Inhibition of insulin fibrillation by osmolytes

We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50-250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies.

Choudhary, S., N. Kishore, and R.V. Hosur, Inhibition of insulin fibrillation by osmolytes: Mechanistic Insights. Sci Rep, 2015. 5: p. 17599.

Betaine accumulates in human lens epithelial cells after exposure to ultraviolet A

Compatible organic osmolytes, such as betaine, myoinositol, and taurine, are involved in antioxidant defense, protein stabilization, and stress responses. This osmolyte strategy requires the expression of specific osmolyte transporters such as betaine (BGT-1), myoinositol (SMIT), and taurine (TAUT). In contrast to the kidney, keratinocytes, and neural cells, few studies have examined osmolytes in human lens epithelial cells (HLECs). We examined the expression of mRNA specific for BGT-1, SMIT, and TAUT in HLECs. In comparison to normoosmotic (305 mOsM) controls, there was a 3-5-fold time-dependent reaction of BGT-1, SMIT, and TAUT mRNA levels in HLECs exposed to hyperosmotic stress (405 mOsM). Maximal responses were obtained for BGT-1, SMIT, and TAUT mRNA expression after 3, 24 and 9 h of hyperosmotic exposure, respectively. This expression was correlated with increased osmolyte uptake. In contrast, hypoosmotic (205 mOsM) stimulation led to a significant efflux of osmolytes. Exposure to ultraviolet A (340-400 nm) radiation significantly stimulated osmolyte uptake. Increased osmolyte uptake was associated with upregulation of mRNA steady-state levels for osmolyte transporters in irradiated cells. These results demonstrate that ultraviolet A radiation leads to the accumulation of compatible organic osmolytes in HLECs as hyperosmotic pressure, which can maintain cellular environmental homeostasis.

Wu, D.Y. and J.S. Zhang, Effect of ultraviolet A exposure on transport of compatible organic osmolytes in human lens epithelial cells. Genet Mol Res, 2015. 14(2): p. 5132-40.

Betaine may protect ocular surface epithelia from MMP-mediated disorders in dry eye disease

PURPOSE: Hyperosmolarity has been recognized as a proinflammatory stress in the pathogenesis of dry eye disease. This study investigated the suppressive effect of osmoprotectants (L-carnitine, erythritol, and betaine) on the production and activity of matrix metalloproteinases (MMPs) in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.
METHODS: Primary HCECs were established from fresh donor limbal tissue explants. The cultures in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with different concentrations of L-carnitine, erythritol, or betaine (2, 10, or 20 mM). The mRNA expression of the MMPs was determined with reverse transcription and quantitative real-time PCR (RT-qPCR). Protein production and activity were evaluated with immunofluorescent staining and gelatin zymography.
RESULTS: Hyperosmotic media (400, 450, or 500 mOsM) significantly stimulated mRNA expression of collagenase MMP-13, gelatinases MMP-9 and MMP-2, stromelysin MMP-3, and matrilysin MMP-7, mostly in an osmolarity-dependent fashion. The stimulated mRNA expression and protein production of these MMPs were significantly but differentially suppressed by L-carnitine, erythritol, or betaine, as evaluated with RT-qPCR and immunofluorescent staining. Interestingly, these osmoprotectants not only suppressed production but also inhibited activation of MMP-9 and MMP-2, as evaluated with gelatin zymography.
CONCLUSIONS: Our findings for the first time demonstrate that osmoprotectants, L-carnitine, erythritol, and betaine, suppress the gene expression, protein production, and enzymatic activity of MMPs in HCECs exposed to hyperosmotic stress. L-carnitine appears to have the broadest and strongest suppressive effect on these MMPs. These osmoprotectants may have potential effects in protecting ocular surface epithelia from MMP-mediated disorders in dry eye disease.

Deng, R., et al., Osmoprotectants suppress the production and activity of matrix metalloproteinases induced by hyperosmolarity in primary human corneal epithelial cells. Mol Vis, 2014. 20: p. 1243-52

Carnitine, erythritol and betaine may have efficacy in reducing innate inflammation in dry eye disease.

Purpose: To explore the effects of osmoprotectants on pro-inflammatory mediator production in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.
Methods: HCECs cultured in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with 2-20 mM of l-carnitine, erythritol or betaine for different time periods. The mRNA expression and protein production of pro-inflammatory markers in HCECs were evaluated by RT-qPCR and ELISA.
Results: Hyperosmolar media significantly stimulated the mRNA and protein expression of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, and chemokines, IL-8, CCL2 and CCL20 in HCECs in an osmolarity dependent manner. The stimulated expression of these pro-inflammatory mediators was significantly but differentially suppressed by l-carnitine, erythritol or betaine. l-Carnitine displayed the greatest inhibitory effects and down-regulated 54-77% of the stimulated mRNA levels of TNF-alpha (down from 12.3-5.7 fold), IL-1beta (2.2-0.9 fold), IL-6 (7.3-2.9 fold), IL-8 (4.6-2.0 fold), CCL2 (15.3-3.5 fold) and CCL20 (4.1-1.5 fold) in HCECs exposed to 450 mOsM. The stimulated protein production of TNF-alpha, IL-1beta, IL-6 and IL-8 was also significantly suppressed by l-carnitine, erythritol and betaine. l-carnitine suppressed 49-79% of the stimulated protein levels of TNF-alpha (down from 81.3 to 17.4 pg/ml), IL-1beta (56.9-29.2 pg/ml), IL-6 (12.8-4.6 ng/ml) and IL-8 (21.2-10.9 ng/ml) by HCECs exposed to 450 mOsM. Interestingly, hyperosmolarity stimulated increase in mRNA and protein levels of TNF-alpha, IL-1beta and IL-6 were significantly suppressed by a transient receptor potential vanilloid channel type 1 (TRPV1) activation inhibitor capsazepine.
Conclusions: l-carnitine, erythritol and betaine function as osmoprotectants to suppress inflammatory responses via TRPV1 pathway in HCECs exposed to hyperosmotic stress. Osmoprotectants may have efficacy in reducing innate inflammation in dry eye disease.

Hua, X., et al., Effects of l-Carnitine, Erythritol and Betaine on Pro-inflammatory Markers in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress. Curr Eye Res, 2014: p. 1-11.

Fenofibrate causes elevation of betaine excretion but not excretion of other osmolytes by healthy adults

BACKGROUND: Cross-sectional data suggest that bezafibrate increases betaine excretion in dyslipidemic patients.
OBJECTIVE: We aimed to demonstrate that fenofibrate induces increased betaine excretion in normal subjects and explore whether other 1-carbon metabolites and osmolytes are similarly affected.
METHODS: Urine was collected from 26 healthy adults before and after treatment with fenofibrate (145 mg/day for 6 weeks). Excretions of betaine, N,N-dimethylglycine, free choline, myo-inositol, taurine, trimethylamine-N-oxide, carnitine, and acetylcarnitine were measured by liquid chromatography with mass spectrometric detection.
RESULTS: Fenofibrate increased the median betaine excretion from 7.5 to 25.8 mmol/mole creatinine (median increase 3-fold), P < .001. The median increase in N,N-dimethylglycine excretion was 2-fold (P < .001). Median choline excretion increased 12% (significant, P = .029). Participants with higher initial excretions tended to have larger increases (P < .001 in all 3 cases). Fenofibrate did not significantly change the median excretions of myo-inositol, taurine, trimethylamine-N-oxide, and carnitine. The excretion of acetylcarnitine decreased 4-fold on treatment, with no correlation between the baseline and after-treatment excretions. Changes in all urine components tested, except trimethylamine-N-oxide, positively correlated with changes in betaine excretion even when the median excretions before and after were not significantly different.
CONCLUSIONS: Fibrates increase betaine, and to a lesser extent N,N-dimethylglycine and choline, excretion. Other osmolytes are not elevated. Because the increase in betaine excretion depends on the baseline excretion, large increases in excretion in the metabolic syndrome and diabetes (where baseline excretions are high) could be expected. Replacement with betaine supplements may be considered.

Lever, M., et al., Fenofibrate causes elevation of betaine excretion but not excretion of other osmolytes by healthy adults. J Clin Lipidol, 2014. 8(4): p. 433-40.

Increased urinary betaine excretions in type 2 diabetes are further increased by fibrate treatment

PURPOSE: Betaine deficiency is a probable cardiovascular risk factor and a cause of elevated homocysteine. Urinary betaine excretion is increased by fibrate treatment, and is also often elevated in diabetes. Does fibrate further increase betaine excretion in diabetes, and does it affect the plasma concentrations and excretions of related metabolites and of other osmolytes?
METHODS: Samples from a previous study of type 2 diabetes were selected if participants were taking bezafibrate (n = 32). These samples were compared with participants matched for age and gender and not on a fibrate (comparator group, n = 64). Betaine, related metabolites, and osmolytes were measured in plasma and urine samples from these 96 participants.
RESULTS: Median urinary betaine excretion in those on bezafibrate was 5-fold higher than in the comparator group (p < 0.001), itself 3.5-fold higher than the median reported for healthy populations. In the bezafibrate group, median dimethylglycine excretion was higher (9-fold, p < 0.001). Excretions of choline, and of the osmolytes myo-inositol, taurine and glycerophosphorylcholine, were not significantly different between groups. Some participants excreted more betaine than usual dietary intakes. Several betaine fractional clearances were >100 %. Betaine excretion correlated with excretions of the osmolytes myo-inositol and glycerophosphorylcholine, and also with the excretion of choline and N,N-dimethylglycine, but it was inconclusive whether these relationships were affected by bezafibrate therapy.
CONCLUSIONS: Increased urinary betaine excretions in type 2 diabetes are further increased by fibrate treatment, sometimes to more than their dietary intake. Concurrent betaine supplementation may be beneficial.

Lever, M., et al., Extreme Urinary Betaine Losses in Type 2 Diabetes Combined with Bezafibrate Treatment are Associated with Losses of Dimethylglycine and Choline but not with Increased Losses of Other Osmolytes. Cardiovasc Drugs Ther, 2014

Betaine mitigates the destabilizing effect of protein crowding

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by cells stressed by dehydration.

Sarkar, M. and G.J. Pielak, An osmolyte mitigates the destabilizing effect of protein crowding. Protein Sci, 2014

Topical application of betaine limited progression of environmentally induced dry eye

Purpose: To evaluate the efficacy of osmoprotectants on prevention and treatment of dry eye in a murine model. Methods: Dry eye was induced in mice using an intelligently controlled environmental system (ICES). Osmoprotectants betaine, L-carnitine, erythritol, or vehicle (PBS) were topically administered to eyes 4 times daily following two schedules: Schedule 1 (modeling prevention): dosing started at the beginning of housing in ICES and lasted for 21 or 35 days; Schedule 2 (modeling treatment): dosing started after ICES-housed mice developed dry eye (Day 21), continuing till Day 35. Treatment efficacy was evaluated for corneal fluorescein staining; corneal epithelial apoptosis by TUNEL and caspase-3 assays; goblet cell numbers by PAS staining; and expression of inflammatory mediators, TNF-alpha, IL-17, IL-6 or IL-1beta using RT-PCR on Days 0, 14, 21 and/or 35.
Results: Compared to vehicle, prophylactic administration of betaine, L-carnitine or erythritol significantly decreased corneal staining and expression of TNF-alpha and IL-17 on Day 21 (Schedule 1). Treatment of mouse dry eye with osmoprotectants significantly reduced corneal staining on Day 35 compared to Day 21 (Schedule 2). Relative to vehicle, L-carnitine treatment of mouse dry eye for 14 days (Day 21 to 35) resulted in a significant reduction in corneal staining, number of TUNEL-positive cells, and expression of TNF-alpha, IL-17, IL-6, or IL-1beta, as well significantly increased number of goblet cells.
Conclusion: Topical application of betaine, L-carnitine or erythritol systematically limited progression of environmentally induced dry eye. L-carnitine can also reduce the severity of such dry eye conditions.

Chen, W., et al., Efficacy of Osmoprotectants on Prevention and Treatment of Murine Dry Eye. Invest Ophthalmol Vis Sci, 2013

Effects of Betaine on Energy Utilization in Growing Pigs - A Review

One of the well known biological functions of betaine is that of a methyl donor. Therefore, betaine may partly replace choline and methionine in the diet. Another widely documented role of betaine is to restore and maintain the osmotic balance. As an organic osmotic compound, betaine regulates the water balance, thus exerting a stabilizing influence on tissue metabolism, particularly within the digestive tract. As a donor of methyl groups necessary for various reactions in the body, betaine is indirectly involved in lipid metabolism. Due to its metabolic functions, betaine is also believed to play a significant role in energy metabolism in pigs. Of particular note are the results of experiments in which a positive effect of betaine supplementation was observed as the energy content of the diet was decreased.

Lipinski, K., et al., Effects of Betaine on Energy Utilization in Growing Pigs - A Review. Annals of Animal Science, 2012. 12(3): p. 291-300

Osmoregulation by betaine involves well-orchestrated gene expression

Betaine critically contributes to the control of hepatocellular hydration and provides protection of the liver from different kinds of stress. To investigate how the hepatocellular hydration state affects gene expression of enzymes involved in the metabolism of betaine and related organic osmolytes we used qRT-PCR gene expression studies in rat hepatoma cells as well as metabolic and gene expression profiling in primary hepatocytes of both wild-type and 5,10-methylenetetrahydrofolate reductase (MTHFR) deficient mice. Anisotonic incubation caused co-ordinated adaptive changes in the expression of various genes involved in betaine metabolism, in particular of betaine homocysteine methyltransferase (BHMT), dimethylglycine dehydrogenase (DMGDH), and sarcosine dehydrogenase (SARDH). The expression of betaine-degrading enzymes was downregulated by cell shrinking and strongly induced by an increase in cell volume under hypotonic conditions. Metabolite concentrations in the culture system changed accordingly. Expression changes were mediated through tyrosine kinases, cyclic nucleotide-dependent protein kinases and JNK-dependent signalling. Assessment of hepatic gene expression using a customised microarray chip showed that hepatic betaine depletion in Mthfr-/- mice was associated with alterations that were comparable to those induced by cell swelling in hepatocytes. In conclusion, the adaptation of hepatocytes to changes in cell volume involves the co-ordinated regulation of betaine synthesis and degradation and concomitant changes in intracellular osmolyte concentrations. The existence of such a well-orchestrated response underlines the importance of cell volume homeostasis for liver function and of methylamine osmolytes such as betaine as hepatic osmolytes.

Hoffmann, L., et al., Osmotic regulation of hepatic betaine metabolism. Am J Physiol Gastrointest Liver Physiol, 2013

Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress

Elevated tear osmolarity is one of the key pathological factors in dry eye leading to ocular discomfort associated with damage to the ocular surface and inflammation. The aim of this study was to determine the capacity of the organic osmolyte, betaine, to act as an osmoprotectant against hypertonic stress-induced human corneal epithelial cell shrinkage and apoptosis using in vitro cell culture models. Human corneal limbal epithelial (HCLE) cells exposed to culture medium for 16 h at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) in the presence or absence of betaine (5 or 10 mM) were evaluated for cell volume changes; cell viability; and apoptosis. Betaine (10 mM) ameliorated hyperosmotically induced reduction of cell volume (from 27% reduction to 11%) and resulted in increased mitochondrial activity (by 17%) and an increase in viable cell numbers (by 12%) compared to controls (exposure to hyperosmotic medium without betaine). Hyperosmotically shocked HCLE cells in the presence of betaine (10 mM) halved the number of damaged cells (apoptotic/necrotic) compared to cells in the absence of betaine. The presence of betaine (at 5 or 10 mM) significantly reduced the activity of caspase-8, -9 and -3/7 and release of TNF-α was also reduced by 34% or 55% after exposure of HCLE to 500 mOsm in the presence of 5 or 10 mM betaine, respectively. Using polyclonal antibody against Betaine/GABA transporter 1 (BGT-1), we detected the presence of BGT-1 in HCLE. We demonstrated that the transport of betaine was facilitated by increased osmolarity. In conclusion, betaine stabilized corneal epithelial cell volume under hyperosmotic stress and limited hyperosmotic stress-induced HCLE apoptosis.

Garrett, Q., et al., Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress. Experimental Eye Research, 2013

Macromolecular crowding regulates assembly of mRNA stress granules after osmotic stress: new role for compatible osmolytes

The massive uptake of compatible osmolytes such as betaine, taurine and myo-inositol is a protective response shared by all eukaryotes exposed to hypertonic stress. Their accumulation results mostly from the expression of specific transporters triggered by the transcriptional factor NFAT5/TonEBP. This allows the recovery of the cell volume without increasing intracellular ionic strength. In this study, we consider the assembly and dissociation of mRNA stress granules (SGs) in hypertonic-stressed cells and the role of compatible osmolytes. In agreement with in vitro results obtained on isolated mRNAs, both macromolecular crowding and a high ionic strength favor the assembly of SGs in normal rat kidney epithelial cells. However, after hours of constant hypertonicity, the slow accumulation in the cytoplasm of compatible osmolytes via specific transporters both reduces macromolecular crowding and the ionic strength, thus leading to the progressive dissociation of SGs. In line with this, when cells are exposed to hypertonicity in order to accumulate large amount of compatible osmolytes, the formation of SGs is severely impaired and cells increase their chances of survival to another hypertonic episode. Altogether, these results indicate that the impact of compatible osmolytes on the mRNA-associated machineries and especially that associated with SGs may play an important role in the cell resistance and adaption to hyperosmolarity in many tissues like kidney and liver.

Bounedjah, O., et al., Macromolecular crowding regulates assembly of mRNA stress granules after osmotic stress: new role for compatible osmolytes. J Biol Chem, 2012. 287(4): p. 2446-58

Variability of plasma and urine betaine in diabetes mellitus and its relationship to methionine load test responses: an observational study

BACKGROUND: Since betaine is an osmolyte and methyl donor, and abnormal betaine loss is common in diabetes mellitus (>20% patients), we investigated the relationship between betaine and the post-methionine load rise in homocysteine, in diabetes and control subjects. The post-methionine load test is reported to be both an independent vascular risk factor and a measure of betaine sufficiency.
METHODS: Patients with type 2 diabetes (n = 34) and control subjects (n = 17) were recruited. We measured baseline fasting plasma and 4-hour post-methionine load (L-methionine, 0.1 mg/kg body weight) concentrations of homocysteine, betaine, and the betaine metabolite N,N-dimethylglycine. Baseline urine excretions of betaine, dimethylglycine and glucose were measured on morning urine samples as the ratio to urine creatinine. Statistical determinants of the post-methionine load increase in homocysteine were identified in multiple linear regression models.
RESULTS: Plasma betaine concentrations and urinary betaine excretions were significantly (p > 0.001) more variable in the subjects with diabetes compared with the controls. Dimethylglycine excretion (p = 0.00014) and plasma dimethylglycine concentrations (p = 0.039) were also more variable. In diabetes, plasma betaine was a significant negative determinant (p > 0.001) of the post-methionine load increase in homocysteine. However, it was not conclusive that this was different from the relationship in the controls. In the patients with diabetes, a strong relationship was found between urinary betaine excretion and urinary glucose excretion (but not with plasma glucose).
CONCLUSIONS: Both high and low plasma betaine concentrations, and high and low urinary betaine excretions, are more prevalent in diabetes. The availability of betaine affects the response in the methionine load test. The benefits of increasing betaine intake should be investigated.

Lever, M., et al., Variability of plasma and urine betaine in diabetes mellitus and its relationship to methionine load test responses: an observational study. Cardiovasc Diabetol, 2012. 11(1): p. 34.

Betaine induces differential and selective conformational stability and enhances aggregation in some proteins in the presence of surfactants

In this study, we extensively report the effect of glycine betaine during the refolding of partially folded bovine alpha-lactalbumin (alpha-LA) in presence of hexadecyl trimethyl ammonium bromide (HTAB), and Ribonuclease A (RNAse A) in presence of sodium dodecyl sulfate (SDS) by different complementary biophysical, light scattering, and microscopic techniques. Though a substantial refolding/compaction was observed in both the studied proteins, the fluorescence studies contradicted the finding obtained from circular dichroism spectroscopy (CD) in case of alpha-LA. CD stopped flow showed extensive presence of intermediates during the refolding of proteins which could potentially lead to aggregation. The aggregates as observed in dynamic light scattering (DLS), in alpha-LA were massive as compared to RNAse A and was directly proportional to betaine concentration. The zeta potential confirmed that the aggregates are a direct manifestation of strong aggregating and/or immense preferential excluding tendency of GB and not because of charge neutralization; however a possible role of conformational change as observed in FTIR spectroscopy cannot be completely ruled out. In contrary though RNAse A showed a substantial refolding, the final state of the folded protein was significantly different from the native state. These findings for alpha-LA and RNAse A were further supported by electron microscopic and thermodynamic studies. We thus propose that betaine has a strong macromolecular excluding tendency, primarily directed to shield the hydrophobic exposure either by refolding or aggregation, and depending on the hydrophobicity of the proteins, the functional restoration of the protein is manifested.

Misra, P.P. and N. Kishore, Glycine betaine: A widely reported osmolyte induces differential and selective conformational stability and enhances aggregation in some proteins in the presence of surfactants. Biopolymers, 2012. 97(12): p. 933-49.

Betaine improves performance under heat stress in rabbit model

The objective of this study was to determine the effects of dietary betaine supplementation on growth performance, carcass characteristics, rectal temperature, respiration rate and immune response of growing rabbits under high ambient temperature.

A total of 120 weaned New Zealand White male rabbits, 6 weeks old, were randomly divided into five experimental treatments (24 each). Animals were fed ad libitum the basal diet supplemented with 0 (control), 250, 500, 750 and 1000 mg betaine/kg diet from 6 to 12 weeks of age. Animals were provided with water freely. The average daily temperature and relative humidity inside the rabbitry were 30.3 ± 0.9 °C and 76.2 ± 2.5%, respectively. Under heat stress conditions, diet significantly increased the body weight and hot carcass weight and significantly reduced the feed conversion. Dietary 1000 and 750 mg betaine/kg increased (P < 0.05) final body weights (2529.1 and 2418.5 g, respectively) compared with the control (2110.3 g). Betaine supplementation ameliorated some of the adverse effects of heat stress on immune responsiveness, rectal temperature and respiration rate. Dietary 250, 500, 750 and 1000 mg betaine/kg led to a decrease in rectal temperature (40.03, 39.85, 39.63 and 39.53 °C, respectively) compared with the control (40.20 °C). The inclusion of 1000 mg betaine/kg in the growing rabbits' diets nearly doubled the humoral and immune responses compared to the controls (P < 0.05) and significantly reduced rectal temperature and respiration rate. Serum T3, T4, total protein, globulin and total lipids were significantly increased while serum glucose concentration was significantly decreased due to dietary betaine.

In conclusion, supplemental dietary betaine enhanced growth performance and humoral and cell-mediated immunity as well as reduced rectal temperature and respiration rate in growing rabbits subjected to heat stress. From an economic point of view, high levels of betaine are not recommended because betaine is reasonably effective at lower, less expensive, doses.

Hassan et al (2011). "Effect of dietary betaine supplementation on growth, carcass and immunity of New Zealand White rabbits under high ambient temperature." Livestock Science 135(2-3): 103-109.

Betaine alleviates heat stress in chickens

Exposing broilers to a high temperature increases water and electrolyte K(+) and Na(+) excretion, which negatively affects the heat dissipation capacity and acid-base homeostasis, resulting in losses in growth performance.

In this experiment, the efficacy of providing oral rehydration therapy and betaine on growth performance, acid-base balance, and water and electrolyte retention was evaluated. A total of 432 one-day-old broiler chicks (Cobb) were allocated to 72 metabolic cages and reared to 31 d of age under standard conditions. From 32 to 41 d of age, chicks were exposed to heat stress (ambient temperature, 32 degrees C) and high RH (80 to 100% RH) for 9 h daily. The ameliorative effects of a 3 x 3 factorial array of treatments administered via drinking water were evaluated in 8 replicates of 6 chicks per cage for each treatment. Two oral rehydration therapy (ORT) fluids, based on either citrate or bicarbonate salts, were added to tap water. In addition, betaine was added to tap water at an inclusion rate of 0, 500, or 1,000 mg/L to complete the array of 9 liquid-based treatments. Growth performance was assessed at 32, 35, and 41 d of age. From 32 to 35 d of age, chicks receiving ORT fluids exhibited improved growth performance, water balance, and electrolyte (K(+), Na(+)) retention. In addition, the physiological response to stress was attenuated, as indicated by lower heterophil-to-lymphocyte ratios and blood glucose concentrations relative to the negative controls. The addition of betaine at an inclusion rate of 500 mg/L improved BW gain. From d 36 to 41, treatments did not significantly influence growth performance, which suggests that chicks receiving tap water were able to compensate and adapt to the heat-stress conditions.

The results demonstrate that the beneficial effects of providing ORT fluids and 500 mg of betaine/L were observed only during the first 4 d of heat exposure. After this period, adaptation to the heat appears to occur, and none of the treatments was successful in improving growth performance.

Sayed and Downing (2011). "The effects of water replacement by oral rehydration fluids with or without betaine supplementation on performance, acid-base balance, and water retention of heat-stressed broiler chickens." Poult Sci 90(1): 157-67.

A review on the clinical significance of betaine

Betaine is an essential osmolyte and source of methyl groups and comes from either the diet or by the oxidation of choline. Its metabolism methylates homocysteine to methionine, also producing N,N-dimethylglycine. Betaine insufficiency is associated with the metabolic syndrome, lipid disorders and diabetes, and may have a role in vascular and other diseases. Betaine is important in development, from the pre-implantation embryo to infancy. Betaine supplementation improves animal and poultry health, but the effect of long-term supplementation on humans is not known, though reports that it improves athletic performance will stimulate further studies. Subsets of the population that may benefit from betaine supplementation could be identified by the laboratory, in particular those who excessively lose betaine through the urine. Plasma betaine is highly individual, in women typically 20-60 [mu]mol/L and in men 25-75 [mu]mol/L. Plasma dimethylglycine is typically < 10 [mu]mol/L. Urine betaine excretion is minimal, even following a large betaine dose. It is constant, highly individual and normally < 35 mmol/mole creatinine. The preferred method of betaine measurement is by LC-MS/MS, which is rapid and capable of automation. Slower HPLC methods give comparable results. Proton NMR spectrometry is another option but caution is needed to avoid confusion with trimethylamine-N-oxide.

Lever and Slow (2010). "The clinical significance of betaine, an osmolyte with a key role in methyl group metabolism." Clinical Biochemistry 43(9): 732-744.

Osmolyte protection against urea denaturation of chymotrypsin

The destabilizing effect of urea on proteins can be counteracted by methylamines (osmolytes), such as trimethylamine N-oxide (TMAO), betaine, and sarcosine. This study compared the counteracting effects of these methylamines on urea-induced denaturation of α-chymotrypsin (CT). They measured the hydrodynamic diameter (dH) and the thermodynamic properties (Tm, ΔH, ΔGU, and ΔCp) with dynamic light scattering (DLS) and differential scanning calorimeter (DSC), respectively. The osmolytes strongly counteracted the urea actions on α-chymotrypsin, with TMAO having the strongest effect.

Venkatesu et al (2009). "Osmolyte Counteracts Urea-Induced Denaturation of alpha-Chymotrypsin." The Journal of Physical Chemistry B 113(15): 5327-5338.

Effect of pH on protein stabilizing property of betaine

This study investigated the pH dependence of the stabilizing effect of betaine on three different proteins - α-lactalbumin (α-LA), lysozyme and ribonuclease-A (RNase-A). They measured Tm (midpoint of denaturation), ΔHm (denaturational enthalpy change at Tm), ΔCp (constant-pressure heat capacity change) and ΔGDo (denaturational Gibbs energy change at 25 °C) of proteins in the presence of different betaine concentrations.

They found that betaine:

- stabilizes RNase-A at all pH values
- has opposite effects on α-LA and lysozyme at high pH and low pH values
- did not significantly change ΔHm and ΔCp

Singh et al (2009). "Glycine betaine may have opposite effects on protein stability at high and low pH values." Biochim Biophys Acta. Epub

Individuality of plasma and urine betaine and DMG

The individuality (within subject consistency) of plasma and urine betaine and N,N-dimethylglycine (DMG) was compared in two groups of 8 males (ages 19 to 40) either over a single day or over an 8 week period. The study found that plasma betaine and urinary betaine excretions are more individual than DMG, and that plasma and urine betaine are highly individual in the general population.

Lever et al (2009). "Plasma and urine betaine and dimethylglycine variation in healthy young male subjects." Clin Biochem 42: 706-12.

Papers 2004-7

Lever et al (2004). "Short and long-term variation of plasma glycine betaine concentrations in humans." Clin Biochem 37(3): 184-90.

Slow et al (2004). "Betaine analogues alter homocysteine metabolism in rats." Int J Biochem Cell Biol 36(5): 870-80.

Lever et al (2005). "Homocysteine, glycine betaine, and N,N-dimethylglycine in patients attending a lipid clinic." Metabolism - Clinical and Experimental 54(1): 1-14.

Slow et al (2005). "The betaine content of New Zealand foods and estimated intake in the New Zealand diet." J Food Comp Anal 18: 473-85.

Lever et al (2007). "An abnormal urinary excretion of glycine betaine may persist for years." Clin Biochem 40(11): 798-801.

Lever et al (2007). "Inter- and intra-individual variations in normal urinary glycine betaine excretion." Clin Biochem 40(7): 447-53.

Lever et al (2007). "Sex differences in the control of plasma concentrations and urinary excretion of glycine betaine in patients attending a lipid disorders clinic." Clin Biochem 40(16-17): 1225-31.

Atkinson et al (2008). "Dietary and supplementary betaine: acute effects on plasma betaine and homocysteine concentrations under standard and postmethionine load conditions in healthy male subjects." Am J Clin Nutr 87(3): 577-585.