Betaine regulates cellular volume and acts as an intracellular osmoprotectant in the adaptation of cells to stress. Hyperosmotic stress and ultraviolet B radiation inhibit cell proliferation and induce apoptosis in human keratinocytes [21, 29]. Osmotic stress signals keratinocytes to differentiate . Betaine prevents hyperosmotically induced and UV radiation-induced cell shrinkage, a hallmark of apoptotic cell death  in normal human keratinocytes  and human HaCaT cells [21, 32]. Dietary and endogenous betaine exists primarily in the liver and kidney but also significantly in other tissues, including skin [19, 33]. Our findings show that betaine is a natural component in the skin, and we are the first to our knowledge to describe that the epidermis contains by number more betaine than dermis. Balanced hydration in the epidermis is needed, for instance, for the optimal differentiation of keratinocytes . Keratinocytes and fibroblasts also express the betaine/γ-amino-n-butyric acid (GABA) transporter BGT-1, the expression of which rises when they are exposed to hyperosmotic conditions or UV irradiation with concomitant increase in the betaine uptake [20, 22, 32]. However, the volunteers in the study were not selected based on any specific exclusion/inclusion criteria, but were women having a mammoplasty. As betaine is used in cosmetic products, a wash out period would have needed to exclude the effect of utilization of betaine containing products in the results. Furthermore, controlling the dietary betaine would have been important in evaluating the effect of diet in the betaine content of the skin. With the scope of this publication, this was not, however, possible, but a simpler approach was selected instead to obtain preliminary results. Therefore, additional studies with more control and increased sample number are warranted.
The increase in TEER at 12 h and 24 h indicates that betaine improves TJ integrity and contributes to the inside-out barrier, and this function is yet another addition to moisturizing function of betaine: maintenance of hydration of the skin through the regulation of TJs by improving water retention . Notably, at 1 h and 2 h, betaine protected cells from the decrease in TEER that was observed with untreated NHEKs. TEER is sensitive to temperature, and the decline in TEER after replacement of the medium might have been due variations in temperature . Osmolytes can be metabolic and cytoprotective under many perturbing conditions, and extreme temperature is a stressor against which osmolytes stabilize cellular function . No consistent changes in TJ protein expression was observed in differentiated NHEKs explaining the effect in TEER; OCLN alone was decreased significantly, but not steadily with all tested betaine concentrations. However, betaine was able to increase the ZO-1 protein and also the differentiation marker involucrin when analyzed with betaine-treated freshly isolated human keratinocytes during the 72-h differentiation. We have previously shown, that the expression of ZO-1 in keratinocytes is regulated by p38 signaling pathway . In experiments with diabetic male mice in relation to protection of blood-testis barrier, betaine has been shown to inhibit p38 phosphorylation and upregulate the protein expressions of ZO-1, OCLN, CLDN-11, N-cadherin, and connexin-43  so it might be a plausible mechanism of action for betaine. However, the increase in ZO-1 in combination with the TEER-increasing effect obtained with the topical modelling of betaine in differentiated NHEKs shown in this study indicate jointly that betaine might strengthen the skin barrier through its capacity to modulate TJ proteins.
The effects of betaine on barrier function in a rat organotypic model of human epidermis in a similar setting has been investigated previously, and no evident changes in histology, epidermal thickness, or proliferation were observed . Likewise, in REK model, no changes in the mRNA expression of OCLN, and ZO-1, could be observed, which indicates that betaine does not have an impact on the studied TJ protein gene regulation in long term exposure of high amount of betaine, but rather affects to TJs are in a protein level, and our finding is in accordance with previous study showing that organic osmolytes affect TJs through protecting the native protein protein conformation . Betaine has been shown to stabilize proteins from abiotic stress through its high water binding capacity and ability to retain inorganic ions around itself .
In a model in which human skin is explanted to a SCID mouse and with cultured human skin equivalent (HSE), the intercellular diffusion of subcutaneous, or basolateral, tracer stops at the level of TJs—specifically, at the level of ZO-1 or OCLN . When the human skin explant and the HSE model are irradiated with UVB, the tracer passes through ZO-1- and OCLN-rich sites, highlighting the importance of TJs in the intercellular permeability barrier to small molecules and the function of UVB as cause of TJ dysfunction . We also examined the permeation of topical mannitol after UVB exposure (30 mJ/cm2) in the REK model but failed to observe any differences between the control, UVB-treated, and UVB-plus-betaine-treated samples. In a previous study with REK model, betaine was able to rescue UVB induced permeability increase , but the difference is that we utilized cells with mature TJs instead of developing TJs, and developing TJs might be more sensitive to the effects of UVB  The stratum corneum, just above the TJs, is the barrier to water movement across the skin , and it has been challenging to demonstrate the function of TJs in the outside-in permeability barrier due to difficulties in accessing the TJs below the stratum corneum . Cornified envelope lipids are key determinants of the types of substances that can be absorbed from the skin surface through diffusion, and these substances are typically nonpolar and less than 500 Da . Mannitol is a 182 Da highly polar sugar alcohol that requires an enhancer for its penetration , and it is typically used as a low permeation standard in percutaneous absorption models. We suspect that this property explains in part the lack of an effect; thus, different tracers, such as caffeine with known permeability characteristics, could be used in these types of assays instead [45, 46]. The differences in the permeability of human versus rat skin should be, however, taken into account, as caffeine is more readily absorbed through rat skin than human skin .
TJ proteins OCLN, CLDN-1, and CLDN-4 both at mRNA and protein level are not downregulated on exposure of keratinocytes to UVB irradiation [38, 40]. Similarly, we observed no change in the mRNA expression of the TJ components CLDN-4, ZO-1, and OCLN when REK cultures were exposed to UVB. Although OCLN was downregulated at certain concentrations of topical betaine in the NHEK model, the changes were not consistent. TJs and adherens junctions below them, are highly dynamic and regulated by an intricate network of phosphorylation and dephosphorylation reactions by specific protein kinases and phosphatases [48, 49], by translocation of the TJ proteins in and out of these structures , and by modulation of the actin cytoskeleton, which is linked to TJs . Betaine did not affect the protein levels of adherens junction components E-cadherin or β-catenin in freshly isolated keratinocytes, nor CD147 or TGF-β2 mRNA levels in the REK model. CD147, also known as basigin and EMMPRIN, associates with monocarboxylate transporters, interacts with integrins, and forms a complex with CD44, the major hyaluronan receptor . At least 3 TGF-β isoforms have been identified: TGF-β1, TGF-β2, and TGF-β3. TGF-β2 is a growth factor that functions in highly malignant and invasive phenotypes of skin cancers . It is also enhanced by UV radiation , but we did not see any changes in the expression of these genes—not even in the control treatment. Betaine has anti-inflammatory effects [55, 56], but whether betaine alleviates the inflammatory responses that arise from extensive UV irradiation , is still unknown. Nevertheless, it has been shown that in keratinocytes UV irradiation induces osmolyte transporters and that osmolytes are taken up inside the cells after UV irradiation, and that is part of the mechanisms by which cells act against harmful effects of UV radiation [20, 32].
In this study, there was no clinical intervention with topical or oral betaine, and there is no knowledge whether the donors utilized any topical creams containing betaine. It is also possible that the betaine in the epidermis originates from the biosynthetic route from choline, but most likely it originates from the uptake from the extracellular sources, which has been suggested to be the primary source of betaine . Thus, without topical application, the betaine in the epidermis most likely originates from the diet via systemic circulation in the blood [19, 33].