Katsuyuki Mukai

Mechanism of Visceral Fat Reduction in Tsumura Suzuki Obese, Diabetes (TSOD) Mice Orally Administered β-Cryptoxanthin from Satsuma Mandarin Oranges (Citrus unshiu Marc)

The carotenoid β-cryptoxanthin (β-CRX) is abundant in Satsuma mandarins (Citrus unshiu Marc). Several studies have shown a relationship between Satsuma mandarin consumption and a low risk of several diseases, for example, diabetes, gout, and hypertension, suggesting β-CRX involvement in disease prevention. We investigated the effect of β-CRX on mildly obese males. β-CRX administration reduced visceral adipose tissue, body weight, and abdominal circumference. However, the detailed mechanism by which β-CRX mediates these changes remains unknown. To identify this mechanism, we used an obese model mouse (TSOD). Oral β-CRX administration repressed body weight, abdominal adipose tissue weight, and serum lipid concentrations in TSOD; these results are identical to previous human trial results. β-CRX administration significantly repressed adipocyte hypertrophy. Gene expression analysis strongly indicated that β-CRX can alter cytokine secretion and cell proliferation. These results suggest that β-CRX derived from Satsuma mandarins can help prevent obesity by repressing hypertrophy of abdominal adipocytes.

4,8-Sphingadienine and 4-hydroxy-8-sphingenine activate ceramide production in the skin

BackgroundIngestion of glucosylceramide improves transepidermal water loss (TEWL) from the skin, but the underlying mechanism by which a small amount of dietary glucosylceramide can vastly improve skin conditions remains unclear. In a previous report, glucosylceramides were shown to be digested to sphingoids, which were shown to be absorbed through the intestinal epithelium. Based on these observations, we hypothesized that sphingoids are the key molecules facilitating endogenous ceramide production. In this study, we assessed the effect of 4,8-sphingadienine (d18:2) and 4-hydroxy-8-sphingenine (t18:1), derived from konjac glucosylceramide, on stimulating ceramide production.MethodsKonjac glucosylceramide acidolysis was performed using hydrochloric acid; the resulting d18:2 and t18:1 were fractionated by column chromatography. Real-time quantitative RT-PCR was performed to assess the effect of d18:2 and t18:1 on gene expression in normal human epidermal keratinocytes, while their effect on the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)γ, was measured using a receptor-cofactor assay system. The effect of d18:2 and t18:1 on stimulating ceramide production was evaluated using HPTLC analysis in a 3-dimensional human skin model.ResultsWe noted the upregulation of genes related to de novo ceramide synthesis as well as of those encoding the elongases of very long-chain fatty acids by d18:2 and t18:1, but not by glucosylceramide and 4-sphingenine. Both these sphingoids also facilitated the expression of PPARβ/δ and PPARγ; moreover, they also demonstrated ligand activity for PPARγ. These results indicated that d18:2 and t18:1 promote the differentiation of keratinocytes. Analysis of the lipids within the 3-dimensional human skin model indicated that treatment with d18:2 and t18:1 not only upregulated gene expression but also increased ceramide production.ConclusionsThe sphingoids d18:2 and t18:1 activated genes related to de novo ceramide synthesis and increased ceramide production, whereas glucosylceramide and 4-sphingenine could not. These results suggest that the effect of dietary glucosylceramides on the skin is mediated by d18:2 and t18:1.

In Vivo and in Vitro Studies on the Absorption Characteristics of β-Cryptoxanthin in the Intestine

β-Cryptoxanthin (β-CRX) is a carotenoid abundantly present in the Satsuma mandarin (Citrus unshiu Marc.), one of the most popular fruits in Japan, and it is reported to have several health benefits. Although it is thought to have higher bioavailability than other carotenoids, the usual daily intake is small, and a good method to improve its bioavailability is needed. Hence we studied the effect of emulsification on the absorption characteristics of β-CRX in the intestine. Human trials showed that its serum transfer efficiency was statistically higher in the emulsified formulation than in fresh Satsuma mandarin juice. Caco-2 permeability studies indicated that emulsifiers preferentially accelerate the absorption of the non-esterified form of β-CRX, suggesting that emulsification is more effective for free β-CRX. This information might be useful to improve the efficiency of β-CRX serum transfer, as well as to increase the health benefits of β-CRX.

Beta-Cryptoxanthin, a Novel Carotenoid Derived from Satsuma Mandarin, Prevents Abdominal Obesity

Satsuma mandarin ( Citrus unshiu Marc.), a unique Japanese citrus species, is one of the most β-cryptoxanthin (β-CRX)-rich foods in the world. This chapter describes a study in which continuous oral administration of satsuma mandarin-derived β-CRX reduced the body weight of obese mice. Furthermore, β-CRX intake caused a significant reduction in visceral fat. A human clinical trial to evaluate the efficacy of β-CRX was carried out in mildly obese men: results revealed that β-CRX decreased visceral fat, body weight, and waist circumference. Further investigation using 3T3-L1 preadipocytes revealed that β-CRX prevents preadipocyte maturation, adipocyte hypertrophy, and lipid accumulation in mature adipocytes. We also found these effects to be canceled by the addition of LE540, a pan-agonist of the retinoic acid receptor (RAR). These results indicate that β-CRX prevents adipocyte hypertrophy by downregulating peroxisome proliferator-activated receptor gamma (PPAR-γ) via RAR and suppressing mastocytosis. This suggests that β-CRX is effective in obesity prevention and in improving the symptoms of metabolic syndromes.

Chemoenzymatically prepared konjac ceramide inhibits NGF-induced neurite outgrowth by a semaphorin 3A-like action

Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid bases in the intestine. However, ceramide is only found in trace amounts in food plants and thus is frequently taken as GlcCer in a health supplement. In the present study, we successfully prepared konjac ceramide (kCer) using endoglycoceramidase I (EGCase I). Konjac, a plant tuber, is an enriched source of GlcCer (kGlcCer), and has been commercialized as a dietary supplement to improve dry skin and itching that are caused by a deficiency of epidermal ceramide. Nerve growth factor (NGF) produced by skin cells is one of the itch factors in the stratum corneum of the skin. Semaphorin 3A (Sema 3A) has been known to inhibit NGF-induced neurite outgrowth of epidermal nerve fibers. It is well known that the itch sensation is regulated by the balance between NGF and Sema 3A. In the present study, while kGlcCer did not show an in vitro inhibitory effect on NGF-induced neurite outgrowth of PC12 cells, kCer was demonstrated to inhibit a remarkable neurite outgrowth. In addition, the effect of kCer was similar to that of Sema 3A in cell morphological changes and neurite retractions, but different from C2-Ceramide. kCer showed a Sema 3A-like action, causing CRMP2 phosphorylation, which results in a collapse of neurite growth cones. Thus, it is expected that kCer is an advanced konjac ceramide material that may have neurite outgrowth-specific action to relieve uncontrolled and serious itching, in particular, from atopic eczema.

Konjac Ceramide (kCer) Regulates NGF-Induced Neurite Outgrowth via the Sema3A Signaling Pathway

The tuber of the konjac plant is a source enriched with GlcCer (kGlcCer), and has been used as a dietary supplement to improve the dry skin and itching that are caused by a deficiency of epidermal ceramide. Previously, we showed chemoenzymatically prepared konjac ceramide has a neurite-outgrowth inhibitory effect that is very similar to that of Sema3A and is not seen with animal-type ceramides. While, it has been unclear whether kCer may act on Sema3A or TrkA signaling pathway. In the present study, we showed kCer induces phosphorylation of CRMP2 and microtubules depolymerization via Sema3A signaling pathway not TrkA. It is concluded that kCer may be a potential Sema3A-like agonist that activates Sema3A signaling pathway directly.

Characterization of Konjac Ceramide (kCer) Binding to Sema3A Receptor Nrp1

Konjac ceramide (kCer) can be prepared by a chemoenzymatic method as previously published (Usuki, S.; Tamura, N.; Sakai, S.; Tamura, T.; Mukai, K.; Igarashi, Y. Biochem. Biophys. Rep. 5, 160-167 (2016)). Thus prepared kCer showed an activation effect on Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerizaion, resulting in opposing NGF-induced neurite outgrowth. In the present study, we have shown that kCer is a potential Sema3A-like ligand that has a competitive effect on Sema3A binding to a cell surface receptor Nrp1, but animal-type ceramides have no effect on Sema3A binding to Nrp1. In addition, kCer showed a direct molecular interaction with Nrp1, but animal-type ceramides, C16Cer, C18Cer, and C24Cer show no specific bindings to Nrp1. Further, kCer showed an additive effect to activate the Sema3A signaling pathway together with low-dose Sema3A but a reversed effect to inhibit this pathway when combined with high-dose Sema3A.