Generation of intestinal chemosensory cells from nonhuman primate organoids.

Several gastrointestinal epithelial cells are involved in taste signal transduction. Although rodent tissues are extensively used as a human gut model, recent studies show that the chemical sensing system in rodents differs from that in humans. Nonhuman primates in biomedical research are valuable animal models to advance our understanding of biological responses in humans. The 3D organoid culture produces functional gastrointestinal epithelial cells in vitro and can be generated from animal and human tissues. Here, we report the generation of intestinal chemosensory cells from nonhuman primates, macaques, using an organoid culture system. We were able to maintain macaque intestinal organoids in the proliferation medium for more than six months. Upon switching to differentiation medium, we observed a drastic change in organoid morphology and chemosensory cell marker protein expression. This switch from proliferation to differentiation was confirmed by transcriptome analysis of the duodenum, jejunum, and ileum organoids. We further observed that the supplementation of culture media with interleukin (IL)-4 or the Notch inhibitor dibenzazepine (DBZ) accelerated terminal cell differentiation into chemosensory cells. Overall, we generated monkey intestinal organoids for the first time. These organoids are suitable for studying the function of primate chemosensory cells.

Branched-chain amino acids regulate hyaluronan synthesis and PPARα expression in the skin.

We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in branched-chain amino acid catabolism, on hyaluronan synthesis in mice. The skin levels of hyaluronan and the gene expression levels of hyaluronan synthase (Has)2, Has3, and peroxisome proliferator-activated receptor-α were significantly lower in the BDK-knockout group than in the wild-type group.

Interleukin-4 Promotes Tuft Cell Differentiation and Acetylcholine Production in Intestinal Organoids of Non-Human Primate.

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.

Ngn3-Positive Cells Arise from Pancreatic Duct Cells.

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.

Negative effects of a low-quality protein diet on wound healing via modulation of the MMP2 activity in rats.

Protein malnutrition is largely associated with a delay or failure of the healing process. However, the effect of dietary protein quality on wound healing is largely unknown. This study aimed to reveal the effect of dietary protein quality on wound healing and elucidate the regulatory mechanisms in a rat model of full-thickness cutaneous wounds. Rats were fed a normal diet for a week, and then they were divided into three groups that were fed the following diet for the experimental period: casein diet, gluten diet and gluten + lysine diet. The gluten diet significantly decreased body weight and wound healing compared with the casein diet, but this effect was reversed by supplementation with lysine. The numbers of leukocytes were significantly higher in the skin of the gluten group than those in the casein group. The wounded skin tissues of the gluten group showed lower amounts of collagen deposition compared with that in the casein group. Our results also showed that both matrix metalloproteinase (MMP) 2 activity and MMP14 mRNA levels were significantly increased in the skin of the gluten group, compared with the casein group. In summary, this study suggests low-quality protein diets have negative effects on wound healing via modulation of MMP2 activity in rats.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Wound fluid of rats fed protein-free diets delays wound healing through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Adequate nutrition is required to maintain healthy skin integrity, and malnourished patients with poor protein diet often experience delayed wound healing. Understanding the cellular mechanisms of protein malnutrition will justify the importance of optimal protein diets in health and disease defense. Therefore in the present study, we examined the effects of changes in wound fluid composition and its function caused by protein malnutrition on wound healing. Rats were fed a control (CO; 20% protein) diet or a protein-free (PF) diet for 2 weeks; we then created full-thickness wounds on the dorsolateral skin. On day 5 after wounding, frozen sections of the wounds were created to investigate the state of granulation tissues, and wound fluid obtained from the rats was collected to examine variations in cytokine levels and its function. Wound closure was significantly delayed from day 4 until total wound closure in rats fed a PF diet. Thickness of granulation tissue, which is composed of mainly dermal fibroblasts, and Ki67 immunohistochemical staining were significantly decreased in rats fed PF diets. PF diets decreased insulin-like growth factor (IGF)-I, which promotes wound healing, and increased IGF-binding protein-1, which inhibits IGF-I bioavailability, in wound fluid. Wound fluid obtained from rats fed a PF diet suppressed dermal fibroblast proliferation. Furthermore, the wound fluid remarkably decreased the phosphorylation level of IGF-I receptor ß (IGF-IR) and extracellular signal-regulated kinase (ERK)(1/2) in dermal fibroblasts. These results show that wound fluid of rats fed PF diets delays wound healing by inhibiting granulation tissue formation through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Starvation reduces hyaluronan synthesis by suppressing TGF-ß1/IGF-I signaling in rat skin.

Although starvation has been reported to influence the functions of various tissues, its effects on the skin are not well understood. In this study, we investigated the effect of starvation on hyaluronan synthesis in rat skin. Starvation reduced hyaluronan synthesis in the skin. Starvation also decreased the skin mRNA expression of transforming growth factor (TGF)-ß1, which enhances the gene expression of rhas2 and rhas3. The serum levels of insulin-like growth factor (IGF)-I, which enhances rhas2, rhas3, and TGF-ß1 mRNA expression, in the starvation group were considerably lower than those in the control (CO) group. IGF-IR phosphorylation was substantially lower in the starvation group compared with the CO group. These findings suggest that starvation reduces hyaluronan synthesis in the skin by suppressing TGF-ß1/IGF-I signaling. Abbreviations: HAS: hyaluronan synthase; IGF-I: insulin-like growth factor-I; IGFBP-1: insulin-like growth factor binding protein-1; TGF-ß1: transforming growth factor-ß1; TBST: tris buffered saline containing 0.5% (v/v) Tween 20; HABP: hyaluronic acid binding protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Protein-restricted maternal diet during lactation decreases type I and type III tropocollagen synthesis in the skin of mice offspring.

We investigated the effects of a low protein (LP) maternal diet during lactation on type I and III tropocollagen synthesis in infant mouse skin. The LP diet decreased the levels of type I and III tropocollagen proteins and COL1A1 and COL3A1 mRNA. Thus, the protein composition of the maternal perinatal diet may influence the skin health of offspring.

Branched-chain amino acids regulate type I tropocollagen and type III tropocollagen syntheses via modulation of mTOR in the skin.

Branched-chain amino acids (BCAAs) exhibit many physiological functions. However, the potential link and mechanism between BCAA and skin function are unknown. We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in BCAA catabolism, on type I and III tropocollagen syntheses in mice. Leucine and isoleucine levels were significantly lower in the skin of BDK-KO mice compared with wild-type mice. No changes in valine concentrations were observed. The levels of type I and III tropocollagen proteins and mRNAs (COL1A1 and COL3A1) were significantly lower in the skin of BDK-KO mice compared with wild-type mice. The phosphorylation of p70 S6 kinase, which indicates mammalian target of rapamycin (mTOR) activation, was reduced in the skin of BDK-KO mice compared with wild-type mice. These findings suggest that deficiencies of leucine and isoleucine reduce type I and III tropocollagen syntheses in skin by suppressing the action of mTOR.

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.

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.

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.

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.

Pseudomonas aeruginosa quorum-sensing signaling molecule N-3-oxododecanoyl homoserine lactone induces matrix metalloproteinase 9 expression via the AP1 pathway in rat fibroblasts.

Quorum sensing is a cell-to-cell communication mechanism, which is responsible for regulating a number of bacterial virulence factors and biofilm maturation and therefore plays an important role for establishing wound infection. Quorum-sensing signals may induce inflammation and predispose wounds to infection by Pseudomonas aeruginosa; however, the interaction has not been well investigated. We examined the effects of the P. aeruginosa las quorum-sensing signal, N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), on matrix metalloproteinase (MMP) 9 expression in Rat-1 fibroblasts. 3OC12-HSL upregulated the expression of the MMP9 gene bearing an activator protein-1 (AP-1) binding site in the promoter region. We further investigated the mechanism underlying this effect. c-Fos gene expression increased rapidly after exposure to 3OC12-HSL, and nuclear translocation of c-Fos protein was observed; both effects were reduced by pretreatment with an AP-1 inhibitor. These results suggest that 3OC12-HSL can alter MMP9 gene expression in fibroblasts via the AP-1 signaling pathway.

Calcitonin gene-related peptide regulates mitogen-activated protein kinase pathway to decrease transforming growth factor ß1-induced hepatic plasminogen activator inhibitor-1 mRNA expression in HepG2 cells.

Transforming growth factor (TGF) ß1-induced plasminogen activator inhibitor (PAI)-1 is one of factors associated with the development of hepatic fibrosis. Calcitonin gene-related peptide (CGRP) shows hepatoprotective effect during hepatic injuries, including fibrosis. However, the effects of CGRP on PAI-1 expression induced by TGFß1 are unknown. In this study, we investigated the effect of CGRP on TGFß1-induced PAI-1 expression and its regulatory mechanisms in HepG2 cells. CGRP inhibited TGFß1-induced PAI-1 expression. H89, a protein kinase A inhibitor, abolished the inhibition of TGFß1-induced PAI-1 expression by CGRP. TGFß1 activated mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase, c-jun NH2-terminal kinase, and p38, and this activation was abolished by CGRP. These results show that the CGRP-induced cAMP/PKA activation suppresses activation of MAPK induced by TGFß1, leading to decreased PAI-1 expression in HepG2 cells.

Acute ultraviolet B irradiation increases cholesterol and decreases Cyp7a1 expression in the liver of female mice.

Recent studies have demonstrated that ultraviolet B (UVB) irradiation impacts both skin and hepatic functions. In this study, we investigated the effects of UVB irradiation on cholesterol metabolism in the liver. Hairless mice were exposed to UVB (1.6 J/cm2) irradiation. Dorsal skin and liver samples were collected 24 h after exposure. Total RNA was extracted from the skin and liver tissues, and used for DNA microarray analysis and real-time polymerase chain reaction (PCR). Hepatic mRNA expression of Cyp7a1 revealed a 4.4-fold decrease in the UVB (+) group compared to that in the UVB (-) group. No differences were observed in the expression of the other genes related to cholesterol metabolism. Additionally, the level of hepatic total cholesterol in the UVB (+) group was significantly higher than in the UVB (-) group. These findings suggest that acute UVB irradiation increases total cholesterol levels and decreases Cyp7a1 expression in the liver.

Calcitonin gene-related peptide upregulates serum amyloid A synthesis through activation of interleukin-6.

We found that calcitonin gene-related peptide (CGRP) enhanced the expression of levels of serum amyloid A (SAA) and interleukin-6 (IL-6) in HepG2. In addition, CGRP-induced SAA1/2 mRNA expression was blocked by an anti-IL-6 neutralizing antibody in HepG2. These results suggest that CGRP promotes SAA synthesis through activation of IL-6 in human hepatocytes.

Accelerating effect of soy peptides containing collagen peptides on type I and III collagen levels in rat skin.

Two weeks of feeding soy peptides containing 2% collagen peptides increased the levels of type I and III tropocollagen and their mRNAs. In contrast, the diet did not increase the mRNA levels of rat hyaluronan synthases, serine palmitoyltransferase (the rate-limiting enzyme of ceramide synthesis), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (the key enzyme of cholesterol synthesis). These results suggest that feeding of soy peptides with collagen peptides specifically enhanced the tropocollagen level in the skin.