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 inhibits vascularization via suppression of Akt in the retinas of streptozotocin-induced hyperglycemic rats

Diabetic retinopathy is a severe microvascular complication amongst patients with diabetes, and is the primary cause of visual loss through neovascularization. Betaine is one of the components of Fructus Lycii. In the present study, the effects of betaine on the expression levels of vascular endothelial growth factor (VEGF) and hypoxiainducible factor (HIF)1alpha in association with the Akt pathway were investigated in the retinas of streptozotocin (STZ)induced diabetic rats using western blot and immunohistochemical analyses. The results of the present study revealed that the expression levels of VEGF, HIF1alpha, and Akt were increased in the retinas of the STZinduced diabetic rats. Betaine treatment attenuated this increase in VEGF and HIF1alpha expression via suppression of diabetesinduced Akt activation in the retinas of the diabetic rats. The results suggested that betaine may potentially be used to delay the onset of complications associated with diabetic retinopathy via inhibition of retinal neovascularization in patients with diabetes.

Kim, Y.G., et al., Betaine inhibits vascularization via suppression of Akt in the retinas of streptozotocin-induced hyperglycemic rats. Mol Med Rep, 2015

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.

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

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

High dietary betaine intake associated with less advanced AMD

Objective
We evaluated monozygotic twin pairs with discordant age-related macular degeneration (AMD) phenotypes to assess differences in behavioral and nutritional factors.

Design
Case series.

Participants
Caucasian male twin pairs from the United States Twin Study of Macular Degeneration.

Methods
Twin pairs were genotyped to confirm monozygosity. Ocular characteristics were evaluated based on fundus photographs using the Wisconsin Grading System and a 5-grade Clinical Age-Related Maculopathy Staging System. We selected twin pairs discordant in each of the following phenotypic categories: Stage of AMD (n = 28), drusen area (n = 60), drusen size (n = 40), and increased pigment area (n = 56). The Wilcoxon signed-rank test and linear regression were used to assess associations between behavioral and nutritional characteristics and each phenotype within discordant twin pairs.

Main Outcome Measures
Differences in smoking and dietary factors within twin pairs discordant for stage of AMD, drusen area, drusen size, and pigment area.

Results
Representative fundus photographs depict the discordant phenotypes. Pack-years of smoking were higher for the twin with the more advanced stage of AMD (P = 0.05). Higher dietary intake of vitamin D was present in the twins with less severe AMD (P = 0.01) and smaller drusen size (P = 0.05) compared with co-twins, adjusted for smoking and age. Dietary intakes of betaine and methionine were significantly higher in the twin with lower stage of AMD (P = 0.009) and smaller drusen area (P = 0.03), respectively.

Conclusions
The twin with the more advanced stage of AMD, larger drusen area, drusen size, and pigment area tended to be the heavier smoker. The twin with the earlier stage of AMD, smaller drusen size and area, and less pigment tended to have higher dietary vitamin D, betaine, or methionine intake. Results suggest that behavioral and nutritional factors associated with epigenetic mechanisms are involved in the etiology of AMD, in addition to genetic susceptibility.

Seddon, J.M., et al., Smoking, dietary betaine, methionine, and vitamin D in monozygotic twins with discordant macular degeneration: epigenetic implications. Ophthalmology, 2011. 118(7): p. 1386-94.