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.     

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.     

Effects of betaine on performance and body composition: a review of recent findings and potential mechanisms

Betaine is a methyl derivative of glycine first isolated from sugar beets. Betaine consumed from food sources and through dietary supplements presents similar bioavailability and is metabolized to di-methylglycine and sarcosine in the liver. The ergogenic and clinical effects of betaine have been investigated with doses ranging from 500 to 9,000 mg/day. Some studies using animal models and human subjects suggest that betaine supplementation could promote adiposity reductions and/or lean mass gains. Moreover, previous investigations report positive effects of betaine on sports performance in both endurance- and resistance-type exercise, despite some conflicting results. The mechanisms underlying these effects are poorly understood, but could involve the stimulation of lipolysis and inhibition of lipogenesis via gene expression and subsequent activity of lipolytic-/lipogenic-related proteins, stimulation of autocrine/endocrine IGF-1 release and insulin receptor signaling pathways, stimulation of growth hormone secretion, increased creatine synthesis, increases in protein synthesis via intracellular hyper-hydration, as well as exerting psychological effects such as attenuating sensations of fatigue. However, the exact mechanisms behind betaine action and the long-term effects of supplementation on humans remain to be elucidated. This review aims to describe evidence for the use of betaine as an ergogenic and esthetic aid, and discuss the potential mechanisms underlying these effects.     

Betaine chemistry,roles, and potential use in liver disease

BackgroundBetaine is the trimethyl derivative of glycine and is normally present in human plasma due to dietary intake and endogenous synthesis in liver and kidney. Betaine is utilized in the kidney primarily as an osmoprotectant, whereas in the liver its primary role is in metabolism as a methyl group donor. In both organs, a specific betaine transporter mediates cellular uptake of betaine from plasma. The abundance of both betaine and the betaine transporter in liver greatly exceeds that of other organs.Scope of reviewThe remarkable contributions of betaine to normal human and animal health are summarized together with a discussion of the mechanisms and potential beneficial effects of dietary betaine supplements on liver disease.Major conclusionsA significant amount of data from animal models of liver disease indicates that administration of betaine can halt and even reverse progression of the disruption of liver function. Betaine is well-tolerated, inexpensive, effective over a wide range of doses, and is already used in livestock feeding practices.General significanceThe accumulated data indicate that carefully controlled additional investigations in humans are merited. The focus should be on the long-term use of betaine in large patient populations with liver diseases characterized by development of fatty liver, especially non-alcoholic fatty liver disease and alcoholic liver disease.     

Increased catabolism of phosphatidylcholine to betaine regulates the liver phosphatidylcholine content in rats fed orotic acid

Feeding a semi-synthetic diet containing 1% orotic acid to rats for one day stimulates the CDPcholine pathway of liver phosphatidylcholine synthesis 4.5-fold without significantly increasing the liver phosphatidylcholine level. The liver betaine level increases 1.6-fold. The present experiments were performed to investigate the source of the increased liver betaine. Orotic acid feeding did not alter the rate of oxidation of 1,2[14C] choline to betaine. After liver phosphatidylcholine was labelled in vivo with 2[14-C]-ethanolamine, over 90% of the choline-derived radioactivity was recovered in liver betaine and this was consistently increased in rats fed orotic acid. It is concluded that the increased synthesis of liver phosphatidylcholine caused by dietary orotic acid is accompanied by an increased rate of liver phosphatidylcholine catabolism, with betaine as the major end-product of the choline moiety.     

Hyperhomocysteinemia induced by guanidinoacetic acid is effectively suppressed by choline and betaine in rats

Rats were fed 25% casein (25C) diets differing in choline levels (0-0.5%) with and without 0.5% guanidinoacetic acid (GAA) or 0.75% L-methionine for 7 d to determine the effects of dietary choline level on experimental hyperhomocysteinemia. The effects of dietary choline (0.30%) and betaine (0.34%) on GAA- and methionine-induced hyperhomocysteinemia were also compared. Dietary choline suppressed hyperhomocysteinemia induced by GAA, but not by methionine, in a dose-dependent manner. GAA-induced enhancement of the plasma homocysteine concentration was suppressed by choline and betaine to the same degree, but the effects of these compounds were relatively small on methionine-induced hyperhomocysteinemia. Dietary supplementation with choline and betaine significantly increased the hepatic betaine concentration in rats fed a GAA diet, but not in rats fed a methionine diet. These results indicate that choline and betaine are effective at relatively low levels in reducing plasma homocysteine, especially under the condition of betaine deficiency without a loading of homocysteine precursor.     

Red beet and betaine as ingredients in diets of rainbow trout (Oncorhynchus mykiss): effects on growth performance,nutrient retention and flesh quality

The objective of the study was to evaluate the impact of different concentrations of dietary red beet and betaine on the growth performance and fish flesh quality of rainbow trout. Therefore, a control diet was compared with four diets in which two levels of red beet (14% and 28%) and betaine (0.9% and 1.63%) were incorporated in combination. The study was set up with an average body weight of 69 ± 2.2 g and finished when fish reached commercial weight (175–250 g) after 105 d. The impact of the diets was studied based on the growth performance, biometric indexes, proximal composition, protein and fat retention efficiencies and apparent nutrient digestibility by fish reared on a recirculation system. Further estimates were the effect of red beet and betaine on the flesh proximate composition and quality of the final product (water activity, colour, texture, thiobarbituric acid reactive substances and sensory characteristics). Results showed that inclusion of 14% red beet and 0.9% betaine did not affect growth, nutritive or biometric parameters and nutrient retention when compared with the control diet. However, higher levels of red beet and betaine had negative effects on growth and nutritive parameters. The tested ingredients enhanced quality parameters regardless of the concentration used. After feeding the red beet and betaine, fish flesh showed lower water activity and better textural and colour properties than the control and also a dose-dependent effect on lipid oxidation was observed.     

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4–8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [¹³C₁]methionine and [²H₃-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 μg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70–80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.     

Betaine prevents ethanol-induced oxidative stress and reduces total homocysteine in the rat cerebellum

Oxidative stress is a hypothesis for the association of reactive oxygen species with cerebrovascular and neurodegenerative diseases. Thus, we examined whether oral betaine can act as a preventive agent in ethanol-induced oxidative stress on the cerebellum of rats. Thirty-two adult male Sprague–Dawley rats were divided into four equal groups (control, ethanol, betaine, and betaine plus ethanol) with different dietary regimens and were followed up for 1 month. Total homocysteine (tHcy) of plasma and cerebellum homogenate was determined by an Axis^® homocysteine EIA kit, and antioxidant enzyme (glutathione peroxidase (GPx), SOD, and CAT) activities of cerebellum homogenate were measured chemically by a spectrophotometer. Lipid peroxidation of cerebellum was shown by the measurement of thiobarbituric reactive substances (TBARS) via a spectrophotometer. Ethanol-induced hyperhomocysteinemia was manifested by an increase in the concentrations of tHcy in the plasma and cerebellum homogenates of the ethanol group, while ethanol-induced oxidative stress was indicated via an increase in lipid peroxidation marker (TBARS) in cerebellum homogenates of ethanol-treated rats. In contrast, betaine prevented hyperhomocysteinemia and oxidative stress in the betaine plus ethanol group as well as the betaine group. The results of the present investigation indicated that the protective effect of betaine is probably related to its ability to strengthen the cerebellum membrane cells by enhancement of antioxidant enzyme activity principally GPx, while the methyl donor effect of betaine to reduce hyperhomocysteinemia has been explained previously and confirmed in the present study.     

Betaine aids in the osmoregulation of duodenal epithelium of broiler chicks, and affects the movement of water across the small intestinal epithelium in vitro.

In Experiment 1, the water holding capacity of broiler chick intestinal tissue was studied in vitro. The chicks were fed with corn-based diets with or without a 0.2% betaine supplementation in the drinking water. Slices from duodenum and jejunum were incubated in iso-osmotic (300 mM) or hyperosmotic saline (600 mM) with or without 10 mM betaine. The water volume of tissue slices was studied by adding tritiated water in the incubation medium while [14C]inulin was used to correct for the adherent water. After 30 min of incubation, by which time the steady-state of tritium influx had been achieved, the 3H and 14C-activities of the tissue slices were measured. The ileal and duodenal tissues incubated in the hyperosmotic saline accumulated less tritium than those incubated in iso-osmotic saline. Duodenal slices incubated in hyperosmotic saline with the presence of betaine showed a tritium content similar to slices incubated in iso-osmotic saline. The data suggest that the presence of betaine helped the duodenal, but not jejunal, epithelium to maintain water balance in hyperosmotic conditions. The dietary betaine supplementation diminished the differences between the incubation treatments in duodenal, but not in ileal tissue. In Experiment 2, the same double labeling method, but with shorter incubation times, was used to assess the rate of water flux from the incubation medium to duodenal or jejunal slices. The dietary treatments (as in Experiment 1) had little effect on the results. Betaine in the hyperosmotic saline significantly decreased the rate of tritium accumulation into the tissue slices, indicating that betaine slowed down the influx of water to the epithelium. We suggest that betaine affects the movement of water across the intestinal epithelium and has a role in the osmoregulation of small intestine of broiler chicks.     

Amelioration of D-galactosamine-induced acute liver injury in rats by dietary supplementation with betaine derived from sugar beet molasses

The effects of betaine supplementation on D-galactosamine-induced liver injury were examined in terms of hepatic and serum enzyme activities and of the levels of glutathione and betaine-derived intermediates. The rats induced with liver injury showed marked increases in serum enzyme activity, but those receiving dietary supplementation of 1% betaine showed enzyme activity levels similar to a control group without liver injury. Administration of betaine also increased both hepatic and serum glutathione levels, even following D-galactosamine injection. The activity of glutathione-related enzymes was markedly decreased following injection of D-galactosamine, but remained comparable to that of the control group in rats receiving 1% betaine. The concentrations of hepatic S-adenosyl methionine and cysteine showed similar trends to that observed for hepatic glutathione levels. These results indicate that 1% betaine has a hepatoprotective effect by increasing hepatic and serum glutathione levels along with glutathione-related enzyme activities in rats.     

Choline status is not a reliable indicator of moderate changes in dietary choline consumption in premenopausal women

For the prevention of liver dysfunction in women, a choline adequate intake of 425 mg/day was established. To date, the relationship between dietary choline intake and plasma concentrations of choline moieties remains relatively unexplored. As an extension of our previous work, this 14-week controlled feeding study investigated the relationship between moderate changes in dietary choline intake and blood indicators of status. The influences of folate intake and the methylenetetrahydrofolate reductase (MTHFR) C677T genotype were also considered. Healthy premenopausal women (n=45, 18-46 years) with the MTHFR 677CC (n=28) or TT (n=17) genotype consumed a folate-restricted diet for 2 weeks followed by randomization to one of four dietary treatments (n=6-9/group) differing in total choline (344-486 mg/day), betaine (122-349 mg/day) and/or folate (400-800 microg dietary folate equivalents/day) content for 12 weeks. Responses to treatment were assessed as changes in the plasma levels of choline moieties (i.e., betaine, choline, phosphatidylcholine and sphingomyelin) and/or leukocyte global DNA methylation between pretreatment (Week 2) and posttreatment (Week 14) values. No significant changes were detected in the measured variables in response to dietary increases in choline (i.e., 41% increase) or betaine (i.e., 286% increase) intake. However, the MTHFR C677T genotype, alone or together with a diet, influenced betaine (P=.03) and phosphatidylcholine (P=.03). These data suggest that choline status is not a reliable indicator of moderate changes in dietary choline intake possibly due to the engagement of compensatory mechanisms. In addition, the MTHFR C677T genotype appears to influence the direction and use of choline moieties in this group of women.     

Effects of feeding diets containing essential oils and betaine to heat-stressed growing-finishing pigs

This study was to evaluate the effects of dietary essential oils (EO) and betaine on growth performance, nutrient digestibility and serum hormones in growing-finishing pigs under heat stress conditions. A total of 96 crossed pigs [(Landrace × Yorkshire) × Duroc] with an initial body weight (BW) of 24.7 ± 0.27 kg were used in an 18-week trial. Pigs were randomly allocated to four treatments according to BW and gender. There were six replication pens in each treatment, with four pigs (two barrows and two gilts) per pen. Treatment groups were: (1) control group (CON), basal diet + 23°C for 24 h; (2) heat stress group (HC) with basal diet + 37°C for 9 h, 23°C for 15 h; (3) group HEO, HC with 0.01% EO; (4) group HBE, HC with 0.1% betaine. During the overall period, groups HEO and HBE had higher (p < 0.05) average daily gain than group HC. At week 6, group HC had a lower apparent total tract digestibility (ATTD) of dry matter (DM) (p < 0.05), but at week 12, this group had lower ATTD of DM, nitrogen and gross energy than group HEO (p < 0.05). At week 12 and 18, dietary EO decreased (p < 0.05) serum cortisol and norepinephrine concentration. At week 18, dietary EO and betaine decreased (p < 0.05) epinephrine concentration. Conclusively, dietary EO may be a potential nutritional strategy to alleviate heat stress in growing-finishing pigs.     

Purification and characterization of a glycine betaine binding protein from Escherichia coli

A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000. The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine. This periplasmic protein has now been purified to homogeneity. Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein. The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport. The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.     

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.     

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.     

Betaine improves growth, but does not induce whole body or hepatic palmitate oxidation in swine (Sus scrofa domestica)

Dietary betaine may reduce carcass fat in growing pigs. We explored the effects of betaine on short-term growth and in vivo and in vitro fatty acid oxidation. Pigs were housed in metabolism crates and fed diets containing either 0% (control), 0.125% or 0.5% betaine at 80% of ad libitum energy intake. Fatty acid oxidation was measured during intravenous infusions of 1-(13)C-palmitate and in hepatocytes incubated in the presence or absence of betaine and carnitine. CO2 and palmitate isotopic enrichments were determined by mass spectrometry. Pigs consuming 0.125% and 0.5% betaine for at least 9 days had growth rates that were 38% and 12% greater than controls, respectively. Feed efficiency was also improved with betaine. Fasting increased palmitate oxidation rates 7-8-fold (P < 0.01), but betaine had no effect in either the fed or fasted state (P > 0.1). For hepatocytes, carnitine but not betaine enhanced palmitate oxidation. This response suggests that previously observed reduction in adipose accretion must be via a mechanism other than oxidation. Betaine had no effect on plasma non-esterified fatty acids or urea nitrogen. Under the confinement conditions in this study, dietary betaine improved animal growth responses, but it had no apparent effect on either whole body or hepatic fatty acid oxidation.     

Cloning of a Na(+)- and Cl(-)-dependent betaine transporter that is regulated by hypertonicity.

Many hypertonic bacteria, plants, marine animals, and the mammalian renal medulla are protected from the deleterious effects of high intracellular concentrations of electrolytes by accumulating high concentrations of the nonperturbing osmolyte betaine. When kidney-derived Madin-Darby canine kidney (MDCK) cells are cultured in hypertonic medium, they accumulate betaine to 1,000 times its medium concentration. This results from induction by hypertonicity of high rates of betaine transport into cells. We have isolated a cDNA (BGT-1) encoding a renal betaine transporter by screening an MDCK cell cDNA library for expression of a betaine transporter in Xenopus oocytes. The cDNA encodes a single protein of 614 amino acids, with an estimated molecular weight of 69 kDa. The deduced amino acid sequence exhibits highly significant sequence and topographic similarity to brain gamma-amino-n-butyric acid (GABA) and noradrenaline transporters, suggesting that the renal BGT-1 is a member of the brain GABA/noradrenaline transporter gene family. Expression in oocytes indicates that the BGT-1 protein has both betaine and GABA transport activities that are Cl(-)- as well as Na(+)-dependent and functionally similar to betaine and GABA transport in MDCK cells. Northern hybridization indicates that transporter mRNA is localized to the kidney medulla and is induced in MDCK cells by hypertonicity.     

The osmolyte betaine promotes protein misfolding and disruption of protein aggregates

In this work the effect of betaine on the structure and aggregation of the GST-GFP fluorescent fusion protein was studied by different complementary techniques, including electron microscopy, dynamic light scattering, circular dichroism, and FTIR spectroscopy. Although osmolytes are known to be protein stabilizers in vivo, the effect of betaine on the structure and aggregation of our model protein was found to be strictly concentration dependent. We demonstrated that, by changing betaine concentration, it was possible to tune the formation of protein soluble assemblies and insoluble aggregates, as well as to disaggregate preformed aggregates. In particular, at a critical concentration of betaine between 5 and 7.5 mM, the protein precipitated into macroscopic prefibrillar structures, rich in intermolecular beta-sheets, which were found to bind thioflavine T and to be inaccessible to protease. Instead, at higher betaine concentration (10-20 mM) the misfolded protein lost its fluorescence, but formed soluble assemblies with hydrodynamic radius of about 16 nm. These structures displayed a reduced propensity to further aggregate under thermal treatment. In addition, betaine at this high concentration was also found to disrupt large preformed aggregates, obtained under different conditions, into protein soluble assemblies. It is the first time that a disaggregation process has been described for a chemical chaperone. A mechanism for the betaine concentration-dependent effect on protein misfolding, aggregation, and disaggregation is proposed and its possible physiological implications are discussed.