藏羚羊与藏绵羊心肌、骨骼肌中肌红蛋白含量和乳酸脱氢酶活性的比较

为了探讨藏羚羊(Pantholops hodgsonii)对低氧环境的适应机制。以生活在同海拔(4 300 m)的藏绵羊(Tibetan Sheep)为对照,用分光光度法测定2种动物心肌、骨骼肌中肌红蛋白(myoglobin,Mb)含量、乳酸(lactic acid,LD)含量及乳酸脱氢酶(lactate dehydrogenase,LDH)活力。结果显示,藏羚羊心肌和骨骼肌中Mb含量明显高于藏绵羊(P0.05),但心肌和骨骼肌的Mb含量无差别(P0.05),而藏绵羊心肌Mb含量明显高于骨骼肌(P0.05);藏羚羊心肌和骨骼肌中LD含量及LDH活力明显低于藏绵羊(P0.05),且2种动物心肌中的LDH活力均明显低于其骨骼肌(P0.01)。结果表明,藏羚羊尽管生活在高寒缺氧地区,其心肌和骨骼肌细胞仍然能得到丰富的氧供应,并非处于缺氧状态,这可能是通过增加心肌和骨骼肌中Mb的含量,提高其在低氧环境获取和储存氧的能力,从而提高有氧获能水平。与之相反,藏绵羊尽管也生活在高寒缺氧地区,但其心肌和骨骼肌中Mb含量相对于藏羚羊较低,且LD含量和LDH活力较高,说明其心肌和骨骼肌细胞内氧供不如藏羚羊丰富,提示藏绵羊可能主要以糖酵解获能。我们推测这种差异可能与两种动物不同的运动习性密切相关,且认为藏羚羊较高的Mb含量可能是其适应高原缺氧条件的分子基础之一。     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.     

Effect of Dichloroacetate on Regional Energy Metabolites and Pyruvate Dehydrogenase Activity During Ischemia and Reperfusion in Gerbil Brain

The objective of this study was to determine whether administration of dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), improves recovery of energy metabolites following transient cerebral ischemia. Gerbils were pretreated with DCA, and cerebral ischemia was produced using bilateral carotid artery occlusion for 20 min, followed by reperfusion up to 4 h. DCA had no effect on the accumulation of lactic acid and the decrease in ATP and phosphocreatine (PCr) during the 20-min insult, nor on the recovery of these metabolites measured at 20 and 60 min reperfusion. However, at 4 h reperfusion, levels of ATP and PCr were significantly higher in DCA-treated animals than in controls, as PCr exhibited a secondary decrease in caudate nucleus of control animals. PDH was markedly inhibited at 20 min reperfusion in both groups, but was reactivated to a greater extent in DCA-treated animals at 60 min and 4 h reperfusion. These results demonstrate that DCA had no effect on the initial recovery of metabolites following transient ischemia. However, later in reperfusion, DCA enhanced the postischemic reactivation of PDH and prevented the secondary failure of energy metabolism in caudate nucleus. Thus, inhibition of PDH may limit the recovery of energy metabolism following cerebral ischemia.     

鱼类乳酸脱氢酶同工酶聚丙烯酰胺凝胶电泳技术研究

本文对用聚丙烯酰胺凝胶电泳（ＰＡＧＥ）分离鱼类乳酸脱氢酶（ＬＤＨ）同工酶的技术进行了研究。结果表明：该方法优于淀粉凝胶电泳及琼脂糖凝胶电泳分析鱼类ＬＤＨ同工酶,具有较高的分辩率。     

Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues

Abstract: We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.     

中国林蛙乳酸脱氢酶多基因系统及基因间连锁关系的研究

（１）用聚丙烯酰胺凝胶电泳对山西产中国林蛙４个地理群的３３３只林蛙进行了分析,结果表明：中国林蛙的ＬＤＨ由ＬＤＨ－Ａ,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ３个基因决定。ＬＤＨ－Ａ是单态座位,ＬＤＨ－Ｂ和ＬＤＨ－Ｃ均为多态座位,每个多态座位均有两个等位基因。ＬＤＨ－Ｂ与ＬＤＨ－Ｃ呈紧密连锁关系。认为ＬＤＨ－Ｃ是ＬＤＨ－Ｂ的重复产物。（２）热稳定性、尿素处理稳定性及组织特异性研究表明：ＬＤＨ对温度和尿素处理稳定性顺序为Ａ４＞Ｂ’４＞Ｂ４＞Ｃ４。Ａ４在骨骼肌和肝脏等组织中活力最大,Ｂ４在心肌和卵巢中活力最强,ＬＤＨ－Ｃ主要在眼球和卵巢中表达。（３）Ｌｄｈ－ｂ和Ｌｄｈ－ｂ＇在不同地理群间呈差异分布,随着纬度的增高,Ｌｄｈ－ｂ在种群中的频率增大。     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Activity of Lactate Dehydrogenase in Serum and Cerebral Cortex of Immature and Mature Rats after Hypobaric Hypoxia

In our previous studies we have found both an increase of lipid peroxidation damage (expressed as levels of thiobarbituric acid-reactive substances) in brain and plasma lactate concentration in 21-day-old rats after a 30-min exposure to hypobaric hypoxia. Pretreatment of rats with l-carnitine decreased both parameters. The aim of our present study was to determine if the l-carnitine-dependent decrease of plasma lactate could be due to a modification of lactate dehydrogenase (LDH) activity. We followed brain and blood serum LDH activity of 14-, 21- and 90-day-old Wistar rats. We found an increase of brain LDH activity with age. However, we did not observe any significant differences in LDH activity after exposure to hypobaric hypoxia or l-carnitine pretreatment. In contrast to brain, serum LDH activity did not show any clear age-dependence. The hypoxia exposure increased LDH activity of 21-day-old rats only. Pretreatment of rats with l-carnitine decreased serum LDH activity of 21- and 90-day-old rats probably due to membrane stabilizing role of l-carnitine. In conclusions, acute hypobaric hypoxia and/or l-carnitine pretreatment modified serum but not brain LDH activity.     

急性淋巴细胞白血病患儿血清25羟维生素D3、乳酸脱氢酶、白细胞介素-6水平与危险度分层和预后不良的关系研究

摘要 目的：探讨急性淋巴细胞白血病（ALL）患儿血清25羟维生素D3（25-OH-D3）、乳酸脱氢酶（LDH）、白细胞介素-6（IL-6）水平与危险度分层和预后不良的关系。方法：选择2018年6月至2019年8月在我院住院治疗的ALL患儿60例为病例组,另选取同期到我院健康查体的同龄健康儿童60例为对照组。采用酶联免疫吸附试验（ELISA）检测血清25-OH-D3和IL-6水平,采用全自动生化分析仪检测血清LDH水平,对比病例组与对照组血清25-OH-D3、LDH、IL-6水平,对比不同危险度分层ALL患儿血清25-OH-D3、LDH、IL-6水平,分析其与危险度分层的相关性。ALL患儿进行规范化治疗,根据治疗结果分为预后良好组和预后不良组,对比预后良好组和预后不良组临床特征及血清25-OH-D3、LDH、IL-6水平,采用COX比例风险回归模型分析预后不良的危险因素。结果：病例组患儿血清25-OH-D3水平低于对照组,血清LDH、IL-6水平高于对照组（P＜0.05）。低危型、中危型ALL患儿血清25-OH-D3水平高于高危型,且低危型高于中危型（P＜0.05）;低危型、中危型ALL患儿血清LDH、IL-6水平低于高危型,且低危型低于中危型（P＜0.05）。预后不良组高危型患儿及白细胞计数＞100×10⁹/L的患儿所占比例、血清LDH及IL-6水平高于预后良好组（P＜0.05）,血清25-OH-D3水平低于预后良好组（P＜0.05）。血清25-OH-D3水平与ALL患儿危险度分层呈负相关（P＜0.05）,血清LDH、IL-6水平与ALL患儿危险度分层呈正相关（P＜0.05）。危险度分层及血清25-OH-D3、LDH、IL-6是ALL患儿预后不良的危险因素（P＜0.05）。结论：ALL患儿血清25-OH-D3、LDH、IL-6水平异常改变,与危险度分层相关,是预后不良的危险因素。     

腹腔注射腺病毒pMultiRNAi-Ldhc对高原鼠兔骨骼肌Ldhc基因的沉默效果

腹腔注射是一种简单且方便给药的方式,为了验证腹腔注射腺病毒pMultiRNAi-Ldhc对高原鼠兔(Ochotonacurzoniac)骨骼肌Ldhc基因沉默的可行性,将27只高原鼠兔分为干扰组、空壳组和空白对照组,每组各9只个体,干扰组和空壳组分别注射0.65ml腺病毒pMultiRNAi-Ldhc和腺病毒pMultiRNAi-NS,空白对照组注射等量生理盐水,注射后7d检测骨骼肌中Ldhc基因mRNA和蛋白的表达水平,测定了乳酸脱氢酶(LDH)活性和乳酸(LD)及三磷酸腺苷(ATP)的含量。结果表明,与空白对照组相比,干扰组在mRNA和蛋白水平上,Ldhc基因表达分别降低了41.73%和15.76%;乳酸脱氢酶(LDH)活性、乳酸(LD)和ATP含量分别降低了23.98%、51.08%和19.29%。结果说明,腹腔注射腺病毒pMultiRNAi-Ldhc能有效沉默骨骼肌中Ldhc基因表达。     

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-¹⁴C]lactate, [1-¹⁴C]glucose, or [6-¹⁴C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with V_max = 360 μmol/g protein/h and K_m = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO₂. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO₂ and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.     

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD^−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD^−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.     

A Temporary Local Energy Pool Coupled to Neuronal Activity: Fluctuations of Extracellular Lactate Levels in Rat Brain Monitored with Rapid-Response Enzyme-Based Sensor

Abstract: A successfully developed enzyme-based lactate microsensor with rapid response time allows the direct and continuous in vivo measurement of lactic acid concentration with high temporal resolution in brain extracellular fluid. The fluctuations coupled to neuronal activity in extracellular lactate concentration were explored in the dentate gyrus of the hippocampus of the rat brain after electrical stimulation of the perforant pathway. Extracellular glucose and oxygen levels were also detected simultaneously by coimplantation of a fast-response glucose sensor and an oxygen electrode, to provide novel information of trafficking of energy substances in real time related to local neuronal activity. The results first give a comprehensive picture of complementary energy supply and use of lactate and glucose in the intact brain tissue. In response to acute neuronal activation, the brain tissue shifts immediately to significant energy supply by lactate. A local temporary fuel "reservoir" is established behind the blood-brain barrier, evidenced by increased extracellular lactate concentration. The pool can be depleted rapidly, up to 28% in 10–12 s, by massive, acute neuronal use after stimulation and can be replenished in ∼20 s. Glutamate-stimulated astrocytic glycolysis and the increase of regional blood flow may regulate the lactate concentration of the pool in different time scales to maintain local energy homeostasis.     

高温胁迫对烟草叶绿体NADPH脱氢酶复合体活性的促进

为探讨叶绿体NAD(P)H脱氢酶复合体(NDH)在植物抵御热胁迫中的生理意义,比较了烟草ndhJK基因缺失突变体(ΔndhJK)和野生型对50℃高温胁迫的响应.高温下,野生型中一条NBT-NADPH氧化还原酶活性带有所增加,免疫印迹分析确定了此活性染色带是NDH亚复合体,该活性带中的NDH-K表达量也在热胁迫条件下明显地增加.与ΔndhJK相比,在高温胁迫下,野生型中远红光诱导的P700 氧化速率明显地变慢,而远红光关闭后的P700暗还原速率则显著地变快,表明高温促进NDH介导的围绕光系统I的循环电子传递.根据这些结果推测,在热胁迫条件下野生型中对NADPH底物专一的NDH活性的增加可能有利于减少NADPH的积累,减轻叶绿体间质的过度还原.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

G6PD缺陷通过Caspase-3和PARP-1诱导人黑色素瘤细胞凋亡

葡糖-6-磷酸脱氢酶(G6PD)在许多肿瘤细胞中高表达,但其发生的作用机理目前仍然不明确.以正常人表皮黑色素细胞(HEM)、野生型人黑色素瘤A375细胞(A375-WT)和G6PD缺陷的A375细胞(A375-G6PDΔ)为对象,经real-time PCR、Western印迹和紫外分光光度法分析显示,A375-WT细胞的mRNA、G6PD蛋白和G6PD活性分别是HEM细胞的1.89倍(P0.05)、6.86倍(P0.01)和2.30倍(P0.05).Annexin V/PI流式细胞仪和Western印迹测定表明,A375-G6PDΔ的凋亡率是A375-WT的5.10倍(P0.01),活化半胱氨酸蛋白酶3(caspase-3)增高1.84倍(P0.01)以及89 kD多聚二磷酸腺苷核糖聚合酶-1(PARP-1)生成增加2.87倍(P0.01).分光光度法分析显示,A375-G6PDΔ的NADPH和GSH分别降低了72.30%(P0.01)和27.39%(P0.05),并伴有75.43%的H2O2增高(P0.01).结果提示,G6PD在黑色素瘤细胞中高表达和高活性,而敲减G6PD表达通过caspase-3和PARP-1信号诱发人黑色素瘤细胞凋亡,这为深入揭示黑色素瘤的发生机理提供了新思路。     

NMDA Receptor Involvement in Toxicity to Dopamine Neurons In Vitro Caused by the Succinate Dehydrogenase Inhibitor 3-Nitropropionic Acid

Abstract: Exposure of mesencephalic dopamine neurons to an irreversible inhibitor of succinate dehydrogenase (SDH), 3-nitropropionic acid (3-NPA), for 24 h on day 12 in vitro, produced a dose-dependent loss of high-affinity dopamine uptake when measured 48 h following 3-NPA removal. ATP concentrations in the cultures were reduced by 57% after 3 h of treatment with the highest concentration of 3-NPA tested (500 µ M ). To determine whether glutamate receptors mediated the dopamine toxicity by 3-NPA, cultures were examined for their sensitivity to excitatory amino acid-induced toxicity. Mesencephalic cultures exposed to either 100 µ M NMDA or kainate, on day 12 for 24 h, showed complete loss of dopamine uptake following 48 h of recovery. The NMDA and non-NMDA antagonists, MK-801 (1 µ M ) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 15 µ M ), completely prevented the effects of NMDA or kainate, respectively, when present at the time of toxin exposure. In cultures treated with 3-NPA, MK-801, but not CNQX, significantly attenuated the loss of dopamine uptake. Direct measurement of the effect of 3-NPA on SDH activity showed that 3-NPA dose-dependently inhibited SDH in vitro in a manner commensurate with the loss of dopamine uptake by 3-NPA. MK-801 had no effect on basal SDH activity or on 3-NPA inhibition of SDH. These data are consistent with the interpretation that metabolic inhibition in dopamine neurons can trigger a secondary excitotoxicity that is mediated by NMDA receptors.     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

Inhibition of Glutamate Dehydrogenase in Brain Mitochondria and Synaptosomes by Mg2+ and Polyamines: A Possible Cause for Its Low In Vivo Activity

Abstract: Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 m M and NADH levels less than 0.08 m M . The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (>2.0 m M ) and NADH (>0.32 m M ). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.     

Lactate Contributes to Glyceroneogenesis and Glyconeogenesis in Skeletal Muscle by Reversal of Pyruvate Kinase

Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase, whereas another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle, but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol, and lactate under a fed or fasted state. Only one of the three substrates was ¹³C-labeled in each experiment. After 3 h of normal awake activity, the animals were sacrificed, and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of ¹³C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in the skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected.