas concentraciones de glucosa para su crecimiento indiferenciado en cultivo. La lnd en dosis de 0.2 mM disminuyó significativamente el crecimiento celular y la formación de colonias en agar; con la interrupción del tratamiento se observó el restablecimiento del crecimiento celular en 24 horas. El tratamiento con lnd produce la redistribución de los glicoconjugados y el receptor de la manosa, sin afectar en forma drástica la síntesis de glucógeno ni la de proteínas. Estas posiblemente sean las causas de la rápida reversibilidad del tratamiento.

Lonidamine (1-[ 2,4-dichlorophenyl methyl]-1H indazole-3-carboxylic acid), lnd, is an antitumoral drug acting on mitochondria and glucose metabolism. Cell growth and metabolic effects of lnd and drug post-treatment effect were investigated in undifferentiated HT-29 human colonic carcinoma cell line which requires high glucose medium concentration for growth. 0.2 mM lnd significantly decreased cell spreading and growth in monolayer or agar cell culture. After drug treatment cell growth was reestablished to control value within 24 h. Ind modified glycoconjugates and mannose-receptor distribution (analyzed by confocal microscopy), while glucose-glycogen and protein synthesis were not affected, these being the possible reasons for the fast reversible effect.

Modulatory effect of Gynandropsis gynandra L. on glucose metabolizing enzymes in aflatoxin B1-induced hepatocellular carcinoma in rats.

The modulation of glucose-metabolizing enzymes activities play a vital role in the depletion of energy metabolism and leads to inhibition of cancer growth. In the present study, the effect of Gynandropsis gynandra L. extract on aflatoxin B1 (AFB1)-induced hepatocellular carcinoma (HCC) was studied on glucose-metabolizing enzymes in rats. A significant increase (p < 0.001) in the activities of the key glycolytic enzymes viz., hexokinase and phosphoglucoisomerase, with a significant decrease (p < 0.001) in the gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase were observed in HCC-bearing rats, when compared with the control. Administration of G. gynandra extract caused a significant decrease in the activities of glycolytic enzymes and an increase in the gluconeogenic enzymes activities to near normal values. Thus, findings suggest the G. gynandra extract has a definite modulating role on the key enzymes of glucose metabolism in HCC. The modulatory effect may be due to the phytoactive constituents present in the extract of G. gynandra.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 æmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 æmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18 percent, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Metabolic dysregulation and inhibition of spermatogenesis by gibberellic acid in rat testicular cells.

Enzymatic and histological change in the testicular cells of rats treated orally and intradermally for 45 days with gibberellic acid (GBA) in independent studies is reported. Assay of hexokinase (HK), acid phosphatase (AcP) and alkaline phosphatase (AkP) in rat testicular tissue homogenate preparations yielded results that suggested changes in these enzyme activities relative to their respective controls. Histological studies showed loss of germ cells, derangement of the germinal cells, and reduction in the size of the seminiferous tubules and dystrophy of Leydig cells. More importantly decreased sperm count in the lumen was observed. A dysregulatory role is thus established for GBA in rat testicular cell function. This compound may serve as an inhibitor of testicular cell function.

Comparative evaluation of hexokinase isoenzyme typing and antigen detection for differentiation of Entamoeba histolytica and Entamoeba dispar.

Entamoeba histolytica, the causative organism of invasive amebiasis is a potential pathogen, while asymptomatic infection is caused by E. dispar. Differentiation of the species is not possible on the basis of morphological characters by microscopic examination. In the present study an attempt has been made to differentiate E. histolytica from E. dispar in 45 isolates obtained from culture and direct stool samples respectively on the basis of hexokinase isoenzyme analysis and Tech Lab ELISA. A 100% correlation was found between these two techniques. However, Tech Lab E. histolytica antigen detection test was found to be both rapid and technically simple. Its use in diagnosis and epidemiological studies is recommended.

Biochemical studies on hypoglycemic effect of Aavirai kudineer: a herbal formulation in alloxan diabetic rats.

Aavirai Kudineer (AK) is an herbal decoction of seven botanical drugs, cited in the Gunapadam; a Tamil Siddha medical text. The anti-diabetic efficacy of this formulation was evaluated using alloxan-induced diabetic and normal rats. Glucose tolerance was observed within 1 hr in AK-treated rats (10 ml/kg body ) as compared to control. A significant decrease in the severe hyperglycemia characteristic of alloxan diabetes was noted after 15 days of AK treatment. Further AK treatment reversed the elevated urea, creatinine, cholesterol and decreased protein values to near normal levels. Assay of glycogen content and chief carbohydrate-metabolizing enzymes, viz. hexokinase, glucose-6-phosphatase and fructose 1,6 diphosphatase in the liver of diabetic and AK-treated diabetic rats clearly ascertains the hypoglycemic efficacy of this formulation. The mode of action of this herbal formulation remains to be elucidated.

Estudio comparativo de dos equipos de glicemia capilar / Comparative study of 2 devices for capilary blood glucose measurement

En el presente estudio se probaron los equipos Super glucocard II y b-Glucose analyzer para glicemia capilar en una población adulta (n=70) y se compararon los resultados con el método tradicional del laboratorio (hexoquinasa realizado en el equipo Hitachi 917tm). Se evaluó la precisión y linearidad, los efectos del hematocrito y del ácido ascórbico. Se utilizaron los métodos estadísticos de regresión lineal simple, la prueba t de Student con una significancia de p<0,05 y la comparación de métodos por Bland-altman. Los resultados fueron evaluados bajo los criterios de la National Committee for Clinical Laboratory standards (NCCLS) para equipos de glicemia capilar. Los resultados demostraron una correlación de 0.952 y 0,975 respectivamente, encontrándose diferencias significativas entre glicemias de los equipos y del método de referencias (p<0,01). Ningún valor mayor de 100 mg/dl estuvo fuera del rango de concordancia aceptado para glicemias capilares, según los criterios de la NCCLS. El coeficiente de variación fue menor de 6 por ciento en los dos equipos, para distintos niveles de glicemia. Ambos presentaron exelente linearidad entre 50 y 400 mg/dl y se demostró que el nivel de hematocrito puede influir en las determinaciones. El ácido ascórbico demostró ser un interferente en ambos equipos

Effect of protein malnutrition on the glycolytic and glutaminolytic enzyme activity of rat thymus and mesenteric lymph nodes

The activity of important glycolytic enzymes (hexokinase, phosphofructokinase, aldolase, phosphohexoseisomerase, pyruvate kinase and lactate dehydrogenase) and glutaminolytic enzymes (phosphate-dependent glutaminase) was determined in the thymus and mesenteric lymph nodes of wistar rats submited to protein malnutrition (6 percent protein in the diet rather than 20 percent) from conception to 12 weeks after birth. The wet weight (g) of the thymus and mesenteric lymph nodes decreased due to protein malnutrition by 87 percent (from 0.30 + 0.05 to 0.04 + 0.01) and 75 percent (0.40 + 0.04 to 0.10 + 0.02), respectively. The protein content was reduced only in the thymus from 102.3 + 4.4 (control rats) to 72.6 + 6.6 (malnourished rats). The glycolytic enzymes were not affected by protein malnutrition, but the glutaminase activity of the thymus and lymph nodes was reduced by halfing in protein-malnourished rats as compared to controls. This fact may lead to a decrease in the cellularity of the organ and thus in its size, weight and protein content.

Activation of hexokinase by mild insulin dependent diabetes mellitus in rat erythrocytes.

In order to reveal sequel of events responsible for increase in red cell cytosolic glucose-6 phosphate (G-6P) content of diabetic patients the enzyme producing and transforming G-6P were assayed. Increase in the activity of hexokinase and decrease in phosphoglucoisomerase activity was observed in mild insulin dependent diabetes mellitus (mIDDM) rat erythrocytes. Increase and decrease in activity of hexokinase and phosphoglucoisomerase respectively will increase the cytosolic glucose-6 phosphate content. Thus any substance which autoregulate the activity of hexokinase and maintains critical level of G-6P necessary for generation of ATP and coenzymes (NADPH & NAD+) in the prevailing hyperglycemic state can be a potential therapeutic agent for diabetic patients.

detection of hepatic glucose phosphorylation enzymes in two strains of mice and their relation to thioacetamide effect on the liver

Hexokinase isoenzymes in the liver of two inbred strains of mice [C3H/He and C58] have been determined initially. Four isoenzymes catalyzing phosphorylation of glucose and designated GK, HKI, HKII and HKIII, were resolved by high performance liquid chromatography on DEAE-cellulose columns of liver extracts from C3H/He and C58 mice. The enzymes were identified by their activity and in a series of immunological tests including immunoelectrophoresis and immunoprecipitation. In untreated mice, the glucokinase [GK] activity was predominant and the three hexokinases [HKI, HKII and HKIII] detected were characterized by low activities. However, intraperitoneal injection of thiocetamide resulted in a significant decrease in the glucokinase activity and in an increase in hexokinases activity, especially that of hexokinases II and III. While confirming the presence of hexokinase III in mice livers, the present work also provides for a significant improvement in the detection of hexokinase III in mice and presents an interesting observation on the effect of thiocetamide on the liver

Activation of yeast trehalase by heat shock

1. Activation of Saccharomyces cerevisiae trehalase by heat shock was shown in all strains tested, including mutants in which the reponse to a glucose signal was absent. A low concentration of cAMP favored the response as seen in 2nd log cells or in ras2 and cyr1ts mutant strains. The heat shock effect upon trehalse activity was not observed under conditions of catabolite repession. 2 Neither hexokinase PII nor the heat shock protein hsp26 seemed to be involve in the axtivation of trehalase by heat shock. However, mutant strains deleted in the polyubiquitin gene showed only a 2-fold activation of the enzyme while in control strains a 5-to 7-fold irreversible activation was observed. 3. An alternative mechanism of trehalase activation by removal of an inhibitor through ligation with ubiquitin is discussed. Activation by cAMP-independent phosphorylation is also considered