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 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.

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.

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.

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.

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.

Antidiabetic-Like Effects of Naringenin-7-O-glucoside from Edible Chrysanthemum 'Kotobuki' and Naringenin by Activation of the PI3K/Akt Pathway and PPARγ.

Obesity is directly associated with cancer, cardiovascular injury, hypertension, and type 2 diabetes. To date, Yamamoto identified that hot water extracts of edible Chrysanthemum (EC) induced cell size reduction, up-regulation of adiponectin expression, and glucose absorption inhibition in 3T3-L1 cells during adipocyte differentiation. Furthermore, EC showed antidiabetic effects such as improvement in insulin resistance and the down-regulation of the blood glucose level and liver lipid content in type 2 diabetes model mice. In this study, we attempted to identify the antidiabetic components in EC. The methanol fraction from EC that showed relatively strong biological activity was purified by chromatography to obtain acacetin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, and naringenin-7-O-glucoside. Among the isolated compounds and their aglycones, naringenin (NA) and naringenin-7-O-glucoside (NAG) up-regulated the intracellular accumulation of lipid and adiponectin-secretion and down-regulated the diameter of 3T3-L1 cells during adipocyte differentiation. Because the PPARγ antagonist BADGE and PI3K/Akt inhibitors wortmannin and LY29004 inhibited the intracellular lipid accumulation by NA and NAG associated with adipogenesis, it was considered that NA and NAG showed the above-mentioned activities via the activation of PPARγ as well as phosphorylation of the PI3K/Akt pathway.

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.

A Method for Identifying Mouse Pancreatic Ducts.

Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancreatic ducts. First, we aimed to visualize pancreatic ducts using black, waterproof fountain pen ink. We injected the ink into pancreatic ducts through the bile duct. The flow of ink was observed in pancreatic ducts, revealing their precise architecture. Next, to visualize pancreatic ducts in live animals, we injected fluorescein-labeled bile acid, cholyl-lysyl-fluorescein into the mouse tail vein. The fluorescent probe clearly marked not only the bile duct but also pancreatic ducts when observed with a fluorescent microscope. To confirm whether the pancreatic duct labeling was successful, we performed PDL on Neurogenin3 (Ngn3)-GFP transgenic mice. As a result, acinar tissue is lost. PDL tail pancreas becomes translucent almost completely devoid of acinar cells. Furthermore, strong activation of Ngn3 expression was observed in the ligated part of the adult mouse pancreas at 7 days after PDL.

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.

mTOR inhibition by rapamycin increases ceramide synthesis by promoting transforming growth factor-ß1/Smad signaling in the skin.

Although mammalian target of rapamycin (mTOR) mediates a wide variety of biological functions, little information is available on the effect of mTOR on the functions of skin cells. In this study, we investigated effects of mTOR inhibition by rapamycin on ceramide synthesis in the skin of rats and human keratinocytes and its regulatory mechanisms. The phosphorylation of p70 S6 kinase, which indicates mTOR activation, was induced in the skin of rats fed a high-fat diet, but this abnormality was reversed by supplementation with rapamycin. Ceramide levels and the mRNA levels of serine palmitoyltransferase (SPT) and transforming growth factor (TGF)-ß1 were suppressed in the skin of rats fed high-fat diets, but this abnormality was reversed by supplementation with rapamycin. TGF-ß1-induced SPT mRNA expression was blocked by SB525334, an inhibitor of TGF-ß1-induced Smad2/3 nuclear localization, in human keratinocytes. Rapamycin-induced SPT mRNA expression was blocked by an anti-TGF-ß1 antibody or SB525334 in human keratinocytes. These results show that mTOR inhibition by rapamycin increases ceramide synthesis by promoting TGF-ß1/Smad signaling in the skin.

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.

Transforming growth factor-ß1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in keratinocytes.

In this study, we investigated the effect of TGF-ß1 on cholesterol synthesis in human keratinocytes. TGF-ß1 increased the level of cholesterol and the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in human keratinocytes. These results show that TGF-ß1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in human keratinocytes.

Effects of Various 5,7-Dihydroxyflavone Analogs on Adipogenesis in 3T3-L1 Cells.

We studied the effects of twelve 5,7-dihydroxyflavone analogs on adipogenesis in 3T3-L1 cells. Among the compounds, luteolin, diosmetin, and chrysoeriol partly inhibited adipogenesis by blocking the accumulation of triacylglycerol in the cells. Conversely, tricetin facilitated triacylglycerol accumulation in the cells. The induction of lipogenesis or lipolysis may depend on the number and bonding position of hydroxyl or methoxy groups on the B ring of 5,7-dihydroxyflavone. The mRNA expression levels of adipogenic and lipogenic genes were suppressed by luteolin treatment in the cells, while the mRNA levels of lipolytic genes were not affected. However, the expression levels of the adipogenic, lipogenic, and lipolytic genes, except for adipocyte protein 2 (aP2), were not affected by the addition of tricetin. Moreover, luteolin suppressed glucose transporter type 4 (GLUT4) gene and protein levels. These results indicate that luteolin decreased triacylglycerol levels in 3T3-L1 cells during adipogenesis through the suppression of adipogenic/lipogenic and GLUT4 genes and GLUT4 protein.

Hydrocellular foam dressing increases the leptin level in wound fluid.

Hydrocellular foam dressing (HCF) absorbs excessive wound fluid, which contains various cytokines and growth factors, and ensures a moist environment to promote wound healing. However, the molecular mechanisms underlying the wound fluid component changes induced by HCF are poorly understood. In the present study, we examined the effect of HCF on wound healing and the associated regulatory mechanisms in relation to variations in cytokine levels in the wound fluid. We created full-thickness wounds on the dorsolateral skin of rats and collected the resulting wound fluid samples. HCF was immersed in a plate containing the wound fluids. HCF was then removed and the excess wound fluid remaining in the plate was examined by cytokine array and enzyme-linked immunosorbent assay. We also used a rat model and human dermal fibroblast cultures to examine the effect of wound fluid component changes during the wound healing process. Upon treatment with HCF, leptin levels were upregulated in the wound fluid. Fibroblast proliferation was enhanced and the effect was suppressed in the presence of leptin antagonist. In our in vivo model, HCF increased wound contraction compared with film dressings and this positive effect of HCF was suppressed by addition of leptin antagonist. Our results suggest that dermal fibroblast proliferation is upregulated by HCF due to increased leptin level at the wound surface, and these effects promote wound healing. We believe that the present study contributes to furthering the understanding of the mechanisms underlying the effects of HCF-induced wound healing.

Chrysanthemum promotes adipocyte differentiation, adiponectin secretion and glucose uptake.

The adipose tissue is an endocrine organ, and its endocrine function is closely related to type 2 diabetes. Edible Chrysanthemum morifolium Ramat. (ECM) possesses several biological properties; however, its effect on adipocytes remains unclear. We investigated the effect of the hot water extract of ECM (HW-ECM) on 3T3-L1 adipocytes. HW-ECM enhanced adipocyte differentiation, adiponectin secretion, and glucose uptake in 3T3-L1 cells. It also increased the mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ), a regulator of adipocyte differentiation, adiponectin transcription, and GLUT4 expression. In addition, HW-ECM increased the mRNA levels of CCAAT/enhancer-binding protein-delta (C/EBPδ), which induces PPARγ expression, but not C/EBPß, during early adipocyte differentiation. These results suggest that HW-ECM enhances adipocyte differentiation, adiponectin secretion, and glucose uptake through C/EBPδ-induced PPARγ expression. These effects of HW-ECM on adipocytes suggest that HW-ECM is potentially beneficial for type 2 diabetes.

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.

Hydrocellular foam dressings promote wound healing associated with decrease in inflammation in rat periwound skin and granulation tissue, compared with hydrocolloid dressings.

The effects of modern dressings on inflammation, which represent the earliest phase of wound healing, are poorly understood. We investigated the effects of modern hydrocellular foam dressings (HCFs) on wound healing and on the gene expression levels of the inflammatory markers--interleukin (IL)-1ß, IL-6, and IL-10--in rat periwound skin and granulation tissue by quantitative reverse transcription-polymerase chain reaction. HCF absorbed significantly higher volume of water than hydrocolloid dressing (HCD) and increased the contraction of wounds. Polymorphonuclear neutrophils were massively infiltrated to the wound edge and boarded between granulation and dermis in the HCD group. IL-1ß, IL-6, and IL-10 mRNA levels were significantly decreased in the periwound skin around the wounds and granulation tissue covered with HCF. These findings suggest that HCF may promote wound healing along with decrease in inflammation by reducing gene expression levels of IL-1ß, IL-6, and IL-10.

Perfluorooctanoic acid binds to peroxisome proliferator-activated receptor γ and promotes adipocyte differentiation in 3T3-L1 adipocytes.

We examined the effect of perfluorooctanoic acid (PFOA) on adipose cells using 3T3-L1 adipocytes and found that PFOA increased adipocyte differentiation, triglyceride accumulation, and the mRNA level of factors related to adipocyte differentiation. In addition, PFOA bound to peroxisome proliferator-activated receptor γ (PPAR γ). These results suggest that PFOA promotes adipocyte differentiation as a PPAR γ ligand.