Antioxidant Defense of Betaine Against Oxidative Stress Induced by Ethanol in the Rat Testes

Oxidative stress is one of the factors associated with decline in fertility and betaine has been shown to bear antioxidant and methyl donor properties in our recent studies. Thus, we designed the present study to examine antioxidant and methyl donor abilities of betaine in oxidative stress induced by ethanol in the rat testes. The adult male Sprague-Dawley rats were divided into four experimental groups and treated daily for 2?months as follows: control, ethanol (4?g/kg, orally), betaine (1.5?% of total diet, orally), and betaine plus ethanol (betaine, 1.5?% of total diet and after 120?min, ethanol 4?g/kg). Sperm motility and concentration significantly increased in betaine group when compared to the ethanol?Ctreated rats. The main antioxidant enzyme (GPx) activity significantly increased (in order compensatory) in ethanol-treated rats when compared to betaine group while, antiperoxidative enzyme (CAT) activity significantly increased in betaine plus ethanol group as compared to ethanol-treated rats. Total homocysteine (tHcy) and TBARS concentration (as a lipid peroxidation marker) also significantly decreased in betaine and betaine plus ethanol groups as compared to ethanol-treated rats. Overall, methyl donor and antioxidant properties of betaine are promising and reduce the elevated tHcy and TBARS concentrations in betaine plus ethanol group. Therefore, betaine might be used as a potential therapy in hyperhomocysteinemia and oxidative stress induced by ethanol in alcoholism.     

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic Methanoarchaeon Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance ¹³C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in M. portucalensis.     

Dietary betaine accumulates in the liver and intestinal tissue and stabilizes the intestinal epithelial structure in healthy and coccidia-infected broiler chicks

The aim of this experiment was to study the patterns of betaine accumulation into intestinal tissue, liver and plasma of broiler chicks with or without coccidial infection. The chicks were raised on a corn-based, low-betaine diet with or without 1000 ppm betaine supplementation and with or without intestinal microparasite (Eimeria maxima) challenge to the age of 21 days. Plasma, liver, intestinal tissue and digesta of non-challenged (NC) birds and plasma and intestinal tissue of coccidiosis challenged (CC) birds were analysed for betaine content. NC birds were also analyzed for homocysteine in plasma and S-adenosylmethionine (S-AM) in liver. The jejunal epithelium was histologically examined for the presence of coccidia and the crypt-villus ratio was measured. Dietary betaine supplementation decreased the plasma homocysteine concentration but had no effect on liver S-AM of NC birds. The data suggest that chicks on a low-betaine diet accumulate betaine into the intestinal tissue. When the diet was supplemented with betaine, betaine accumulated heavily into liver and to a lesser degree into intestinal tissue. The concentration of betaine in jejunal and ileal digesta was low suggesting that dietary betaine was mainly absorbed from the proximal small intestine. The coccidial challenge decreased the concentration of betaine in the liver, but greatly increased that in the intestinal tissue. The crypt-villus ratio was decreased by the dietary betaine supplementation in healthy and challenged chicks, suggesting that dietary betaine both protects the jejunal villi against coccidial infection and also stabilizes the mucosal structure in healthy broiler chicks. These results support our earlier findings suggesting that betaine is likely to act as an important intestinal osmolyte in broiler chicks.     

外源甜菜碱对低温胁迫下香蕉内源甜菜碱合成的影响

以巴西香蕉(MusaAAA Giant Cavendish cv.Brazil)幼苗为试验材料,用不同浓度外源甜菜碱(BT)预处理香蕉幼苗后,置于人工气候箱中模拟低温(7℃)胁迫,分别测定香蕉叶片和根系内源甜菜碱的含量和甜菜碱合成关键酶甜菜碱醛脱氢酶(BADH)活性,以探讨外源甜菜碱对香蕉叶片和根系内源甜菜碱合成的影响.结果显示:7℃低温胁迫16 h后,10 mg/L外源甜菜碱即可极显著提高香蕉幼苗叶片BADH活性,叶片内源BT含量也同步极显著增加,低温胁迫24h后根系内源甜菜碱的含量虽显著高于常温对照,其BADH活性却无显著提升.同时,香蕉幼苗叶片内源BT含量的积累与叶片BADH活性的提高具有显著正相关关系,与根系内源BT含量的增加呈极显著正相关关系,与外源BT浓度无显著相关性.研究表明,外源甜菜碱可促进低温胁迫下香蕉内源甜菜碱的合成和积累,叶片和根系均具有合成内源BT的能力.     

Uptake of arsenic-betaines by the mussel Mytilus edulis

Mussels (Mytilus edulis) were exposed to trimethyl(carboxymethyl)arsonium bromide (arsenobetaine, C-1 betaine), trimethyl(2-carboxyethyl)arsonium bromide (C-2 betaine), or trimethyl(3-carboxypropyl)arsonium bromide (C-3 betaine). Arsenic was accumulated by the mussels in all cases but the efficiency of uptake decreased with the number of methylene units in the carboxyalkyl group. Arsenobetaine (C-1 betaine) was the most readily accumulated, followed by the C-2 betaine (70% as efficient as arsenobetaine) and the C-3 betaine (∼7%). Chromatographic analysis (HPLC-ICPMS) of extracts of the mussels demonstrated that the arsenic compounds were accumulated uncahanged. A 46-day depuration period which followed exposure did not significantly reduce the arsenic concentration in any of the three groups. Comparison with previous data on accumulation of arsenic compounds by M. edulis indicates that uptake may be influenced by the presence of a quaternary arsonium group and the zwitterionic nature of the arsenic-betaines.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

Detection of the osmoregulator betaine in methanogens

Trimethyl glycine (glycine betaine) was detected by 13C nuclear magnetic resonance spectroscopy at high intracellular concentrations in several methanogens (Methanogenium cariaci, "Methanogenium anulus" AN9, Methanohalophilus zhilinae, Methanohalophilus mahii, and Methanococcus voltae) grown on marine media containing yeast extract. 13C labeling studies with Methanogenium cariaci suggested that the betaine which accumulated inside the cells was not synthesized de novo but was transported in from the medium. Proof of such a transport system was provided by growing Methanogenium cariaci on yeast-free medium supplemented with betaine. Under these conditions, betaine was the dominant osmoregulator.     

Betaine: New Oxidant in the Stickland Reaction and Methanogenesis from Betaine and l-Alanine by a Clostridium sporogenes-Methanosarcina barkeri Coculture

Growing and nongrowing cells of Clostridium sporogenes fermented betaine with l-alanine, l-valine, l-leucine, and l-isoleucine as electron donors in a coupled oxidation-reduction reaction (Stickland reaction). For the substrate combinations betaine and l-alanine and betaine and l-valine balance studies were performed; the results were in agreement with the following fermentation equation: 1 R- CH(NH(2))-COOH + 2 betaine + 2 H(2)O --> 1 R-COOH + 1 CO(2) + 1 NH(3) + 2 trimethylamine + 2 acetate. Growth and production of trimethylamine were strictly dependent on the presence of selenite in the medium. With cell suspensions it was shown that C. sporogenes was unable to catabolize betaine as a single substrate. Betaine, however, was reduced to trimethylamine and acetate under an atmosphere of molecular hydrogen. For the reduction of betaine by cell extracts of C. sporogenes, dimercaptans such as 1,4-dithiothreitol could serve as electron donors. No betaine reductase activity was detected in cells grown in a complex medium without betaine. The pH optimum of betaine reductase was at pH 7.3. When C. sporogenes was cocultured with Methanosarcina barkeri strain Fusaro on betaine together with l-alanine, an almost complete conversion of the two substrates to CH(4), NH(3), and presumably CO(2) was observed.     

Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities

Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O(2)(-)) or hydrogen peroxide (H(2)O(2)). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes.     

Detection of the osmoregulator betaine in methanogens.

Trimethyl glycine (glycine betaine) was detected by 13C nuclear magnetic resonance spectroscopy at high intracellular concentrations in several methanogens (Methanogenium cariaci, "Methanogenium anulus" AN9, Methanohalophilus zhilinae, Methanohalophilus mahii, and Methanococcus voltae) grown on marine media containing yeast extract. 13C labeling studies with Methanogenium cariaci suggested that the betaine which accumulated inside the cells was not synthesized de novo but was transported in from the medium. Proof of such a transport system was provided by growing Methanogenium cariaci on yeast-free medium supplemented with betaine. Under these conditions, betaine was the dominant osmoregulator.     

The accumulation and synthesis of betaine in winter skate (Leucoraja ocellata)

The present study investigated aspects of betaine metabolism in an elasmobranch fish, the winter skate (Leucoraja ocellata). Based on the level of choline dehydrogenase (ChoDH) activity, the liver and kidney appear to be the major sites of betaine synthesis and the mitochondrial localization of ChoDH and betaine aldehyde dehydrogenase (BADH) indicates that the metabolic organization of betaine synthesis in winter skate is similar to other vertebrates. Food deprivation did not affect white muscle betaine content, and prolonged starvation (70 days) appeared to decrease the total hepatic betaine synthetic capacity. There was no decrease in ChoDH or BADH activity at the mitochondrial level with starvation, suggesting any decrease is due to catabolism of hepatic reserves rather than downregulation of betaine synthesis. Skates fed a high betaine diet (frozen squid approximately 55 micromol g(-1)) had elevated white muscle betaine content compared to those fed a low betaine diet (frozen herring <2 micromol g(-1)); however, high dietary betaine intake did not affect the activity of betaine synthesizing enzymes in liver. Acclimation to elevated salinity (120 and 130% seawater) did not result in an increase in white muscle betaine content. Taken as a whole, the present data suggest that diet is a major determinant of muscle betaine in the winter skate and that betaine is of marginal importance as an intracellular osmolyte in this species.     

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD.

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 μM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 μM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaine by oxidation of choline through glycine betaine aldehyde (choline----glycine betaine aldehyde----glycine betaine; B. Landfald and A.R. Str?m, J. Bacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codAB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aldehyde dehydrogenase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betaine uptake or low-affinity choline uptake, both of which were osmotically regulated.     

Glycine betaine in beer as an antimutagenic substance against 2-chloro-4-methylthiobutanoic acid, the sanma-fish mutagen

Beer can inhibit the mutagenicity of the sanma-fish mutagen, 2-chloro-4-methylthiobutanoic acid (CMBA) in Salmonella typhimurium TA100 and TA1535. The antimutagenic component was isolated from beer and identified as glycine betaine, a compound known to be distributed widely in plants and animals including humans. Beer also contains components that interfere the antimutagenic action of glycine betaine. Glycine betaine seems to antagonize CMBA in a specific manner, since several other direct-acting mutagens tested were not subject to inhibition by glycine betaine. CMBA was stable in the presence of glycine betaine under neutral conditions. Since a treatment of Salmonella with glycine betaine before the bacteria was exposed to CMBA resulted in inhibition of the mutagenesis, the antimutagenic action of glycine betaine may be taking place inside the cells. These observations suggest that the mutagenic action of CMBA may be modified by the presence of both extracellular and intracellular glycine betaine.     

Intestinal uptake of betaine in vitro and the distribution of methyl groups from betaine, choline, and methionine in the body of broiler chicks

The efficiency of betaine absorption into small intestinal slices of broiler chicks was studied in vitro with 14C-labeled betaine. The relative proportion of Na+-coupled betaine uptake, as well as the total uptake capacity was larger in the duodenum than in the jejunum. Dietary betaine increased the Na+-coupled uptake in the duodenum. In in vivo-experiments, methyl-14C-labeled betaine, methionine, or choline was fed to broiler chicks. Betaine appeared in the blood more rapidly, and reached a higher total concentration than choline or methionine. The data suggest that choline and methionine were associated with plasma lipoproteins whereas betaine remained free in the plasma. The label distribution in liver, kidney, and intestinal tissues was studied 24 h after label ingestion. Most of the label from betaine was found in the aquaeous phase in the muscle, while in the liver and jejunum the label from betaine was distributed more evenly between the aquaeous, lipid, and protein phases. Label from choline accumulated in the lipid fraction, particularly so in the liver, whereas label from methionine showed a more variable distribution pattern. The distribution results are interpreted in terms of specific roles of betaine, choline, and methionine in methyl group metabolism.     

Receptor sites for alanine,proline, and betaine in the palatal taste system of the puffer,Fugu pardalis

Summary To elucidate the relative independence of the receptor sites for amino acids and betaine in the gustatory system of fish, the neural responses from the ramus palatinus facialis innervating the anterior palate of the puffer, Fugu pardalis, were recorded.There were observed independent amino acidsensitive and betaine-sensitive fibers.Cross-adaptation to pairs of stimulant was studied. The pair stimulants were applied reciprocally, i.e. after adapting with one stimulus the second stimulus was applied and then reversed. There were observed 3 types of cross-effects for the pairs of stimulants tested: (1) a reciprocal profound cross-adaptation; (2) no distinct cross-adaptation; and (3) a reciprocal enhancement of the response between betaine and alanine or glycine. Such an enhancement between betaine and alanine occurred in the amino acid-sensitive fibers, suggesting that betaine has an enhancing effect on the amino acid receptors.The present results suggest at least 3 different groups of receptor sites for the stimulants tested: (1) alanine sites for alanine, glycine and sarcosine; (2) proline sites for proline and dimethylglycine; and (3) betaine sites for betaine and dimethylglycine.     

Preliminary Genetic Studies of the Phenotype of Betaine Deficiency in Zea mays L

Glycinebetaine-deficient inbreds of Zea mays do not exhibit a general deficiency of nitrogenous solutes; the total free amino acid levels of betaine-deficient lines are not significantly less than those of inbreds which exhibit >100-fold higher betaine levels. Betaine-deficient inbreds are characterized by extremely low betaine: total free amino acid ratios (<0.0015). Highly significant correlations are demonstrated between the expected mid-parent and observed betaine:amino acid ratios of 30 hybrids of known pedigree. In 12 hybrids constructed from a betaine-deficient male parent (inbred 1506), the observed betaine:amino acid ratios of the hybrids are proportional to the betaine:amino acid ratios of the female parents (r = 0.83). Two hybrids, 1146 × 1074 and 1146 × 1506, were chosen for further genetic analysis. The common female parent (1146) and inbred 1074 both exhibit betaine:amino acid ratios of 0.090, a value which is approximately 90-fold greater than the betaine:amino acid ratio of inbred 1506. Hybrid 1146 × 1074 exhibits almost exactly twice the betaine:amino acid ratio of hybrid 1146 × 1506. If inbred 1506 is homozygous recessive for a single nuclear gene responsible for the phenotype of betaine deficiency, and if inbreds 1146 and 1074 are homozygous dominant for this allele, then this twofold difference in betaine:amino acid ratio must be associated with the homozygous dominant and heterozygous conditions, respectively, for 1146 × 1074 and 1146 × 1506. Evidence is presented from both greenhouse and field evaluations of F₂ populations of these hybrids that a single nuclear recessive gene is most likely responsible for the phenotype of betaine-deficiency in inbred 1506. Approximately 25% of the F₂ segregants from 1146 × 1506 exhibited extremely low betaine:amino acid ratios (<0.0015), whereas 0% of the F₂ segregants from 1146 × 1074 exhibited this phenotype. The segregation patterns with respect to betaine:amino acid ratio suggest a 1:2:1 segregation ratio for homozygous recessive:heterozygous:homozygous dominant individuals within the 1146 × 1506-F₂ population.     

Metabolism of betaine as a carbon source by an osmotolerant bacterium isolated from the weed rhizosphere

An isolate of an osmotolerant rhizobacterium has been obtained from a weed rhizosphere which showed tolerance up to 1.0 M NaCl. The isolate has been subjected to growth analysis in a medium which contained 10 mM betaine as the sole carbon source. It was observed that betaine could be used as the sole carbon source for the growth of salt-tolerant rhizobacteria under NaCl-stress at 1.0 M concentration. Interestingly, it was found that betaine at 100 mM concentration suppressed the growth of salt-tolerant rhizobacteria. The growth of the osmotolerant rhizobacterium was stimulated when it was grown in a medium containing both glucose and betaine, demonstrating that betaine was an osmoprotectant. The presence of glucose at 10 mM concentration, however, did not alleviate the growth-suppressive effect of betaine at 100 mM concentration. The osmoprotective effect of betaine was demonstrated by the fact that the addition of betaine at different time intervals enhanced the growth accordingly. However, the growth-suppressive effect of betaine at 100 mM concentration was also noticeable when betaine was added at different time intervals.