Identification of Reg3ß-producing cells using IL-22-stimulated enteroids.

Reg3ß, a lectin, displays antibacterial activity. This study investigated Reg3ß-expressing cells using IL-22-stimulated enteroids. IL-22 stimulation elevated the mRNA and protein levels of Reg3ß. IL-22 also increased the mRNA levels of CD133 (a transit-amplifying cell marker) and lysozyme (a Paneth cell marker). Immunohistochemistry showed partial colocalization of Reg3ß- and lysozyme-positive cells, suggesting that Paneth cells are one of Reg3ß-producing cells.

The Effect of Methanolic Valeriana officinalis Root Extract on Adipocyte Differentiation and Adiponectin Production in 3T3-L1 Adipocytes.

Adipose tissue is an endocrine organ and its endocrine function is closely associated with type 2 diabetes mellitus. Valeriana officinalis (Valerian) exerts some physiological effects; however, its influence on adipocytes remains unclear. We investigated the effect of methanolic Valerian root extract (Vale) on 3T3-L1 adipocytes. Vale (1, 10, and 100 µg/mL) dose-dependently promoted adipocyte differentiation with increasing lipid accumulation. In addition, Vale significantly increased the mRNA levels in genes associated with adipocyte differentiation, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α , and adipocyte protein 2, in dose-dependent manner. Vale also significantly enhanced mRNA and protein levels in adiponectin. A PPARγ antagonist assay and a PPARγ binding assay revealed that Vale-induced increased adipocyte differentiation and adiponectin production were partly associated with direct binding to PPARγ. Valerenic acid, a characteristic component in Valerian, also demonstrated the ability to induce adipocyte differentiation and adiponectin secretion, suggesting that it is one of the functional components in Vale.

Effect of essential amino acids on enteroids: Methionine deprivation suppresses proliferation and affects differentiation in enteroid stem cells.

We investigated the effects of essential amino acids on intestinal stem cell proliferation and differentiation using murine small intestinal organoids (enteroids) from the jejunum. By selectively removing individual essential amino acids from culture medium, we found that 24 h of methionine (Met) deprivation markedly suppressed cell proliferation in enteroids. This effect was rescued when enteroids cultured in Met deprivation media for 12 h were transferred to complete medium, suggesting that Met plays an important role in enteroid cell proliferation. In addition, mRNA levels of the stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) decreased in enteroids grown in Met deprivation conditions. Consistent with this observation, Met deprivation also attenuated Lgr5-EGFP fluorescence intensity in enteroids. In contrast, Met deprivation enhanced mRNA levels of the enteroendocrine cell marker chromogranin A (ChgA) and markers of K cells, enterochromaffin cells, goblet cells, and Paneth cells. Immunofluorescence experiments demonstrated that Met deprivation led to an increase in the number of ChgA-positive cells. These results suggest that Met deprivation suppresses stem cell proliferation, thereby promoting differentiation. In conclusion, Met is an important nutrient in the maintenance of intestinal stem cells and Met deprivation potentially affects cell differentiation.

Combined effects of soy isoflavones and milk basic protein on bone mineral density in hind-limb unloaded mice.

We examined whether the combination of isoflavone and milk basic protein both are reported to be effective for bone metabolism, prevents bone loss induced by skeletal hind-limb unloading in mice. Female ddY strain mice, aged 8 weeks, were divided into six groups (n = 6-8 each): (1) normally housed group, (2) loading group, (3) hind-limb unloading group fed a control diet, (4) hind-limb unloading group fed a 0.2% isoflavone conjugates diet, (5) hind-limb unloading group fed a 1.0% milk basic protein diet, and (6) hind-limb unloading group fed a 0.2% isoflavone conjugates and 1.0% milk basic protein diet. After 3 weeks, femoral bone mineral density was markedly reduced in unloading mice. The combination of isoflavone and milk basic protein showed cooperative effects in preventing bone loss and milk basic protein inhibited the increased expression of osteogenic genes in bone marrow cells in unloading mice. These results suggest that the combination of soy isoflavone and milk basic protein may be useful for bone health in subjects with disabling conditions as well as astronauts.

Hot water extracts of edible Chrysanthemum morifolium Ramat. exert antidiabetic effects in obese diabetic KK-Ay mice.

In this study, the antidiabetic effects of a hot water extract of edible Chrysanthemum morifolium Ramat. (HW-ECM) were investigated in type 2 diabetic mice. HW-ECM improved blood glucose levels and insulin resistance and increased adiponectin mRNA expression in adipose tissues and protein concentrations in the plasma. Moreover, it increased adipose mRNA and protein expressions of peroxisome proliferator-activated receptor γ (PPARγ), a regulator of adiponectin transcription, and mRNA expression of its downstream target genes. It also reduced the adipose cell size and attenuated the mRNA expression of pro-inflammatory adipocytokines in adipose tissues. These data presumably indicate a hypoglycemic mechanism of HW-ECM, involving increased PPARγ expression, decreased the adipocyte sizes, and suppression of chronic inflammation in adipose tissues. Finally, elevated adiponectin levels lead to amelioration of insulin resistance and the corresponding hypoglycemic effects. Therefore, HW-ECM indicates its potential as a functional food for type 2 diabetes.

Effects of daidzein and kiwifruit on bone mineral density and equol production in ovariectomised rats.

In this study, we investigated the synergistic effects of daidzein (Dz) and kiwifruit on bone and equol production in ovariectomised (OVX) rats. Female Sprague-Dawley rats were randomly assigned to one of five groups: sham operated, OVX control, OVX fed 0.1% Dz-supplemented diet (OVX + Dz), OVX fed 0.1% Dz and green kiwifruit (GRK)-supplemented diet (OVX + Dz + GRK) and OVX fed 0.1% Dz and gold kiwifruit (GOK)-supplemented diet (OVX + Dz + GOK). There were no significant differences in whole body and femur bone mineral density (BMD) among groups at week 8. BMD in the OVX group significantly decreased at week 8; however, BMD in the OVX + Dz + GRK was not significantly different from baseline in the end of the study. However, supplementation with kiwifruit did not affect urinary equol concentrations, urinary ratios of equol to Dz and the composition of caecal microbiota. These results suggest that the combination of Dz and GRK may slightly reduce bone loss caused by oestrogen deficiency but does not affect equol production.

Cooperative effects of soy isoflavones and carotenoids on osteoclast formation.

Osteoclasts play a major role in bone resorption. Several functional food components, such as soy isoflavones and carotenoids, are reported to inhibit osteoclast formation. However, the cooperative effect of functional foods or their constituents on bone metabolism has not been clarified. This study aimed to investigate the cooperative effect of soy isoflavones and carotenoids on osteoclast formation in vitro using cultures of RAW264 and bone marrow cells in the presence of receptor activator of nuclear factor κ-B ligand. In RAW264 cells, treatment with soy isoflavones (genistein or equol) or carotenoids (ß-carotene) suppressed osteoclast formation. At 10 µM, genistein and equol inhibited RAW264 cell proliferation but did not affect cell viability. When 10 µM genistein or equol was combined with 0.1 µM ß-carotene, we observed an additive suppressive effect on osteoclast differentiation. Similar results were observed with bone marrow cell cultures. We found that 10 µM of zeaxanthin or lutein suppressed osteoclast formation singly, and further enhanced the suppressive effects of daidzein or genistein when administered in combination. These results suggest that the combination of soy isoflavones and carotenoids have an enhanced suppressive effect on osteoclast formation. This knowledge might be important in planning diet for bone health.

Marked phenotypic differences of endurance performance and exercise-induced oxygen consumption between AMPK and LKB1 deficiency in mouse skeletal muscle: changes occurring in the diaphragm.

LKB1 phosphorylates members of the AMP-activated protein kinase (AMPK) family. LKB1 and AMPK in the skeletal muscle are believed to regulate not only fuel oxidation during exercise but also exercise capacity. LKB1 was also required to prevent diaphragm fatigue, which was shown to affect exercise performance. Using mice expressing dominant negative (DN) mutants of LKB1 and AMPK, specifically in the skeletal muscle but not in the heart, we investigated the roles of LKB1 and AMPK activity in exercise performance and the effects of these kinases on the characteristics of respiratory and locomotive muscles. In the diaphragm and gastrocnemius, both AMPK-DN and LKB1-DN mice showed complete loss of AMPKα2 activity, and LKB1-DN mice showed a reduction in LKB1 activity. Exercise capacity was significantly reduced in LKB1-DN mice, with a marked reduction in oxygen consumption and carbon dioxide production during exercise. The diaphragm from LKB1-DN mice showed an increase in myosin heavy chain IIB and glycolytic enzyme expression. Normal respiratory chain function and CPT I activity were shown in the isolated mitochondria from LKB1-DN locomotive muscle, and the expression of genes related to fiber type, mitochondria function, glucose and lipid metabolism, and capillarization in locomotive muscle was not different between LKB1-DN and AMPK-DN mice. We concluded that LKB1 in the skeletal muscle contributes significantly to exercise capacity and oxygen uptake during exercise. LKB1 mediated the change of fiber-type distribution in the diaphragm independently of AMPK and might be responsible for the phenotypes we observed.

Possible role of S-equol on bone loss via amelioration of inflammatory indices in ovariectomized mice.

S-equol is a natural metabolite of the soy isoflavone, daidzein, produced by intestinal bacteria. S-equol has been shown to have greater estrogenic activity than other soy isoflavones and prevent bone loss in post-menopausal women. Estrogen regulates both bone remodeling and hemopoiesis in the bone marrow, these processes that communicate closely with each other. In this study, we investigated the effect of S-equol on bone mass and gene expression of bone marrow cells in ovariectomized (OVX) mice. Female ddY strain mice, aged 12 weeks, were either sham operated or OVX. The OVX mice were randomly divided into two groups: (1) OVX control and (2) OVX fed a 0.06% (w/w) S-equol supplemented diet. After 2 weeks, the trabecular bone volume of the femoral distal metaphysis was markedly reduced in OVX mice. However, treatment with equol was observed to ameliorate this. Expression of inflammatory-, osteoclastogenesis- and adipogenesis-related genes was increased in OVX mice compared with sham mice, and equol was observed to suppress their expression. The present study demonstrates that equol might ameliorate bone loss caused by estrogen deficiency through regulating hemopoiesis and production of inflammatory cytokines in bone marrow cells.

Serum cholesterol reduction by feeding a high-cholesterol diet containing a lower-molecular-weight polyphenol fraction from peanut skin.

Feeding a high-cholesterol diet with a water-soluble peanut skin polyphenol fraction to rats reduced their plasma cholesterol level, with an increase in fecal cholesterol excretion. The hypocholesterolemic effect was greater with the lower-molecular-weight rather than higher-molecular-weight polyphenol fraction. This effect was possibly due to some oligomeric polyphenols which reduced the solubility of dietary cholesterol in intestinal bile acid-emulsified micelles.

Valerenic Acid Promotes Adipocyte Differentiation, Adiponectin Production, and Glucose Uptake via Its PPARγ Ligand Activity.

Although valerenic acid (VA) is an important marker compound for quantitative assessment of Valeriana officinalis products, little is known about its potential effects on adipocytes. We investigated the effects of VA on adipocyte differentiation, adiponectin production, and glucose uptake using 3T3-L1 adipocytes. The results showed that VA promoted adipocyte differentiation and increased the gene expression of adipogenesis and glucose uptake-related proteins, including peroxisome proliferator-activated receptor gamma (PPARγ), cytosine-cytosine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα), adiponectin, and glucose transporter 4 (GLUT4). Additionally, cell cultures treated with VA had elevated adiponectin secretion and glucose uptake. The PPARγ luciferase assay indicated VA as a partial agonist of PPARγ, while the analysis using its antagonist, GW9662, and a docking simulation between PPARγ and VA revealed the binding site of VA as likely adjacent to the Ω loop pocket of PPARγ. Taken together, these results demonstrate that VA acts as a PPARγ partial agonist to promote adipocyte differentiation, adiponectin production, and glucose uptake.

Effect of exercise intensity and AICAR on isoform-specific expressions of murine skeletal muscle PGC-1α mRNA: a role of ß2-adrenergic receptor activation.

There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1ß-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10-30 m/min for 30 min). Preinjection of ß2-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of ß2-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via ß2-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.

Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids.

Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1ß, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00487-y.

Anorexic action of fusarenon-x in the hypothalamus and intestine.

There is a lack of information available on the anorexic action of fusarenon-x (FX), which is a sesquiterpenoid mycotoxin. In this study, we investigated the changes in the hypothalamus and small intestine related to appetite after oral FX exposure. The time-course change of food intake after oral FX exposure (0.5, 1.0, and 2.5 mg/kg bw) in B6C3F1 mice showed that 2.5 mg/kg bw of FX significantly suppressed food intake during 3-6 h compared to the control. Furthermore, the total food intake for 24 h was lower in the group exposed to FX than in the control. The FX exposure (2.5 mg/kg bw for 3 h) significantly increased mRNA levels of anorexic hormones (pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcription (CART)) without changing the mRNA levels of orexigenic hormones. In addition, FX exposure indicated significantly higher mRNA levels of possible downstream targets of anorexic POMC neurons, such as the melanocortin 4 receptor (MC4R), brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB), in the hypothalamus compared to the control. FX exposure also significantly increased the mRNA level of inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)) and activated nuclear factor-kappa B (NF-κB), which is a regulatory factor for POMC in the hypothalamus. In the intestine, FX exposure did not affect the mRNA level of anorexic peptide YY but significantly elevated that of anorexic cholecystokinin (CCK) and regulatory factors for CCK (calcium-sensing receptor (CaSR), the transient receptor potential ankyrin-1 channel (TRPA1), and transient receptor potential cation channel subfamily M member 5 (TRPM5)). These results suggest that FX sequentially induces inflammatory cytokine expression, NF-κB activation, and POMC expression in the hypothalamus. FX also induces CCK expression in the intestine possibly via induction of CaSR, TRPM5, and TRPA1 expression. These changes will eventually lead to the anorexic action of FX.

Interleukin-4 suppresses the proliferation and alters the gene expression in enteroids.

Interleukin (IL)-4 is known as a cytokine mainly involved in allergy and inflammation, but recent studies have suggested that IL-4 plays a part in the differentiation process of various cells. Since the effect of IL-4 on intestinal epithelial cells, particularly cryptic cells including stem cells, is poorly understood, we investigated IL-4-induced changes in intestinal epithelial cells using mouse jejunal organoids called enteroids. IL-4 treatment decreased cell proliferation, the expression of the stem cell markers leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and olfactomedin 4 (Olfm4), and Lgr5-positive cells in enteroids. Among the differentiation markers, IL-4 significantly decreased the gene expression levels of the Paneth cell markers lysozyme 1 (Lyz1) and regenerating islet-derived protein 3 gamma (Reg3γ). A fluorescent immunostaining showed that IL-4 attenuated the emission and fluorescence intensity derived from lysozyme, which is enriched in Paneth cells. These results suggest that functional changes in Paneth cells caused by IL-4 may contribute to the reduction in Lgr5-positive cells and proliferative activity. IL-4 may affects gut function by altering the proliferation and the gene expression in enteroids.

Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure.

The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.

Adenosine stimulates hepatic glycogenolysis via adrenal glands-liver crosstalk in mice.

Adenosine signaling is involved in glucose metabolism in hepatocytes and myocytes in vitro. However, no information is available regarding the effect of adenosine on glucose metabolism in vivo. Thus, we examined how extracellular adenosine acts on glucose metabolism using mice. Subcutaneous injections of adenosine (10, 25, and 50 mg/kg bodyweight) dose-dependently increased blood glucose levels, with the peak occurring at 30 min post injection. At 30 min after adenosine injection (25 mg/kg bodyweight), glycogen content in the liver, but not the skeletal muscle, was significantly decreased. Hepatic glycogen depletion by fasting for 12 h suppressed the increase of blood glucose levels at 30 min after adenosine injection. These results suggest that adenosine increases blood glucose levels by stimulating hepatic glycogenolysis. To investigate the effect of adenosine on the adrenal gland, we studied the glycogenolysis signal in adrenalectomized (ADX) mice. Adenosine significantly increased the blood glucose levels in sham mice but not in the ADX mice. The decrease in hepatic glycogen content induced by adenosine in the sham mice was partially suppressed in the ADX mice. The level of plasma corticosterone, the main glucocorticoid in mice, was significantly increased in the sham mice by adenosine but its levels were low in ADX mice injected with either PBS or adenosine. These results suggest that adenosine promotes secretion of corticosterone from the adrenal glands, which causes hepatic glycogenolysis and subsequently the elevation of blood glucose levels. Our findings are useful for clarifying the physiological functions of adenosine in glucose metabolism in vivo.

Anorexic action of deoxynivalenol in hypothalamus and intestine.

Although deoxynivalenol (DON) suppresses food intake and subsequent weight gain, its contribution to anorexia mechanisms has not been fully clarified. Thus, we investigated the anorexic actions of DON in the hypothalamus and intestine, both organs related to appetite. When female B6C3F1 mice were orally exposed to different doses of DON, a drastic anorexic action was observed at a dose of 12.5 mg/kg body weight (bw) from 0 to 3 h after administration. Exposure to DON (12.5 mg/kg bw) for 3 h significantly increased the hypothalamic mRNA levels of anorexic pro-opiomelanocortin (POMC) and its downstream targets, including melanocortin 4 receptor, brain-derived neurotrophic factor, and tyrosine kinase receptor B; at the same time, orexigenic hormones were not affected. In addition, exposure to DON significantly elevated the hypothalamic mRNA levels of proinflammatory cytokines (IL-1ß, TNF-α, and IL-6) and activated nuclear factor-kappa B (NF-κB), an upstream factor of POMC. These results suggest that DON-induced proinflammatory cytokines increased the POMC level via NF-κB activation. Moreover, exposure to DON significantly enhanced the gastrointestinal mRNA levels of anorexic cholecystokinin (CCK) and transient receptor potential ankyrin-1 channel (TRPA1), a possible target of DON; these findings suggest that DON induced anorexic action by increasing CCK production via TRPA1. Taken together, these results suggest that DON induces anorexic POMC, perhaps via NF-κB activation, by increasing proinflammatory cytokines in the hypothalamus and brings about CCK production, possibly through increasing intestinal TRPA1 expression, leading to anorexic actions.

Effect of Kiwifruit on Bone Resorption in Ovariectomized Mice.

Kiwifruit is a good source of dietary components and has beneficial effects for health. In this study, we investigated the effects of two types of kiwifruit, green kiwifruit (GRK) and gold kiwifruit (GOK), on bone metabolism in ovariectomized (OVX) mice. Seven-week-old female Balb/c-strain mice were divided into four groups: sham-operated (sham) group, OVX group, and OVX mice that were fed a GRK-supplemented diet or GOK-supplemented diet. Freeze-dried GRK and GOK were prepared and added in the diet at a concentration of 3 g/100 g. After 9 wk, the mice were sacrificed, and the serum, uterus, and femurs were obtained. Final body weight did not differ significantly among the four groups. Compared to the sham group, uterine weight was significantly lower and serum C-terminal telopeptide of type I collagen (CTx) levels and receptor activator of NF-κB ligand (RANKL) mRNA expression of the whole femur were significantly higher in the OVX group. Compared to the OVX group, GRK, but not GOK, reduced serum CTx concentrations and RANKL mRNA expression of the whole femur without changes in uterine weight. These results suggest that the GRK inhibited bone resorption, which might be due to a decrease in RANKL mRNA expression in OVX mice.

Combined Effects of Soy Isoflavones and ß-Carotene on Osteoblast Differentiation.

Soy isoflavones, genistein, daidzein and its metabolite equol, as well as ß-carotene have been reported to be effective for maintaining bone health. However, it remains to be elucidated whether combining soy isoflavones with ß-carotene is beneficial to bone formation. This study investigated the combined effect of soy isoflavones and ß-carotene on the differentiation of MC3T3-E1 preosteoblastic cells. Daidzein and genistein alone did not affect cell growth but increased alkaline phosphatase (ALP) activity. Beta-carotene alone inhibited cell growth and markedly enhanced ALP activity. Soy isoflavones combined with ß-carotene resulted in higher ALP activity than treatment with isoflavones or ß-carotene alone. We observed significant main effects of ß-carotene on the enhanced expression of Runx2, ALP, and ostepontin mRNA, whereas there was a significant main effect of soy isoflavones on the expression of osterix mRNA. To investigate how ß-carotene affected osteoblast differentiation, MC3T3-E1 cells were treated with retinoic acid receptor (RAR) pan-antagonist combined with ß-carotene. Osteopontin and ALP mRNA expression levels, which were increased following treatment with ß-carotene, were significantly suppressed by the RAR pan-antagonist. This suggests treatment with ß-carotene enhanced early osteoblastic differentiation, at least in part via RAR signaling. These results indicate that a combination of isoflavones and ß-carotene may be useful for maintaining a positive balance of bone turnover by inducing osteoblast differentiation.