Involvement of astroglial glutamate–glutamine shuttle in modulation of the jaw‐opening reflex following infraorbital nerve injury

To evaluate the mechanisms underlying orofacial motor dysfunction associated with trigeminal nerve injury, we studied the astroglial cell activation following chronic constriction injury (CCI) of the infraorbital nerve (ION) immunohistochemically, nocifensive behavior in ION‐CCI rats, and the effect of the glutamine synthase (GS) blocker methionine sulfoximine (MSO) on the jaw‐opening reflex (JOR), and also studied whether glutamate–glutamine shuttle mechanism is involved in orofacial motor dysfunction. GFAP‐immunoreactive (IR) cells were observed in the trigeminal motor nucleus (motV) 3 and 14 days after ION‐CCI, and the nocifensive behavior and JOR amplitude were also strongly enhanced at these times. The number of GS‐ and GFAP‐IR cells was also significantly higher in ION‐CCI rats on day 7. The amplitude and duration of the JOR were strongly suppressed after MSO microinjection (m.i.) into the motV compared with that before MSO administration in ION‐CCI rats. After MSO administration, the JOR amplitude was strongly suppressed, and the duration of the JOR was shortened. Forty minutes after m.i. of glutamine, the JOR amplitude was gradually returned to the control level and the strongest attenuation of the suppressive effect of MSO was observed at 180 min after glutamine m.i. In addition, glutamine also attenuated the MSO effect on the JOR duration, and the JOR duration was extended and returned to the control level thereafter. The present findings suggest that astroglial glutamate–glutamine shuttle in the motV is involved in the modulation of excitability of the trigeminal motoneurons affecting the enhancement of various jaw reflexes associated with trigeminal nerve injury.     

Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis

DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, AG at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.     

NO sensitizes rat hepatocytes to proliferation by modifying S-adenosylmethionine levels

BACKGROUND & AIMS: Liver regeneration is a fundamental response of this organ to injury. Hepatocyte proliferation is triggered by growth factors, such as hepatocyte growth factor. However, hepatocytes need to be primed to react to mitogenic signals. It is known that nitrous oxide (NO), generated after partial hepatectomy, plays an important role in hepatocyte growth. Nevertheless, the molecular mechanisms behind this priming event are not completely known. S-adenosylmethionine (AdoMet) synthesis by methionine adenosyltransferase is the first step in methionine metabolism, and NO regulates hepatocyte S-adenosylmethionine levels through specific inhibition of this enzyme. We have studied the modulation of hepatocyte growth factor-induced proliferation by NO through the regulation of S-adenosylmethionine levels. METHODS: Studies were conducted in cultured rat hepatocytes isolated by collagenase perfusion, which triggers NO synthesis. RESULTS: The mitogenic response to hepatocyte growth factor was blunted when inducible NO synthase was inhibited; this process was overcome by the addition of an NO donor. This effect was dependent on methionine concentration in culture medium and intracellular S-adenosylmethionine levels. Accordingly, we found that S-adenosylmethionine inhibits hepatocyte growth factor-induced cyclin D1 and D2 expression, activator protein 1 induction, and hepatocyte proliferation. CONCLUSIONS: Together our findings indicate that NO may switch hepatocytes into a hepatocyte growth factor-responsive state through the down-regulation of S-adenosylmethionine levels.     

Restoration of brain protein synthesis in mature and aged rats by a DA agonist, piribedil

Summary— Brain ageing affects numerous cerebral metabolic pathways such as cerebral glucose consumption or protein synthesis rate. The pharmacological effect of a mixed D₁-D₂ dopaminergic agonist, piribedil, on this last metabolism is reported. Cerebral Protein Synthesis Rate (CPSR) was measured by the [³⁵S]L-methionine autoradiographic procedure in 38 main brain regions of 11 and 26-month-old Wistar rats after a 2-month treatment per os at 9 and 30 mg/kg/day with piribedil. Mean decrease of CPSR was −21% during the 15-month ageing we followed, with important local variations. Mean CPSR increased with the two treatments, +25% in mature and +35% in aged rats. Treatments restored CPSR of aged rats to the exact mature subjects levels in quite all the brain regions. No dose-effect or asymetrical modification was statistically revealed for the two treatments. Metabolic increases involved particularly central brain gray structures, especially some DA-targeted brain nuclei concerned with behaviour and learning. This effect argued for a general metabotrophic effect of D₁-D₂ dopamine stimulation of the brain. The original pattern of local ageing of brain protein synthesis in rat was also incidentally reported. This was the first direct report of a wide and effective metabolic activation of CPSR in the brain during ageing by a curative dopaminergic agonist treatment.     

Metal active site elasticity linked to activation of homocysteine in methionine synthases

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.     

Regulation of methionine adenosyltransferase activity by the glutathione level in rat liver during ischemia-reperfusion

10 (CoQ₁₀) was administered to rats prior to ischemia, both the reduction in the MAT activity and hepatic GSH levels induced by ischemia–reperfusion were protected. Our findings suggest that CoQ₁₀ may posttranslationally regulate the MAT activity via the changes in the GSH level in the liver. (Received for publication of Aug. 31, 1998; accepted on May 28, 1999)     

Relationship between dietary and supplemental intake of folate,methionine, vitamin B6 and folate receptor α expression in ovarian tumors

Because folate receptor α (FRα) is frequently over‐expressed in epithelial ovarian tumors, we hypothesized that its association with folate may differ by FRα expression or by the timing of intake. We examined the association between folate and other cofactors in 152 ovarian cancers evaluated for FRα expression from the Nurses' Health Study. Multivariate odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. FRα expression was higher in serous invasive and advanced stage ovarian tumors. Recent dietary folate intake ≥ 300 μg/day compared to < 300 μg/day was associated with decreased risk of developing ovarian cancer (OR = 0.62; 95%CI 0.40–0.96). There was suggestion of an increased risk with total folate (dietary and supplemental) (OR=1.42; 95%CI 0.94–2.14 for past and OR = 1.53; 95%CI 0.99–2.37 for recent intake). These results did not vary by FRα status of the tumor. Methyl group score, a marker of high dietary folate and methionine intake but low alcohol consumption, was inversely associated with risk of ovarian cancer (OR = 0.44; 95%CI 0.23–0.84 for past and OR=0.46; 95%CI 0.24–0.88 for recent intake). There were no clear individual associations between methionine, vitamin B₆, or multivitamin use and ovarian cancer risk overall or by FRα tumor status. Our data do not support the hypothesis that the relationship between factors involved in one‐carbon metabolism and ovarian cancer risk differs by FRα status of the tumor.     

Effects of low and high doses of fenofibrate on protein,amino acid,and energy metabolism in rat

A feared adverse effect of dyslipidaemia therapy by fibrates is myopathy. We examined the effect of fenofibrate (FF) on protein and amino acid metabolism. Rats received a low (50 mg/kg, LFFD) or high (300 mg/kg, HFFD) dose of FF or vehicle daily by oral gavage. Blood plasma, liver, and soleus and extensor digitorum longus muscles were analysed after 10 days. The FF‐treated rats developed hepatomegaly associated with increased hepatic carnitine and ATP and AMP concentrations, decreased protein breakdown, and decreased concentrations of DNA and triglycerides. HFFD increased plasma ALT and AST activities. The weight and protein content of muscles in the HFFD group were lower compared with controls. In muscles of the LFFD group there were increased ATP and decreased AMP concentrations; in the HFFD group AMP was increased. In both FF‐treated groups there were increased glycine, phenylalanine, and citrulline and decreased arginine and branched‐chain keto acids (BCKA) in blood plasma. After HFFD there were decreased levels of branched‐chain amino acids (BCAA; valine, leucine and isoleucine), methionine, and lysine and increased homocysteine. Decreased arginine and increased glycine concentrations were found in both muscles in FF‐treated animals; in HFFD‐treated animals lysine, methionine, and BCAA were decreased. We conclude that FF exerts protein‐anabolic effects on the liver and catabolic effects on muscles. HFFD causes signs of hepatotoxicity, impairs energy and protein balance in muscles, and decreases BCAA, methionine, and lysine. It is suggested that increased glycine and decreased lysine and methionine levels are due to activated carnitine synthesis; decreased BCAA and BCKA levels are due to increased BCAA oxidation.     

郑州市中原区汉族育龄女性MTHFR和MTRR基因多态性

目的利用基因检测技术筛查出育龄女性叶酸利用能力差的高风险人群,指导育龄女性合理补充叶酸。方法通过荧光定量PCR,对2017年12月至2018年10月采集的2652例郑州市中原区育龄女性进行5,10-亚甲基四氢叶酸还原酶(MTHFR)C667T、A1298C位点和甲硫氨酸合酶还原酶(MTRR)A66G位点的基因型测定,并与其他地区人群基因型和等位基因频率分布进行比较,然后根据基因型综合判断育龄女性叶酸代谢能力强弱,由此得出育龄女性是否有叶酸补充风险及风险级别。结果检测的MTHFR C667T位点中,野生型(CC)、杂合突变型(CT)及纯合突变型(TT)分别占15.05%、46.60%和38.35%,突变基因T的基因频率为61.65%;不同年龄组(≤30和>30岁)均以杂合突变型为主。MTHFR A1298C位点中分别为74.55%、23.49%和1.96%,突变基因C的基因频率为13.71%;不同年龄组(≤30和>30岁)均以野生型为主。MTRR A66G位点中分别为57.43%、36.73%和5.84%,突变基因G的基因频率为24.21%;不同年龄组(≤30和>30岁)均以野生型为主。结论郑州地区育龄女性MTHFR C677T基因多态性分布与地域有相关性,超过50%以上的育龄女性携带高风险基因。因此,对叶酸代谢基因多态性检测高风险孕妇合理增补叶酸剂量,可进一步降低新生儿出生缺陷。     

An improved mouse model that rapidly develops fibrosis in non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine‐/choline‐deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long‐term feeding with a high‐fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1–14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Masson's trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.