Trypsinization of chick glial cells before seeding: Effects on energy metabolism enzymes and glutamine synthetase

In order to test the possible involvement of surface proteins on some metabolical aspects of chick glial cell differentiation in culture, perturbations were induced on the glial cell surface membrane by limited trypsinization before seeding. The developmental changes of enzymes involved in the energy metabolism of the cell: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), hexokinase (HK), lactate dehydrogenase (LDH), enolase as well as glutamine synthetase (GS) were determined in trypsin treated cells and controls. The total protein and DNA content per dish was higher in treated cells than in controls, however the protein ratio towards DNA remained unchanged. The levels of GS, GDH, LDH, and enolase activities were significantly enhanced after trypsin treatment of the cells compared to controls. The enhanced value of total LDH activity is essentially the result of the increase of M subunit containing isoenzymes. Considering that a higher level of GS activity characterizes some maturation of the glial cells (as observed during the maturation of the chick brain) it is apparent that modifications of cell surface located factors, by trypsin treatment, induce differentiation phenomena at the functional state of the glial cells in culture. This may indicate that interactions located at the cell surface are involved in the modulation of key enzymes of the energy metabolism pathway.     

Glutamine synthetase and energy metabolism enzymes in cultured chick glial cells: Modulation by dibutyryl cyclic AMP,hydrocortisone, and trypsinization

Modifications induced by dibutyryl cyclic AMP (diBcAMP) and hydrocortisone in the energy metabolism of chick astroblasts in culture have been investigated. DiBcAMP does not modify the levels of enolase, malate dehydrogenase (MDH), total lactate dehydrogenase (LDH) and glutamine synthetase (GS) activities in these cultured glial cells. However, these cells can be sensitized to the nucleotide analog by trypsinization before seeding. The phenomenon affects specifically GS activity and the synthesis, with an inhibitory effect, of the H subunit of LDH. Addition of hydrocortisone to the culture medium stimulates MDH and GS activities of the cells; trypsinization accentuates the stimulatory effect on GS. This hormone also modifies the synthesis of H and M subunits of LDH in a positive and negative way respectively. The phenomenon is increased by trypsin treatment. The present studies indicate clearly that hydrocortisone generates in cultured chick glial cells metabolic modifications qualitatively different from those obtained by diBcAMP. It is suggested that trypsin treatment, by altering some protein constituents of the cell surface, modifies the adhesiveness of different cell types present in the cell suspension after dissociation of the brain and thus leads to select, in culture, a specific astroglial subpopulation.     

体外培养骨髓巨核细胞脱氢酶活性的细胞化学观察

本文采用液体培养体系结合酶细胞化学方法,对体外培养不同发育阶段的小鼠肾髓巨核细胞乳酸脱氢酶、苹果酸脱氢酶、谷氨酸脱氢酶的活性变化进行了动态观察。在9天培养期间,巨核细胞的增殖数在5—7天达到高峰,并随时间有不同程度分化。对培养3、5、7、9天的巨核细胞进行酶细胞化学研究,结果表明,巨核细胞在发育成熟前,三种酶活性均有增高。提示巨核细胞在分化过程中,糖酵解及三羧酸循环代谢均有增强。     

Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures

In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex. Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery. GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period. The magnitude of GS reduction by hypoxia depends on the age of the cells in culture. Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered. No modification of the MDH level was observed. The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia. Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue. Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.     

The effects of n-3 polyunsaturated fatty acids by gavage on some metabolic enzymes of rat liver

In this experimental study, the effect of fish n-3 fatty acids was studied on the some important enzymes of carbohydrate metabolism, hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) in rat liver. Wistar albino rats of experimental group (n= 9) were supplemented fish omega-3 fatty acids (n-3 PUFA) as 0.4 g/kg bw. by gavage for 30 days in addition to their normal diet. Isotonic solution was given to the control group (n= 8) by the same way. At 30th day, the rats were killed by decapitation under ether anesthesia, autopsied and liver was removed. Spectrophotometric methods were used to determine the activities of above-mentioned enzymes in the liver. The n-3 PUFA caused increases in the activities of HK, G6PD, LDH, and MDH in comparison with control. These increases were statistically significant (P < 0.01) except 6PGD activity. As a result, n-3 PUFA may regulate the metabolic function of liver effectively by increasing HK, G6PD, 6PGD, LDH, and MDH enzyme activities of rat liver when added in enough amounts to the regular diet.     

Postnatal development of the energy supplying enzyme pattern in the rat brain

The activity of seven enzymes connected with energy-supplying metabolism was followed from the second day of life till adulthood (87th day). The enzymes selected were: 1. Triosephosphate dehydrogenase (TPDH), 2. Lactate dehydrogenase (LDH), 3. Glycerol-3-phosphate: NAD dehydrogenase (GPDH), 4. Hexokinase (HK), 5. Malate: NAD dehydrogenase (MDH), 6. Citrate syntase (CS) and 7. 3-Hydroxyacyl Co A dehydrogenase. Although some variations occurred, the enzyme profiles were characteristic of those of the nervous tissue from the second day of life onwards until adulthood and displayed relatively high activities of HK, CS and MDH and low activities of TPDH, LDH, GPDH and HOADH. The activities of all enzymes studied here increased during postnatal development and some reached adult values on the 14th day, that of TPDH on the 27th day and HOADH on the 41st day of life. The activities of MDH and GPDH did not attain the adult values still on the 41st day of life. The anaerobic energy supply capacity seems to increase transiently on the 31st day of life, i.e. at a developmental stage where the resistance against hypoxia is known to increase transiently.     

Activity and Isoenzyme Pattern of Lactate Dehydrogenase in Neurons and Astroblasts Cultured from Brains of Chick Embryos

Primary cultures of neurons and glial cells (astroblasts) prepared from brains of 8-day-old and 15-day-old chick embryos, respectively, were grown for periods between 3 and 19 days. Specific activity of lactate dehydrogenase (LDH) increased in both types of cultures as a function of time and was always significantly higher in glial cells than in neurons. Glial cell extracts were found to contain predominantly the anaerobic isoenzymatic form of LDH (LDH-H4), and this pattern did not change over a period of 19 days. Cultured neurons contained predominantly the aerobic isoenzymatic form LDH-H4, and there was a progressive appearance of all other isoenzymes over an 8-day period. These results support the hypothesis of a different energy metabolism in neurons and glia.     

Ontogenetic development of energy-supplying enzymes in rat and guinea-pig heart

The purpose of the present study was to compare the ontogenetic development of the activity of myocardial energy-supplying enzymes in two mammalian species, differing significantly in their level of maturation at birth. The animals were investigated during the late prenatal period and 2, 7, 14, 21, 25, 30, 63, 120 and 730 days after birth in the rat and 2, 21, 84 and 175 days in the guinea-pig. The following enzymes were assayed in the right and left ventricular myocardium: lactate dehydrogenase (LDH, lactate uptake and/or formation), triose phosphate dehydrogenase (TPDH, carbohydrate metabolism), glycerol phosphate dehydrogenase (GPDH, glycerol-P shuttle)), hexokinase (HK, glucose phosphorylation), malate dehydrogenase (MDH, tricarboxylic cycle), citrate synthase (CS, tricarboxylic cycle) and hydroxyacyl-CoA dehydrogenase (HOADH, fatty acid breakdown). The rat heart, highly immature at birth, exhibits three different developmental patterns of energy-supplying enzymes, identical in both ventricles: (i) two mitochondrial enzymes of aerobic metabolism (CS, HOADH) and GPDH have a relatively low activity at the end of prenatal life; thereafter their activity steadily increases, approaching the adult levels between the 3rd and 4th postnatal weeks. A significant decrease was observed between the 4th and 24th months. (ii) MDH and LDH: prenatal values were significantly higher as compared with the 2nd postnatal day; after this period the activities increased up to adulthood (4 months) and decreased during senescence. (iii) The activities of HK and TPDH are characterized by only moderate changes during development. HK differs from all other enzymes by the highest prenatal values, which exceed even adult values. In contradiction to the rat heart, the developmental differences in more mature guinea-pig heart were significantly less pronounced. The only ontogenetic differences observed were the lower activities of enzymes connected with aerobic metabolism at the end of the prenatal period. Our results point to possible differences in the development of adaptive metabolic pathways in animals with different levels of maturation at birth.     

国人睾丸、附睾和输精管的氧化还原酶的组织化学观察

本文收集了１９—３８岁国人正常男性新鲜睾丸、附睾和输精管１３例,进行了氧化还原酶组织化学染色、光镜定位及定性观察。结果表明：睾丸曲细精管和输出小管上皮的ＧＤＨ,ＮＡＤＨＤ,ＮＡＤＰＨＤ,ＳＤＨ,ＧＰＤＨ,ＩＣＤＨ,ＭＤＨ,ＬＤＨ和Ｇ－６－ＰＤＨ９种酶;睾丸间质细胞和附睾管上皮的ＮＡＤＨＤ,ＮＡＤＰＨＤ,ＳＤＨ,ＩＣＤＨ,ＭＤＨ,ＧＤＨ,ＬＤＨ和Ｇ－６－ＰＤＨ８种酶;输精管的ＮＡＤＨＤ,ＮＡＤＰＨＤ,ＩＣＤＨ和ＧＤＨ４种酶的酶活性呈强阳性或极强阳性。提示输出小管和头部附睾管含有的多种氧化还原酶对精子功能成熟有极重要作用。     

Effect of Cortico-Striate Pathway Lesion on the Activities of Enzymes Involved in Synthesis and Metabolism of Amino Acid Neurotransmitters in the Striatum

The activities of several enzymes involved in the metabolism of aspartate and glutamate were measured in striatal (nucleus caudatus and putamen) homogenates 2-3, 6-7, and 35-40 days following frontoparietal and frontal cortical ablation. The activity of glutamine synthetase (GS) was substantially increased (46-48%) on the operated side 6-7 days following the lesion whereas smaller changes were observed at 2-3 and 35-40 days after lesion. In contrast, decreased levels of glutaminase and malate dehydrogenase (MDH) were observed by 6-7 days while no significant change was found at either 2-3 or 35-40 after the lesion. The activities of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD) were elevated after 35-40 days whereas no changes in the levels of either GDH or aspartate aminotransferase (ASAT) were found at 2-3 or 6-7 days after the fronto-parietal decortication. When only the frontal cortex was removed quantitatively similar changes were observed in striatal GS and glutaminase activity. The content of glutamate and glutamine in the denervated striatum followed qualitatively the changes in glutaminase and GS. The results indicate that the degeneration of cortico-striatal terminals causes a profound glial reaction in the striatum, and both glutaminase and MDH are present in relatively high concentrations in the corticostriatal terminals.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

HUNTINGTON''S CHOREA: POST MORTEM ACTIVITY OF ENZYMES INVOLVED IN CEREBRAL GLUCOSE METABOLISM

Abstract— The loss of at least two different cell types in the basal ganglia of the choreic brain led us to examine the activity of enzymes involved in the metabolism of glucose. Cellular ATPase, HK, G6-PDH, PFK., LDH, GDH were measured. Post mortem stability studies indicated that these enzymes were more unstable in human brain than mouse brain. The most stable enzyme was GDH. HK activity appeared to increase after freezing, suggesting release from another compartment. PFK and G6-PDH activity decreased by 70% over the usual time and temperature period for autopsy. In the autopsied brain tissue we were still able to measure significant activities that allowed us to determine the distribution of these enzymes and the similar post mortem handling of control and choreic brain allowed us to compare these two groups.
The activity of HK and G6-PDH was higher in the frontal cortex than in the basal ganglia. Ouabain insensitive ATPase and PFK were higher in the basal ganglia than the frontal cortex.
GDH activity, an enzyme that is very active in glial cells, was increased in the choreic globus pallidus, an area with a very high glial to neuronal cell ratio.
Although there was a wide variation in PFK activity that appeared to be related to the pre-mortem clinical state there was a significant decrease in PFK activity in the putamen of choreic post mortem brain when compared to controls.
These findings do not indicate an absolute defect in any of the enzymes studied in choreic brain but further studies might prove worthwhile.     

Development of glial cells in primary cultures: Energy metabolism and lactate dehydrogenase isoenzymes

Primary cultures of glial cells prepared from brains of newborn rats were grown for periods of 1–5 weeks. After a proliferative phase of between 2 and 3 weeks, the cultures were maintained in stationary phase, during which a significant increase of oxygen consumption and of the activities of lactate dehydrogenase, succinate dehydrogenase, and mitochondrial glycerolphosphate dehydrogenase could be observed. Furthermore, qualitative changes in the lactate dehydrogenase isoenzyme pattern were found with time, characterized by a shift toward an enhanced synthesis of H subunits. A similar development was found in comparing the LDH isoenzyme pattern in the brain of 15-day-old rat embryo with those of newborn and adult rat brains. It is suggested that some aspects of maturation of glial cells in culture are comparable to those occurring in whole brain in vivo, namely a shift towards an enhanced aerobic metabolism.     

OESTROGEN EFFECTS ON BRAIN AND PITUITARY ENZYME ACTIVITIES

Abstract— Ovariectomized female rats were treated daily with oestradiol-17β benzoate for intervals up to one week and enzyme activities were measured in the pituitary and various brain regions. Brain regions were selected for study on the basis of their previously demonstrated content of putative oestradiol receptor sites. (1) Pituitary showed oestrogen-dependent increases in glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and lactic dehydrogenase (LDH), and no change in NADP⁺-dependent isocitric dehydrogenase (ICDH), NADP⁺-dependent malic dehydrogenase (MDH) or hexokinase (HK). MDH and ICDH were elevated in whole hypothalamus. Enzyme activities did not change significantly in whole amygdala, cerebral cortex, or hippocampus. (2) Sub-regions of the preoptic area, hypothalamus and amygdala were dissected to obtain more highly concentrated populations of cells containing putative oestrogen receptor sites. In the basomedial sub-region of hypothalamus, activities of MDH, ICDH and G6PDH were elevated by oestrogen treatment. In the corticomedial sub-region of amygdala, MDH and ICDH were elevated by oestrogen treatment. No change was observed in any of the six enzymes in medial preoptic area. (3) Increases in enzyme activities were related to the total in vivo dose of oestradiol benzoate given. (4) Hypophysectomy or adrenalectomy did not prevent the enzymatic responses to oestrogen. (S) Oestrogen added directly to the enzyme incubation medium did not change enzyme activities. (6) Weight loss in ovariectomized rats due to reduced food intake did not increase enzyme activities. (7) In the pituitary, good correlation was obtained between the known receptor binding properties of various oestrogenic and non-oestrogenic steroids and the elevation in G6PDH activity. The results indicate that oestradiol acts directly to cause changes in activities of some brain and pituitary enzymes. The possibility is discussed that these changes may result from oestrogen interaction with putative receptor sites found in pituitary and certain brain regions.     

Carbohydrate and amino acid metabolism in fasting and aestivating African lungfish (Protopterus dolloi)

The potential importance of carbohydrates and amino acids as fuels during periods of fasting and aestivation in the African lungfish, Protopterus dolloi, were examined. No significant decreases in tissue glycogen levels were observed following 60 days of fasting or aestivation, suggesting lungfish may undergo 'glycogen sparing'. Yet glycogenolysis may be important during aestivation based on the differing responses of two flux-generating enzymes of the glycolytic pathway, hexokinase (HK) and pyruvate kinase (PK). PK is required for glycogen breakdown whereas HK is not. HK activity is significantly down-regulated in the heart and gill tissues during aestivation, while PK activity is sustained. The significant negative correlation between the activity of HK and glucose levels in the heart of aestivating lungfish suggests HK may be regulated by glucose concentrations. There was no indication of anaerobic glycolytic flux during aestivation as lactate did not accumulate in any of the tissues examined, and no significant induction of lactate dehydrogenase (LDH)activity was observed. The increase in glutamate dehydrogenase (GDH) and aspartate aminotransferase (Asp-AT) activities in the liver of aestivating P. dolloi suggests some energy may be obtained via increased aminoacid catabolism, leading to the generation of tricarboxylic acid (TCA) cycle intermediates. These findings indicate the importance of both carbohydrate and amino acid fuel stores during aestivation in aphylogenetically ancient, air-breathing fish.     

粘虫飞行过程中四种相关酶的活性变化

对3日龄粘虫雌蛾吊飞过程中4种相关酶3-羟酰辅酶A脱氢酶(HOAD)、3-磷酸甘油醛脱氢酶(GAPDH)、3-磷酸甘油脱氢酶(GDH)和乳酸脱氢酶(LDH)的研究结果表明,在室内条件下,粘虫在吊飞过程中其能量代谢有以下特点： 在吊飞的初始5 min,所有与糖代谢和脂肪代谢相关的酶活性都快速升高,这段时期脂肪代谢的酶活性也完全被活化,HOAD活性明显增强;但在随后的5～60 min持续吊飞期间与能量代谢有关的酶活性都有所下降,表明此时飞行活性趋于平稳。飞行中的粘虫具有极高的有氧代谢能力,也具备一定的无氧代谢能力。吊飞过程中HOAD∶GAPDH大于1,说明粘虫飞行过程中能源物质利用属于混合型,但动用脂肪比糖类要多。     

Malate Dehydrogenase Activity of Rat Testis: Circadian Rhythm and its Ontogeny

Malate dehydrogenase activity and soluble protein content in testes from rats exposed to a 14:00 h light:10:00 h dark photoperiod, have been determined every two or four hours over a 24 hour period in 5, 15, 25 and 120 day-old rats. By using the Cosinor method, the ontogeny of an unimodal rhythm was studied for MDH activity and soluble protein content in testis. In 5 and 15 day-old rats, the MDH acrophases were recorded around 19:00 h and 17:00 h, respectively. Rats aged 25 and 110 days showed the MDH acrophases during the dark period. An inversion of the MDH circadian rhythms was detected in 25 day-old compared to those of 5 and 15 day-old rats. An inversion of the protein circadian rhythm was also detected at 15 days compared to that at 5 days. These inversions persist in the adult rats. The amplitude of the MDH and protein rhythms reached the lowest value in adulthood. The mean daily value of testicular MDH increased between day 5 and 15, decreasing at day 35 and remaining unchanged until adulthood. The variation of malate dehydrogenase activity, soluble protein content levels, and the circadian rhythm parameters during the maturation process may be related to gonad development.     

Malate Dehydrogenase Activity of Rat Testis: Circadian Rhythm and its Ontogeny

Malate dehydrogenase activity and soluble protein content in testes from rats exposed to a 14:00 h light:10:00 h dark photoperiod, have been determined every two or four hours over a 24 hour period in 5, 15, 25 and 120 day-old rats. By using the Cosinor method, the ontogeny of an unimodal rhythm was studied for MDH activity and soluble protein content in testis. In 5 and 15 day-old rats, the MDH acrophases were recorded around 19:00 h and 17:00 h, respectively. Rats aged 25 and 110 days showed the MDH acrophases during the dark period. An inversion of the MDH circadian rhythms was detected in 25 day-old compared to those of 5 and 15 day-old rats. An inversion of the protein circadian rhythm was also detected at 15 days compared to that at 5 days. These inversions persist in the adult rats. The amplitude of the MDH and protein rhythms reached the lowest value in adulthood. The mean daily value of testicular MDH increased between day 5 and 15, decreasing at day 35 and remaining unchanged until adulthood. The variation of malate dehydrogenase activity, soluble protein content levels, and the circadian rhythm parameters during the maturation process may be related to gonad development.     

不同日龄和吊飞过程中桔小实蝇成虫飞行肌能量代谢相关酶活性的变化

【目的】明确桔小实蝇Bactrocera dorsalis(Hendel)飞行肌对能源物质的利用。【方法】通过生化方法测定了能源物质代谢相关5种酶[3-磷酸甘油醛脱氢酶(GAPDH)、3-磷酸甘油脱氢酶(GDH)、乳酸脱氢酶(LDH)、柠檬酸合酶(CS)和3-羟酰辅酶A脱氢酶(HOAD)]活性的变化。【结果】桔小实蝇成虫中所测的5种酶活性随日龄的变化而变化,4日龄GAPDH,GDH,LDH和CS活性最高,20日龄HOAD活性最高。吊飞过程中,GAPDH,GDH和CS的活性变化基本一致,随吊飞时间的延长活性逐渐升高;LDH和HOAD的活性变化雌、雄虫完全不同。雄虫LDH活性除吊飞2 h外其他时间均高于静息状态,雌虫则始终低于静息状态;雄虫HOAD活性只有吊飞24 h低于静息状态水平,而雌虫吊飞后HOAD活性一直在静息状态水平及以下波动。【结论】桔小实蝇飞行所利用的能源物质包括糖类和脂肪,以糖类能源为主。吊飞过程中,雄虫除可以进行高速有氧代谢以外,还具备一定的无氧代谢能力,而雌虫只进行有氧代谢;雄虫能利用脂肪供给能量,雌虫则几乎不动用脂肪。研究结果为进一步阐明桔小实蝇的迁飞行为机制提供了依据。     

社群等级对金乌贼行为表型及能量代谢的影响

采用实验生态学方法,在室内水槽条件下研究了金乌贼(Sepia esculenta Hoyle,1885)繁殖过程中社群等级的形成对其行为表型和能量代谢的影响,分析测定了不同优势等级雌雄个体腕部肌肉和性腺组织中己糖激酶(Hexokinase, HK)、丙酮酸激酶(Pyruvate kinase, PK)、乳酸脱氢酶(Lactate dehydrogenase, LDH)、苹果酸脱氢酶(Malate dehydrogenase, MDH)、柠檬酸合酶(Citrate synthetase, CS)活性以及乳酸(Lactic acid, LD)含量。结果显示:(1)金乌贼繁殖期不同优势等级雌雄个体之间行为表型具有显著差异,优势雄性个体游动悬浮、争斗时间显著高于劣势个体,而优势雌性个体静止伏底时间高于劣势个体,游动悬浮时间低于劣势雌性;(2)优势雄性个体在争斗过程中主要通过无氧代谢提供能量,而处于游动悬浮状态时通过有氧代谢提供能量。主要表现在优势雄性个体肌肉中无氧代谢酶(PK、HK、LDH)活性显著高于劣势个体(P<0.05),有氧代谢酶(MDH、CS)活性也显著高于劣势个体,雌性个体之...