Mitochondrial activation and the pyruvate paradox in a human cell line

Pyruvate promotes hyperpolarization of the inner mitochondrial membrane. However, in isolated mitochondria, pyruvate could participate in a futile cycle leading to mitochondrial depolarization. Here, we investigated this paradox in intact human cells by measuring parameters reflecting mitochondrial activation in response to 1 mM pyruvate and 5 mM glucose. NAD(P)H levels were elevated similarly by both substrates. Conversely, pyruvate induced a first transient phase of mitochondrial depolarization before the establishment of the expected sustained hyperpolarization. This correlated with kinetics of cytosolic ATP levels exhibiting a first phase decrease followed by an increase. Therefore, pyruvate transiently depolarizes mitochondria and reduces ATP in intact cells.     

Brain metabolism of exogenous pyruvate

Pyruvate given in large doses may be neuroprotective in stroke, but it is not known to what degree the brain metabolizes pyruvate. Intravenous injection of [3-13C]pyruvate led to dose-dependent labelling of cerebral metabolites so that at 5 min after injection of 18 mmoles [3-13C]pyruvate/kg (2 g sodium pyruvate/kg), approximately 20% of brain glutamate and GABA were labelled, as could be detected by 13C nuclear magnetic resonance spectrometry ex vivo. Pyruvate, 9 mmoles/kg, was equivalent to glucose, 9 mmoles/kg, as a substrate for cerebral tricarboxylic acid (TCA) cycle activity. Inhibition of the glial TCA cycle with fluoroacetate did not affect formation of [4-13C]glutamate or [2-13C]GABA from [3-13C]pyruvate, but reduced formation of [4-13C]glutamine by 50%, indicating predominantly neuronal metabolism of exogenous pyruvate. Extensive formation of [3-13C]lactate from [2-13C]pyruvate demonstrated reversible carboxylation of pyruvate to malate and equilibration with fumarate, presumably in neurones, but anaplerotic formation of TCA cycle intermediates from exogenous pyruvate could not be detected. Too rapid injection of large amounts of pyruvate led to seizure activity, respiratory arrest and death. We conclude that exogenous pyruvate is an excellent energy substrate for neurones in vivo, but that care must be taken to avoid the seizure-inducing effect of pyruvate given in large doses.     

Myocardial postischemic injury is reduced by polyADPripose polymerase-1 gene disruption

BACKGROUND: PolyADPribose polymerase (PARP) is activated by DNA strand breaks to catalyze the addition of ADPribose groups to nuclear proteins, especially PARP-1. Excessive polyADPribosylation leads to cell death through depletion of NAD+ and ATP. MATERIALS AND METHODS: In vivo PARP activation in heart tissue slices was assayed through conversion of [33P]NAD+ into polyADPribose (PAR) following ischemia-reperfusion (I/R) and also monitored by immunohistochemical staining for PAR. Cardiac contractility, nitric oxide (NO), reactive oxygen species (ROS), NAD+ and ATP levels were examined in wild type (WT) and in PARP-1 gene-deleted (PARP-1(-/-)) isolated, perfused mouse hearts. Myocardial infarct size was assessed following coronary artery occlusion in rats treated with PARP inhibitors. RESULTS: Ischemia-reperfusion (I/R) augmented formation of nitric oxide, oxygen free radicals and PARP activity. I/R induced decreases in cardiac contractility and NAD+ levels were attenuated in PARP-1(-/-) mouse hearts. PARP inhibitors reduced myocardial infarct size in rats. Residual polyADPribosylation in PARP-1(-/-) hearts may reflect alternative forms of PARP. CONCLUSIONS: PolyADPribosylation from PARP-1 and other sources of enzymatic PAR synthesis is associated with cardiac damage following myocardial ischemia. PARP inhibitors may have therapeutic utility in myocardial disease.     

The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.     

Pyruvate improves redox status and decreases indicators of hepatic apoptosis during hemorrhagic shock in swine

Previous studies have shown that the liver is the first organ to display signs of injury during hemorrhagic shock. We examined the mechanism by which pyruvate can prevent liver damage during hemorrhagic shock in swine anesthetized with halothane. Thirty minutes after the induction of a 240-min controlled arterial hemorrhage targeted at 40 mmHg, hypertonic sodium pyruvate (0.5 g. kg(-1). h(-1)) was infused to achieve an arterial concentration of 5 mM. The volume and osmolality effects of pyruvate were matched with 10% saline (HTS) and 0.9% saline (NS). Although the peak hemorrhage volume increased significantly in both the pyruvate and HTS group, only the pyruvate treatment was effective in delaying cardiovascular decompensation. In addition, pyruvate effectively maintained the NADH/NAD redox state, as evidenced by increased microdialysate pyruvate levels and a significantly lower lactate-to-pyruvate ratio. Pyruvate also prevented the loss of intracellular antioxidants (GSH) and a reduction in the GSH-to-GSSG ratio. These beneficial effects on the redox environment decreased hepatic cellular death by apoptosis. Pyruvate significantly increased the ratio of Bcl-Xl (antiapoptotic molecule)/Bax (proapoptotic molecule), prevented the release of cytochrome c from mitochondria, and decreased the fragmentation of caspase 3 and poly(ADP ribose) polymerase (DNA repair enzyme). These beneficial findings indicate that pyruvate infused 30 min after the onset of severe hemorrhagic shock is effective in maintaining the redox environment, preventing the loss of the key antioxidant GSH, and decreasing early apoptosis indicators.     

不同剂量链脲佐菌素腹腔注射制备大鼠糖尿病心肌病模型的病理学观察

建立糖尿病性心肌病（DCM）大鼠模型,观察不同剂量链脲佐菌素（STZ）单次腹腔注射后大鼠心肌和胰腺的病理学变化。用STZ 50 mg/kg、55 mg/kg、60 mg/kg 3种剂量单次腹腔注射,制备糖尿病大鼠模型;以柠檬酸三钠-柠檬酸缓冲液腹腔注射,作为对照。72 h后,测空腹血糖及做口服葡萄糖耐量实验（OGTT）;3周后,HE染色观察各组大鼠胰腺和心肌形态学变化,Masson三色染色观察心肌纤维化改变。OGTT和空腹血糖显示3组存活大鼠糖尿病均成模;3周末,50 mg/kg和55 mg/kg剂量死亡率为25%;60 mg/kg剂量高,达到75%;HE染色显示55 mg/kg剂量组大鼠胰岛明显萎缩,轮廓不清晰,胰岛细胞数量少,心肌细胞肥大、排列紊乱,细胞间隙增大,并有炎症细胞浸润;50 mg/kg组胰岛和心肌也有变化,但无55 mg/kg组明显。心肌Masson染色显示55 mg/kg组心肌内胶原组织明显增多,排列紊乱,分布不均。55 mg/kg剂量的STZ单次注射大鼠腹腔,造模3周可以建立较明显的DCM模型,可为DCM的组织病理学和实验研究提供一个较好的动物模型。     

Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.     

Pyruvate-fortified cardioplegia suppresses oxidative stress and enhances phosphorylation potential of arrested myocardium

Cardioplegic arrest for bypass surgery imposes global ischemia on the myocardium, which generates oxyradicals and depletes myocardial high-energy phosphates. The glycolytic metabolite pyruvate, but not its reduced congener lactate, increases phosphorylation potential and detoxifies oxyradicals in ischemic and postischemic myocardium. This study tested the hypothesis that pyruvate mitigates oxidative stress and preserves the energy state in cardioplegically arrested myocardium. In situ swine hearts were arrested for 60 min with a 4:1 mixture of blood and crystalloid cardioplegia solution containing 188 mM glucose alone (control) or with additional 23.8 mM lactate or 23.8 mM pyruvate and then reperfused for 3 min with cardioplegia-free blood. Glutathione (GSH), glutathione disulfide (GSSG), and energy metabolites [phosphocreatine (PCr), creatine (Cr), P(i)] were measured in myocardium, which was snap frozen at 45 min arrest and 3 min reperfusion to determine antioxidant GSH redox state (GSH/GSSG) and PCr phosphorylation potential {[PCr]/([Cr][P(i)])}. Coronary sinus 8-isoprostane indexed oxidative stress. Pyruvate cardioplegia lowered 8-isoprostane release approximately 40% during arrest versus control and lactate cardioplegia. Lactate and pyruvate cardioplegia dampened (P < 0.05 vs. control) the surge of 8-isoprostane release following reperfusion. Pyruvate doubled GSH/GSSG versus lactate cardioplegia during arrest, but GSH/GSSG fell in all three groups after reperfusion. Myocardial [PCr]/([Cr][P(i)]) was maintained in all three groups during arrest. Pyruvate cardioplegia doubled [PCr]/([Cr][P(i)]) versus control and lactate cardioplegia after reperfusion. Pyruvate cardioplegia mitigates oxidative stress during cardioplegic arrest and enhances myocardial energy state on reperfusion.     

Zinc inhibits astrocyte glutamate uptake by activation of poly(ADP-ribose) polymerase-1

Several processes by which astrocytes protect neurons during ischemia are now well established. However, less is known about how neurons themselves may influence these processes. Neurons release zinc (Zn2+) from presynaptic terminals during ischemia, seizure, head trauma, and hypoglycemia, and modulate postsynaptic neuronal function. Peak extracellular zinc may reach concentrations as high as 400 microM. Excessive levels of free, ionic zinc can initiate DNA damage and the subsequent activation of poly(ADP-ribose) polymerase 1 (PARP-1), which in turn lead to NAD+ and ATP depletion when DNA damage is extensive. In this study, cultured cortical astrocytes were used to explore the effects of zinc on astrocyte glutamate uptake, an energy-dependent process that is critical for neuron survival. Astrocytes incubated with 100 or 400 microM of zinc for 30 min showed significant decreases in ATP levels and glutamate uptake capacity. These changes were prevented by the PARP inhibitors benzamide or DPQ (3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone) or PARP-1 gene deletion (PARP-1 KO). These findings suggest that release of Zn2+ from neurons during brain insults could induce PARP-1 activation in astrocytes, leading to impaired glutamate uptake and exacerbation of neuronal injury.     

Protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism

Pyruvate, an endogenous metabolite of glycolysis, is an anti-toxicity agent. Recent studies have suggested possible roles for pyruvate in protecting CNS neurons from excitotoxic and metabolic insults. Utilizing cultures derived from embryonic rat cortex, the studies presented in this paper indicate that an astroglia-mediated mechanism is involved in the neuroprotective effects of pyruvate against glutamate toxicity. Glutamate-induced toxicity could be reversed by pyruvate in a mixed culture of cortex cells. Importantly, in pure neuronal cultures from the same tissue, pyruvate failed to protect against glutamate toxicity. Addition of astroglia to the pure neuronal cultures restores the ability of pyruvate to protect neurons from glutamate-induced toxicity. Our results further suggest that pyruvate can induce glia to up-regulate the synthesis of glutathione (GSH), an antioxidant that protects cells from toxins such as free radicals. Taken together, our data suggest that astroglia in mixed cultures are essential for mediating the effects of pyruvate, revealing a novel mechanism by which pyruvate, an important intermediate of tricarboxylic acid cycle in the body, may act to protect neurons from damage during insults such as brain ischemia.     

Dual regulation of microglia and neurons by Astragaloside IV‐mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury

Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS‐IV), in vitro and in vivo. Our results showed that AS‐IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS‐IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS‐IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS‐IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS‐IV may become a novel therapeutic medicine for SCI.     

Severe Hypoglycemia in a Juvenile Diabetic Rat Model: Presence and Severity of Seizures Are Associated with Mortality

It is well accepted that insulin-induced hypoglycemia can result in seizures. However, the effects of the seizures, as well as possible treatment strategies, have yet to be elucidated, particularly in juvenile or insulin-dependent diabetes mellitus (IDDM). Here we establish a model of diabetes in young rats, to examine the consequences of severe hypoglycemia in this age group; particularly seizures and mortality. Diabetes was induced in post-weaned 22-day-old Sprague-Dawley rats by streptozotocin (STZ) administered intraperitoneally (IP). Insulin IP (15 U/kg), in rats fasted (14–16 hours), induced hypoglycemia, defined as <3.5 mM blood glucose (BG), in 68% of diabetic (STZ) and 86% of control rats (CON). Seizures occurred in 86% of STZ and all CON rats that reached hypoglycemic levels with mortality only occurring post-seizure. The fasting BG levels were significantly higher in STZ (12.4±1.3 mM) than in CON rodents (6.3±0.3 mM), resulting in earlier onset of hypoglycemia and seizures in the CON group. However, the BG at seizure onset was statistically similar between STZ (1.8±0.2 mM) and CON animals (1.6±0.1 mM) as well as between those that survived (S+S) and those that died (S+M) post-seizure. Despite this, the S+M group underwent a significantly greater number of seizure events than the S+S group. 25% glucose administered at seizure onset and repeated with recurrent seizures was not sufficient to mitigate these continued convulsions. Combining glucose with diazepam and phenytoin significantly decreased post-treatment seizures, but not mortality. Intracranial electroencephalograms (EEGs) were recorded in 10 CON and 9 STZ animals. Predictive EEG changes were not observed in these animals that underwent seizures. Fluorojade staining revealed damaged cells in non-seizing STZ animals and in STZ and CON animals post-seizure. In summary, this model of hypoglycemia and seizures in juvenile diabetic rats provides a paradigm for further study of underlying mechanisms. Our data demonstrate that severe hypoglycemia (<2.0 mM) is a necessary precondition for seizures, and the increased frequency of these seizures is associated with mortality.     

Red wine prevents brain oxidative stress and nephropathy in streptozotocin-induced diabetic rats

We have studied the effects of red wine on brain oxidative stress and nephropathy in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in Wistar rats with a single intraperitonally injection of STZ (50 mg/kg). Two weeks before and four weeks after injection, red wine was given orally in both normal and diabetic rats. Blood samples were taken from the neck vascular trunk in order to determine the glucose, triglycerides, total cholesterol, HDL-cholesterol (HDL-c), atherogenic index (AI), total protein, blood urea nitrogen (BUN), creatinine, insulin, lipid peroxidation products, reduced glutathione (GSH) and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. As well, we estimated the lipid peroxidtion, GSH and SOD, GSH-Px and catalase activities in brain and renal homogenates, and the excretion of albumin, proteins and glucose in urine over 24 h period. The administration of STZ caused significant increases in levels of glycosuria, proteinuria, albuminuria, glycemia, total cholesterol and AI, as well as in lipid peroxidation products in the brain, plasma and kidney, whereas it decreased the GSH content and SOD, GSH-Px and catalase activities. Treatment with red wine significantly prevented the changes induced by STZ. These data suggested that red wine has a protective effect against brain oxidative stress, diabetic nephropathy and diabetes induced by STZ, as well as it protects against hypercholesterolemia and atherogenic risk.     

Prior hypoglycemia enhances glucose responsiveness in some ventromedial hypothalamic glucosensing neurons

Antecedent insulin-induced hypoglycemia (IIH) reduces adrenomedullary responses (AMR) to subsequent bouts of hypoglycemia. The ventromedial hypothalamus [VMH: arcuate (ARC) + ventromedial nuclei] contains glucosensing neurons, which are thought to be mediators of these AMR. Since type 1 diabetes mellitus often begins in childhood, we used juvenile (4- to 5-wk-old) rats to demonstrate that a single bout of IIH (5 U/kg sc) reduced plasma glucose by 24% and peak epinephrine by 59% 1 day later. This dampened AMR was associated with 46% higher mRNA for VMH glucokinase, a key mediator of neuronal glucosensing. Compared with neurons from saline-injected rats, ventromedial nucleus glucose-excited neurons from insulin-injected rats demonstrated a leftward shift in their glucose responsiveness (EC50 = 0.45 and 0.10 mmol/l for saline and insulin, respectively, P = 0.05) and a 31% higher maximal activation by glucose (P = 0.05), although this maximum occurred at a higher glucose concentration (saline, 0.7 vs. insulin, 1.5 mmol/l). Although EC50 values did not differ, ARC glucose-excited neurons had 19% higher maximal activation, which occurred at a lower glucose concentration in insulin- than saline-injected rats (saline, 2.5 vs. insulin, 1.5 mmol/l). In addition, ARC glucose-inhibited neurons from insulin-injected rats were maximally inhibited at a fivefold lower glucose concentration (saline, 2.5 vs. insulin, 0.5 mmol/l), although this inhibition declined at >0.5 mmol/l glucose. These data suggest that the increased VMH glucokinase after IIH may contribute to the increased responsiveness of VMH glucosensing neurons to glucose and the associated blunting of the AMR.     

Effects of leptin on oxidative stress in healthy and Streptozotocin-induced diabetic rats

Aims/hypothesis It is generally accepted that oxidative stress is responsible for etiology and complications of diabetes. During uncontrolled Type 1 diabetes, plasma leptin levels rapidly fall. However, it is not known whether diabetes-induced hypoleptinemia has any role in oxidative stress related to uncontrolled Type I diabetes. The present study was designed to examine the effects of leptin treatment on plasma lipid peroxidation and reduced glutathion of normal and streptozotocin(STZ)-induced diabetic rats. Methods Diabetes was induced by single injection of Streptozotocin (55 mg/kg bw). One week after induction of diabetes, rats began 5-day treatment protocol of leptin injections of (0.1 mg/kg bw i.p.) or same volume vehicle. At the end of the 5th day, rats were sacrificed by cardiac puncture under anesthesia and their plasma was taken for plasma leptin, malondialdehyde, and reduced glutathione measurements. Results Plasma leptin levels decreased in STZ-induced diabetic rats while plasma glucose, TBARS, and GSH levels increased. Plasma leptin levels were not affected with leptin treatment in both diabetic and non-diabetic rats. The elevation in plasma TBARS associated with STZ diabetes decreased with leptin treatment. Leptin also increased plasma GSH levels in diabetic rats. In non-diabetic rats, treatment with leptin did not change plasma TBARS and GSH levels. Conclusions/interpretations In conclusion, leptin treatment is able to attenuate lipid peroxidation in STZ-diabetic rats, in the onset of diabetes, by increasing the GSH levels without affecting hyperglycemia and hypoleptinemia.     

链脲佐菌素制备糖尿病大鼠模型探讨

目的探讨链脲佐菌素(STZ)配合不同饮食建立糖尿病模型的方法,并对模型大鼠学习记忆能力进行考察,为糖尿病的深入研究及药物开发提供可靠的模型。方法雄性SD大鼠70只,随机分为7组,分别为空白对照组(Ⅰ);高糖高脂膳食组(Ⅱ);0周STZ(30 mg/kg)+高糖高脂膳食组(Ⅲ);0周STZ(30 mg/kg)+常规膳食组(Ⅳ);6周STZ(20 mg/kg)+高糖高脂膳食组(Ⅴ);6周STZ(25 mg/kg)+高糖高脂膳食组(Ⅵ);6周STZ(30 mg/kg)+高糖高脂膳食组(Ⅶ)。采用尾静脉注射STZ配合不同饮食制备糖尿病模型,动态监测模型大鼠血糖的变化,生化方法检测大鼠血脂的改变,放免法检测模型大鼠血清胰岛素、胰高血糖素。Morris水迷宫检测不同造模型条件对大鼠空间学习记忆能力的影响。结果与对照组比较,Ⅲ组大鼠于注射72 h后血糖升高明显(P<0.01),至注射第2周血糖升高达顶点(P<0.01),以后逐渐降低,至观察第10周,血糖维持在15 mmol/L(P<0.05)。IV组大鼠于注射72 h后血糖升高,以后迅速降低,至观察第10周,血糖降低至正常水平。Ⅴ、Ⅵ、Ⅶ组大鼠于注射72 h后显著升高,此后呈波浪式变化;随着注射剂量增加,降低程度减慢。高糖高脂饲料喂养10周后,各组大鼠CHO,TG,LDL-C均增加;Ⅲ、Ⅳ、Ⅴ组大鼠血清INS水平较对照组增高,除IV外,各组胰高血糖素均高于对照组。水迷宫试验结果显示,Ⅶ组潜伏期延长,与对照组比较,具有统计学意义。结论 STZ(30 mg/kg)配合高糖高脂膳食能够快速、稳定的建立糖尿病大鼠模型,高糖高脂膳食组6周后尾静脉注射STZ(30 mg/kg)制备模型,血糖升高显著,血清胰岛素水平降低明显,倾向于1型糖尿病模型。     

Accumulation of pyruvate by changing the redox status in Escherichia coli

Pyruvate was produced from glucose by Escherichia coli BW25113 that contained formate dehydrogenase (FDH) from Mycobacterium vaccae. In aerobic shake-flask culture (K (L) a?=?4.9?min(-1)), the recombinant strain produced 6.7?g pyruvate?l(-1) after 24?h with 4?g sodium formate?l(-1) and a yield of 0.34?g pyruvate?g?glucose(-1). These values were higher than those of the original strain (0.2?g?l(-1) pyruvate and 0.02?g pyruvate?g?glucose(-1)). Based on the reaction mechanism of FDH, the introduction of FDH into E. coli enhances the accumulation of pyruvate by the regeneration of NADH from NAD(+) since NAD(+) is a shared cosubstrate with the pyruvate dehydrogenase complex, which decarboxylates pyruvate to acetyl-CoA and CO(2). The oxygenation level was enough high to inactivate lactate dehydrogenase, which was of benefit to pyruvate accumulation without lactate as a by-product.     

Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34

An inherited deficiency in the frataxin protein causes neurodegeneration of the dorsal root ganglia and Friedreich''s ataxia (FA). Frataxin deficiency leads to oxidative stress and inflammatory changes in cell and animal models; however, the cause of the inflammatory changes, and especially what causes brain microglial activation is unclear. Here we investigated: 1) the mechanism by which frataxin deficiency activates microglia, 2) whether a brain-localized inflammatory stimulus provokes a greater microglial response in FA animal models, and 3) whether an anti-inflammatory treatment improves their condition. Intracerebroventricular administration of LPS induced higher amounts of microglial activation in the FA mouse model vs controls. We also observed an increase in oxidative damage in the form of 8-oxoguanine (8-oxo-G) and the DNA repair proteins MUTYH and PARP-1 in cerebellar microglia of FA mutant mice. We hypothesized that frataxin deficiency increases DNA damage and DNA repair genes specifically in microglia, activating them. siRNA-mediated frataxin knockdown in microglial BV2 cells clearly elevated DNA damage and the expression of DNA repair genes MUTYH and PARP-1. Frataxin knockdown also induced a higher level of PARP-1 in MEF cells, and this was suppressed in MUTYH-/- knockout cells. Administration of the PARP-1 inhibitor PJ34 attenuated the microglial activation induced by intracerebroventricular injection of LPS. The combined administration of LPS and angiotensin II provoke an even stronger activation of microglia and neurobehavioral impairment. PJ34 treatment attenuated the neurobehavioral impairments in FA mice. These results suggest that the DNA repair proteins MUTYH and PARP-1 may form a pathway regulating microglial activation initiated by DNA damage, and inhibition of microglial PARP-1 induction could be an important therapeutic target in Friedreich''s ataxia.     

The Chronological Characteristics of SOD1 Activity and Inflammatory Response in the Hippocampi of STZ-Induced Type 1 Diabetic Rats

Because it appears that oxidative stress and inflammation are implicated with disease pathogenesis in the diabetic brain, many researchers have used streptozotocin (STZ)-induced diabetic animals to study superoxide production and the effects of superoxide scavengers like Cu,Zn-superoxide dismutase (SOD1). However, many studies have been conducted without considering temporal changes after STZ injection. Interestingly, though SOD activities were not significantly different among the groups, SOD1 and 4-hydroxy-2-nonenal (4-HNE) immunoreactivities were significantly enhanced at 3 weeks after an STZ injection (STZ3w) versus only marginal levels in sham controls, whereas microglial activity was remarkably reduced in injected rats at this time. However, SOD1 immunoreactivity and microglial activities were only at the sham level at STZ4w. The present study provides important information concerning cell damage by ROS generated by STZ. Microglial response was found to be inactivated at STZ3w and neuronal cells (NeuN) showed a non-significant tendency to be reduced in number at STZ4w except in the dentate gyrus. We speculated that the above oxidative stress-related events should be accomplished at STZ3w in the brains of STZ-induced diabetes animal models. Therefore, the aim of the present study was to investigate chronological changes in SOD1 immunoreactivity associated with lipid peroxidation and inflammatory responses in the hippocampi of STZ-induced type I diabetic rats.