PNPLA1 knockdown inhibits esterification of γ-linolenic acid to ceramide 1 in differentiated keratinocytes.

Patatin-like phospholipase domain-containing 1 (PNPLA1) is crucial in the esterification of linoleic acid (LA; 18:2n-6) to ω-hydroxy fatty acids (FA) of ceramide 1 (Cer1), the major barrier lipid of the differentiated epidermis. We previously reported that γ-linolenic acid (GLA; 18:3n-6) as well as LA is esterified to Cer1 subspecies with sphingosine (d18:1) or eicosasphingosine (d20:1) amide-linked to two different ω-hydroxy FA (30wh:0; 32wh:1). Here, we further investigated whether PNPLA1 is also responsible for esterification of GLA to these Cer1 subspecies in normal human keratinocytes (NHK). As late/terminal differentiation was induced in NHK, PNPLA1 and differentiation markers were expressed, and LA-esterified Cer1 subspecies (18:2n-6/C30wh:0 or C32wh:0/d18:1; 18:2n-6/C32wh:0/d20:1) were detected, which were further increased with LA treatment. GLA-esterified Cer1 subspecies (18:3n-6/C30wh:0 or C32wh:0/d18:1; 18:3n-6/C32wh:0/d20:1) were detected only with GLA treatment. Specific small interfering RNA-mediated knockdown of PNPLA1 (KDP) in differentiated NHK decreased levels of these LA-esterified Cer1 subspecies overall and of involucrin (IVL), a terminal differentiation marker. Moreover, KDP resulted in lesser LA/GLA responses as characterized by more significant decreases in IVL and LA/GLA-esterified Cer1 subspecies overall and an accumulation of non-esterified ω-hydroxy ceramides, their putative precursors; the decrease of 18:3n-6/C32wh:0/d18:1, the predominant GLA-esterified Cer1 subspecies, specifically paralleled the increase of C32wh:0/d18:1, its corresponding precursor. PNPLA1 is responsible for NHK terminal differentiation and also for esterification of GLA to the ω-hydroxy FA of Cer1.

The influence of dietary patterns on skin bacterial diversity, composition, and co-occurrence relationships at forearm and neck sites of healthy Korean adults.

AIMS: Diet and nutrition are important aspects of skin physiology and health. However, the influence of diet on the bacterial flora of different skin sites is not well understood. Therefore, we investigated the relationship between dietary patterns (DPs) and skin bacterial flora on the forearm (a dry site) and the neck (a sebaceous site) of healthy Korean adults. METHODS AND RESULTS: In metagenomics analysis, Shannon and Simpson indices were higher on the forearm than on the neck and were negatively correlated with the two dominant species, Cutibacterium acnes and Staphylococcus epidermidis, on two skin sites. In addition, the Simpson index of the forearm was positively associated with DP1 (characterized by a high intake of vegetables, mushrooms, meat, fish and shellfish, seaweed, and fat and oil), while that on the neck was negatively associated with DP2 (characterized by a high intake of fast food). A high intake of DP1 was associated with a lower abundance of dominant species, including C. acnes, and higher degrees of the co-occurrence network, whereas a high intake of DP2 was associated with the opposite pattern. CONCLUSIONS: Specific diets may impact both skin bacterial diversity and composition, as well as the co-occurrence of bacteria, which may vary across different skin sites.

Dietary silk protein, sericin, improves epidermal hydration with increased levels of filaggrins and free amino acids in NC/Nga mice.

Epidermal hydration is maintained primarily by natural moisturising factors (NMF), of which free amino acids (AA) are major constituents that are generated by filaggrin degradation. To identify dietary sources that may improve skin dryness of atopic dermatitis (AD), we investigated dietary effects of silk proteins, sericin and fibroin, on epidermal levels of hydration, filaggrins and free AA, as well as PPARγ, peptidylarginine deiminase-3 (PAD3) and caspase-14 proteins involved in filaggrin expression and degradation processes. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or diets with 1 % sericin (group S) or fibroin (group F) for 10 weeks. In group S, epidermal levels of hydration, total filaggrins and total free AA, as well as PPARγ, PAD3 and caspase-14, which were reduced in group CA, were increased to higher or similar levels of a normal control group of BALB/c mice (group C). Furthermore, profilaggrin, a precursor with multiple filaggrin repeats, and three repeat intermediates were increased, while two repeat intermediates and filaggrin were decreased in parallel with increased levels of glutamate and serine, major AA of NMF in group S. Despite increased levels of total filaggrins, total free AA, PPARγ and PAD3, epidermal levels of hydration, glutamate, serine and caspase-14 were not increased, but other minor AA of NMF were highly detected in group F. Dietary sericin improves epidermal hydration in parallel with enhancing profilaggrin expression and degradation into free AA that is coupled with elevated levels of PPARγ, PAD3 and caspase-14 proteins.

Positive Correlation of Triacylglycerols with Increased Chain Length and Unsaturation with ω-O-Acylceramide and Ceramide-NP as Well as Acidic pH in the Skin Surface of Healthy Korean Adults.

Triacylglycerols (TG) play an important role in skin homeostasis including the synthesis of ω-O-acylceramides (acylCER) required for skin barrier formation by providing linoleic acid (C18:2n6). However, the overall relationships of TG species with various ceramides (CER) including CER-NP, the most abundant CER, ω-O-acylCER, and another acylCER, 1-O-acylCER in human SC, remain unclear. Therefore, we investigated these relationships and their influence on skin health status in healthy Korean adults. Twelve CER subclasses including two ω-O-acylCER and two 1-O-acylCER were identified with CER-NP consisting of approximately half of the total CER. The ω-O-acylCER species exhibited positive relationships with TG 52:4 and TG 54:2 containing C18:2, while interestingly, 1-O-acylCER containing ester-linked C14:0 and C16:0 demonstrated positive relationships with TG 46-50 including C14:0 and C16:0, respectively. In addition, CER-NP and CER-NH showed positive correlations with TG 52-54 containing C18:2 or C18:3. A lipid pattern with higher levels of CER including CER-NP and ω-O-acylCER with TG 54 and TG with 5-6 double bonds was related to good skin health status, especially with acidic skin pH. Collectively, TG with increased chain length and unsaturation seemed to improve CER content, and profiles such as higher acylCER and CER-NP improved skin health status by fortifying skin barrier structure.

Glucocerebroside-Containing Milk Concentrated Powder Suppresses Oxidative Stress and Photoaging in the Skin of Hairless Mice.

This study investigated the protective effects of glucocerebroside-containing buttermilk concentrated powder (GCBM) on oxidative stress and photoaging in ultraviolet B (UVB)-irradiated hairless mice. We measured antioxidant enzyme activities, collagen synthesis-related pathways, and moisturizing-related factors in the dorsal skin of mice. We observed that dietary supplementation with GCBM increased antioxidant enzyme activity and decreased pro-inflammatory cytokine expression in the UVB-irradiated dorsal skin. Furthermore, dietary supplementation with GCBM inhibited wrinkle formation by suppressing the JNK/c-FOS/c-Jun/MMP pathway and stimulating the TGF-ßRI/Smad3/procollagen type I pathway. Dietary supplementation with GCBM also increased skin moisturization by stimulating hyaluronic acid and ceramide synthesis in the dorsal skin. Therefore, buttermilk powder supplementation helps prevent photoaging and can be used as an effective component in developing anti-photoaging products.

Borage Oil Enhances Lamellar Body Content and Alters Fatty Acid Composition of Epidermal Ceramides in Essential Fatty Acid-Deficient Guinea Pigs.

Borage oil [BO: 40.9% linoleic acid (LNA) and 24.0% γ-linolenic acid (GLA)] reverses disrupted epidermal lipid barrier in essential fatty acid deficiency (EFAD). We determined the effects of BO on lamellar body (LB) content and LNA and GLA incorporation into epidermal ceramide 1 (CER1) and epidermal ceramide 2 (CER2), major barrier lipids. EFAD was induced in guinea pigs by a diet of 6% hydrogenated coconut oil (HCO) for 10 weeks (group HCO) or 8 weeks followed by 6% BO for 2 weeks (group HCO + BO). LB content and LNA and GLA incorporation into CER1 were higher in group HCO + BO than in group HCO. Small but significant levels of LNA, GLA, and their C20-metabolized fatty acids [dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA)] were incorporated into CER2, where ARA was detected at a level lower than LNA, but DGLA incorporation exceeded that for GLA in group HCO + BO. Dietary BO enhanced LB content and differential incorporation of GLA into CER1 and DGLA into CER2.

Effects of Lactobacillus plantarum CJLP55 on Clinical Improvement, Skin Condition and Urine Bacterial Extracellular Vesicles in Patients with Acne Vulgaris: A Randomized, Double-Blind, Placebo-Controlled Study.

Lactobacillus plantarum CJLP55 has anti-pathogenic bacterial and anti-inflammatory activities in vitro. We investigated the dietary effect of CJLP55 supplement in patients with acne vulgaris, a prevalent inflammatory skin condition. Subjects ingested CJLP55 or placebo (n = 14 per group) supplements for 12 weeks in this double-blind, placebo-controlled randomized study. Acne lesion count and grade, skin sebum, hydration, pH and surface lipids were assessed. Metagenomic DNA analysis was performed on urine extracellular vesicles (EV), which indirectly reflect systemic bacterial flora. Compared to the placebo supplement, CJLP55 supplement improved acne lesion count and grade, decreased sebum triglycerides (TG), and increased hydration and ceramide 2, the major ceramide species that maintains the epidermal lipid barrier for hydration. In addition, CJLP55 supplement decreased the prevalence of Proteobacteria and increased Firmicutes, which were correlated with decreased TG, the major skin surface lipid of sebum origin. CJLP55 supplement further decreased the Bacteroidetes:Firmicutes ratio, a relevant marker of bacterial dysbiosis. No differences in skin pH, other skin surface lipids or urine bacterial EV phylum were noted between CJLP55 and placebo supplements. Dietary Lactobacillus plantarum CJLP55 was beneficial to clinical state, skin sebum, and hydration and urine bacterial EV phylum flora in patients with acne vulgaris.

Oral supplementation of Lithospermum erythrorhizon prevents the development of atopic dermatitis with reducing ceramide degradation in the epidermis of NC/Nga mice.

Lithospermum erythrorhizon Sieb. et Zucc. (LE) is widely used in the treatment of abnormal skin conditions, but its systemic efficacy, especially in atopic dermatitis (AD), is not clear. To examine the systemic efficacy of LE on the clinical manifestation of AD-like skin lesions, NC/Nga mice, a murine model of AD, were fed a control diet (group CA: atopic control) or a diet with a 70% ethanol extract from 5% LE (group LE) for 10 weeks. In group LE, the clinical manifestation of AD-like skin lesions was prevented as the level of serum IgE, epidermal hyperproliferation, and the number and duration of scratching episodes, which were greater in group CA, were significantly reduced to a similar level of the normal control group of BALB/c mice (group C). In addition, the level of ceramides, the major lipid maintaining the epidermal barrier, in the epidermis of group LE was increased, and was inversely associated with a decreased protein level of ceramidase, an enzyme of ceramide degradation. However, the mRNA and the protein levels of serine palmitoyl transferase (enzyme for de novo ceramide synthesis) in groups C, CA and LE did not differ. It was demonstrated that oral supplementation with LE extract prevented the development of atopic dermatitis with reducing ceramide degradation coupled with a low expression of ceramidase protein.

Dietary Patterns Associated with Sebum Content, Skin Hydration and pH, and Their Sex-Dependent Differences in Healthy Korean Adults.

Sebum content, skin hydration and acidic skin pH are major factors in maintaining skin health. Various nutrients are reported to influence skin health, but the effect of dietary patterns (DPs) on skin health is unclear. In this study, we considered the DPs associated with these three skin health parameters in 84 healthy adults aged 19⁻37 years. Dietary intake was assessed using a food frequency questionnaire (FFQ) and skin health parameters were determined on the forehead of each subject. Among the four DPs extracted from the FFQ, DP2, characterized by a high intake of cereals, potatoes and starch, saccharides and fish and shellfish, was negatively associated with skin hydration. DP3, characterized by a high intake of potatoes and starch, seeds and nuts, fruits and eggs, was positively associated with acidic skin pH only before adjusting for potential confounders. On the other hand, DP4, characterized by a low intake of beans, and a high intake of meats, dairy products and beverages and alcohol, was negatively associated with acidic skin pH and positively associated with sebum content. The data stratified by sex revealed a negative association between skin hydration and DP2 in males and a negative association between sebum content and DP3 and a positive association between sebum content and DP4 in females. In conclusion, we demonstrated that specific DPs were associated with sebum content, skin hydration and pH in healthy Korean adults and that those associations were affected by sex.

Enhanced Triacylglycerol Content and Gene Expression for Triacylglycerol Metabolism, Acyl-Ceramide Synthesis, and Corneocyte Lipid Formation in the Epidermis of Borage Oil Fed Guinea Pigs.

Triacylglycerol (TAG) metabolism is related to the acyl-ceramide (Cer) synthesis and corneocyte lipid envelope (CLE) formation involved in maintaining the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil (BO: 40.9% linoleic acid (LNA) and 24.0% γ-linolenic acid (GLA)) in essential fatty acid (EFA) deficiency, lipidomic and transcriptome analyses and subsequent quantitative RT-PCR were performed to determine the effects of borage oil (BO) on TAG content and species, and the gene expression related to overall lipid metabolism. Dietary BO for 2 weeks in EFA-deficient guinea pigs increased the total TAG content, including the TAG species esterified LNA, GLA, and their C20 metabolized fatty acids. Moreover, the expression levels of genes in the monoacylglycerol and glycerol-3-phosphate pathways, two major pathways of TAG synthesis, increased, along with those of TAG lipase, acyl-Cer synthesis, and CLE formation. Dietary BO enhanced TAG content, the gene expression of TAG metabolism, acyl-Cer synthesis, and CLE formation.

Alterations in IL-4, IL-10 and IFN-γ levels synergistically decrease lipid content and protein expression of FAS and mature SREBP-1 in human sebocytes.

When anti-acne alternatives from dietary and plant sources are ingested, systemic alterations of interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ, individually or simultaneously, are induced at a 0.1-10.0-fold (×) range of normal physiological concentrations (1×). However, little is known about the effects of these cytokines on excess sebum, a pathophysiological factor of acne development. In this study, human sebocytes were treated with 0.1-10.0× of IL-4, IL-10, IL-12 and IFN-γ for 3 or 5 days to elucidate the effects on lipid content. Treatment with individual cytokines decreased the lipid content at specific concentrations rather than in a concentration-dependent manner. Specifically, 5.0× of IL-4, 5.0× of IFN-γ (5.0IFN), and 0.5×, 5.0× and 10.0× of IL-10 for 3 days, and 0.5× of IL-4 (0.5IL4) for 5 days decreased lipid content to 87.6-93.0% of the control. Treatment with other concentrations of IL-4, IL-10 and IFN-γ, and 0.1-10.0× of IL-12 did not alter lipid content. Combined treatment with 0.5IL4, 5.0IFN and 0.5× of IL-10 for 3 or 5 days decreased the lipid content more than each individual treatment. However, this effect was more evident after 3 days, in parallel with decreased levels of triglycerides, cholesterol esters and free fatty acids, the major lipid compositions of sebocytes, and decreased protein expression of fatty acid synthase (FAS) and mature sterol response element-binding protein-1 (SREBP-1), the lipogenesis-related factors, without altered cell proliferation. We demonstrated that suppressed IL-4 and IL-10 with enhanced IFN-γ synergistically decreased lipid content and protein expression of FAS and mature SREBP-1 in human sebocytes.

Borage oil restores acidic skin pH by up-regulating the activity or expression of filaggrin and enzymes involved in epidermal lactate, free fatty acid, and acidic free amino acid metabolism in essential fatty acid-deficient Guinea pigs.

Borage oil (BO) reverses a disrupted epidermal lipid barrier and hyperproliferation in essential fatty acid deficiency (EFAD). However, little is known about its effect on skin pH, which is maintained by epidermal lactate, free fatty acids (FFAs), and free amino acids (FAAs) which is generated by lactate dehydrogenase (LDH), secreted phospholipase A2 (sPLA2), or filaggrin degradation with peptidylarginine deiminase-3 (PADI3). We hypothesized that BO restores skin pH by regulating epidermal lactate, FFA metabolism, or FAA metabolism in EFAD. To test this hypothesis, EFAD was induced in guinea pigs by a hydrogenated coconut oil (HCO) diet for 8 weeks, followed by 2 weeks of a BO diet (group HCO + BO). As controls, groups HCO and BO were fed HCO or BO diets for 10 weeks. In group HCO + BO, skin pH, which was less acidic in group HCO, was restored; and epidermal lactate and total FFAs, including palmitate, stearate, linoleate, arachidate, behenate, and lignocerate, were higher than in group HCO. LDH and sPLA2 (mainly the PLA2G2F isoform) activities and protein expressions were similar between groups HCO + BO and BO. Epidermal acidic FAAs, as well as filaggrin and PADI3 protein and mRNA expressions were higher in group HCO + BO than in group HCO. Oleate, total FAAs including other FAAs, and LDH and sPLA2 mRNA expressions were not altered between groups HCO and HCO + BO. Basic FAAs were not altered among groups. Dietary BO restored acidic skin pH and increased epidermal levels of lactate, most FFAs, and acidic FAAs by up-regulating LDH, sPLA2, filaggrin, and PADI3 activities as well as protein or mRNA expressions in EFAD.

Associations among plasma vitamin C, epidermal ceramide and clinical severity of atopic dermatitis.

BACKGROUND/OBJECTIVES: Atopic dermatitis (AD), a chronic inflammatory skin disease, is accompanied by disruption of the epidermal lipid barrier, of which ceramide (Cer) is the major component. Recently it was reported that vitamin C is essential for de novo synthesis of Cer in the epidermis and that the level of vitamin C in plasma is decreased in AD. The objective of this study was to determine the associations among clinical severity, vitamin C in either plasma or epidermis, and Cer in the epidermis of patients with AD. SUBJECTS/METHODS: A total of 17 patients (11 male and 6 female) aged 20-42 years were enrolled. The clinical severity of AD was assessed according to the SCORAD (SCORing Atopic Dermatitis) system. Levels of vitamin C were determined in plasma and biopsies of lesional epidermis. Levels of epidermal lipids, including Cer, were determined from tape-stripped lesional epidermis. RESULTS: The clinical severity of patients ranged between 0.1 and 45 (mild to severe AD) based on the SCORAD system. As the SCORAD score increased, the level of vitamin C in the plasma, but not in the epidermis, decreased, and levels of total Cer and Cer2, the major Cer species in the epidermis, also decreased. There was also a positive association between level of vitamin C in the plasma and level of total Cer in the epidermis. However, levels of epidermal total lipids including triglyceride, cholesterol, and free fatty acid (FFA) were not associated with either SCORAD score or level of vitamin C in the plasma of all subjects. CONCLUSIONS: As the clinical severity of AD increased, level of vitamin C in the plasma and level of epidermal Cer decreased, and there was a positive association between these two parameters, implying associations among plasma vitamin C, epidermal Cer, and the clinical severity of AD.

Structural identification of skin ceramides containing ω-hydroxy acyl chains using mass spectrometry.

The stratum corneum (SC) acts as a barrier that protects organisms against the environment and from transepidermal water loss. It consists of corneocytes embedded in a matrix of lipid metabolites (ceramides, cholesterol, and free fatty acids). Of these lipids, ceramides are sphingolipids consisting of sphingoid bases, linked to fatty acyl chains. Typical fatty acid acyl chains are composed of α-hydroxy fatty acids (A), esterified ω-hydroxy fatty acids (EO), non-hydroxy fatty acids (N), and ω-hydroxy fatty acids (O). Of these, O-type ceramides are ester-linked via their ω-hydroxyl group to proteins in the cornified envelope and can be released and extracted following mild alkaline hydrolysis. Tandem mass spectrometry (MS/MS) analysis of O-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, suggesting that this method could be applied to the structural identification of O-type ceramides. Based on the MS/MS fragmentation patterns of O-type ceramides, comprehensive fragmentation schemes are proposed. In addition, we have also developed a method for identifying and profiling O-type ceramides in the mouse and guinea pig SC. This information may be used to identify O-type ceramides in the SC of animal skin.

Epidermal Hydration Is Improved by Enhanced Ceramide Metabolism in Aged C57BL/6J Mice After Dietary Supplementation of Royal Jelly.

Epidermal hydration is maintained by the epidermal lipid barrier, of which ceramide (Cer) is the major constituent. We examined the dietary effect of royal jelly (RJ) on epidermal hydration in aged mice. Altered Cer metabolism was further determined by measuring epidermal levels of individual Cer, glucosylceramide (GC), and sphingomyelin (SM) species, and of Cer-metabolizing enzymes. Aged C57BL/6J mice were fed a control diet (group AGED) or diets with 1% RJ harvested from two different areas (groups AGED+RJ1:AGED + RJ2) for 16 weeks. Aged C57BL/6J mice with no dietary intervention (the control group: group C) represented the onset of aging. In group AGED, epidermal levels of hydration, Cer1/2/5/6/7, GC-A/B/C/D, SM1/2/3, and ß-glucocerebrosidase (GCase) protein, an enzyme of GC hydrolysis for Cer generation, were lower than in group C; these levels, as well as those of Cer3/4 and acidic sphingomyelinase (aSMase) protein, an enzyme of SM hydrolysis for Cer generation, were higher in group AGED + RJ1 than in group AGED. Despite increases in GC-B, SM1/2/3, and serine palmitoyltransferase2 protein, an enzyme of de novo Cer synthesis, in group AGED + RJ2 to levels higher than in group AGED, epidermal levels of hydration, Cer1-7, GC-A/C/D, GCase, and aSMase proteins were similar in these two groups. Expression of GCase and aSMase mRNAs, and of Cer synthase3 and ceramidase proteins, enzymes of de novo Cer synthesis and degradation, did not differ among groups. Dietary RJ1 improved epidermal hydration by enhancing Cer metabolism with increased levels of all Cer, GC, and SM species, and of GCase and aSMase proteins.

Mass Spectrometric Confirmation of γ-Linolenic Acid Ester-Linked Ceramide 1 in the Epidermis of Borage Oil Fed Guinea Pigs.

Ceramide 1 (Cer1), a Cer species with eicosasphingenine (d20:1) amide-linked to two different ω-hydroxy fatty acids (C30wh:0:C32wh:1), which are, in turn, ester-linked to linoleic acid (LNA; 18:2n-6), plays a critical role in maintaining the structural integrity of the epidermal barrier. Prompted by the recovery of a disrupted epidermal barrier with dietary borage oil [BO: 36.5% LNA and 23.5% γ-linolenic acid (GLA; 18:3n-6)], in essential fatty acid (EFA)-deficient guinea pigs, we further investigated the effects of BO on the substitution of ester-linked GLA for LNA in these two epidermal Cer1 species by LC-MS in positive and negative modes. Dietary supplementation of BO for 2 weeks in EFA-deficient guinea pigs increased LNA ester-linked to C32wh:1/d20:1 and C30wh:0/d20:1 of Cer1. Moreover, GLA ester-linked to C32wh:1/d20:1, but not to C30wh:0/d20:1, of Cer1 was detected, which was further confirmed by the product ions of m/z 277.2 for ester-linked GLA and m/z 802.3 for the deprotonated C32wh:1/d20:1. C20-Metabolized fatty acids of LNA or GLA were not ester-linked to these Cer1 species. Dietary BO induced GLA ester-linked to C32wh:1/d20:1 of epidermal Cer1.

Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes.

Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 µg/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolic-related enzymes, and as a result, could improve overall epidermal barrier function.

Biological significance of essential fatty acids/prostanoids/lipoxygenase-derived monohydroxy fatty acids in the skin.

The skin displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA), an 18-carbon (n-6) PUFA, results in characteristic scaly skin disorder and excessive epidermal water loss. Although arachidonic acid (AA), a 20-carbon (n-6) PUFA, is metabolized via cyclooxygenase pathway into predominantly prostaglandin E2 (PGE2) and PGF2alpha, the metabolism of AA via the 15-lipoxygenase (15-LOX) pathway, which is very active in skin epidermis and catalyzes the transformation of AA into predominantly 15S-hydroxyeicosatetraenoic acid (15S-HETE). Additionally, the 15-LOX also metabolizes the 18-carbon LA into 13S-hydroxyoctadecadienoic acid (13S-HODE), respectively. Interestingly, 15-LOX catalyzes the transformation of dihomo-gamma-linolenic acid (DGLA), derived from dietary gamma-linolenic acid, to 15S-hydroxyeicosatrienoic acid (15S-HETrE). These monohydroxy fatty acids are incorporated into the membrane inositol phospholipids which undergo hydrolytic cleavage to yield substituted-diacylglycerols such as 13S-HODE-DAG from 13S-HODE and 15S-HETrE-DAG from 15S-HETrE. These substituted-monohydroxy fatty acids seemingly exert anti-inflammatory/antiproliferative effects via the modulation of selective protein kinase C as well as on the upstream/down-stream nuclear MAP-kinase/AP-1/apoptotic signaling events.

Increased OPG/RANKL ratio in the conditioned medium of soybean-treated osteoblasts suppresses RANKL-induced osteoclast differentiation.

Soybean is a major dietary source of isoflavones, particularly daidzein and genistein, which stimulate osteoblastic functions that are initiated by binding to estrogen receptor (ER)-α and ER-ß found on osteoblasts. However, coupled with a low expression of ER-α and ER-ß in osteoclasts, the inhibitory effects of soy isoflavones on osteoclast differentiation is likely mediated through paracrine factors produced by osteoblasts. Therefore, in this study, we investigated whether soybean can indirectly inhibit osteoclast differentiation through the modulation of osteoclastic factors produced by osteoblasts. Treatment with soybean extracts increased the levels of osteoprotegerin (OPG) and decreased those of receptor activator of nuclear factor-κB ligand (RANKL) in the conditioned medium (CM) of MC3T3-E1 osteoblasts. Subsequently, the RANKL-induced RAW264.7 osteoclast formation was markedly inhibited by treatment with CM collected from MC3T3-E1 osteoblasts incubated with soybean extracts (S-CM). Similarly, S-CM significantly attenuated the RANKL-induced increase in the mRNA and protein levels of matrix metalloproteinase-9 (MMP-9), a potential biomarker gene of osteoclast differentiation, through the suppression of nuclear factor of activated T cells c1 (NFATc1) activation. Of note, a soybean concentration of 0.001 mg/ml further increased the OPG/RANKL ratio compared to treatment with a 0.1 mg/ml soybean concentration and was overall, more effective at inhibiting RANKL-induced osteoclast formation and MMP-9 expression. Taken together, our data demonstrate that treatment with soybean extracts stimulates the secretion of OPG and inhibits that of RANKL, thus inhibiting RANKL-induced osteoclast differentiation through the suppression of NFATc1 activation.