Hydrophobic Components in Light-Yellow Pulp Sweet Potato (Ipomoea batatas (L.) Lam.) Tubers Suppress LPS-Induced Inflammatory Responses in RAW264.7 Cells via Activation of the Nrf2 Pathway.

Sweet potato is a crop that is widely consumed all over the world and is thought to contribute to health maintenance due to its abundant nutrients and phytochemicals. Previous studies on the functionality of sweet potatoes have focused on varieties that have colored pulp, such as purple and orange, which contain high levels of specific phytochemicals. Therefore, in the present study, we evaluated the anti-inflammatory effects of light-yellow-fleshed sweet potatoes, which have received little attention. After freeze-drying sweet potatoes harvested in 2020, extracts were prepared from the leaves, stems, roots, and tubers in 100% ethanol. Mouse macrophage-like cell line RAW264.7 cells were cultured with 10 µg/mL of the extracts and induced lipopolysaccharide (LPS)-stimulated inflammation. Of the extracts, the tuber extracts showed the highest suppression of LPS-induced interleukin-6 (IL-6) gene expression and production in RAW264.7, which was attributed to the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress response pathway. In addition, preparative high-performance liquid chromatography (HPLC) experiments suggested that hydrophobic components specific to the tuber were the main body of activity. In previous studies, it has been shown that the tubers and leaves of sweet potatoes with colored pulp exhibit anti-inflammatory effects due to their rich phytochemicals, and our results show that the tubers with light-yellow pulp also exhibit the effects. Furthermore, we were able to show a part of the mechanism, which may contribute to the fundamental understanding of the treatment and prevention of inflammation by food-derived components.

Effects of Additional Granola in Children's Breakfast on Nutritional Balance, Sleep and Defecation: An Open-Label Randomized Cross-Over Trial.

The contribution of breakfast to daily nutrient intake is low, particularly among children, at only about 20%, and it is difficult to determine whether children are receiving adequate nutrients at breakfast. Although alterations in breakfast content are considered to affect lifestyle habits such as sleep and defecation, there have been few intervention studies in children. The relationship between nutritional balance, dietary intake, and lifestyle habits in children remains unclear. We conducted an intervention study on elementary school children's breakfasts and observed the effects of improving the nutritional balance of breakfast on sleep parameters and defecation status. An intervention study was conducted with 26 elementary school students in Tokyo. The study design was an open-label randomized cross-over trial. Subjects consumed their usual breakfast during the control period and a granola snack containing soy protein in addition to their usual breakfast during the intervention period. Questionnaires regarding breakfast, sleep, and bowel movements were administered during each period. Based on the answers to these questionnaires, we compared the nutritional sufficiency of macronutrients, vitamins, and minerals (29 in total), as well as changes in sleep parameters and defecation status. The additional consumption of granola snacks increased the breakfast intake of 15 nutrients. The changes were particularly significant for iron, vitamin B1, vitamin D, and dietary fiber. During the intervention, sleep duration was decreased and wake-up time became earlier. In terms of defecation, the intervention did not change stool characteristics, but the frequency of defecations per week increased on average by 1.2 per week. These results suggest that the nutritional balance and the amount of breakfast are linked to sleep and defecation and that improving breakfast content can lead to lifestyle improvements in children.

Effects of intake of four types of snack with different timings on postprandial glucose levels after dinner.

PURPOSE: It has been reported that the consumption of fruit granola (FG), mulberry leaves, and barley cookies as an afternoon snack suppresses the postprandial increase in glucose levels at dinner. However, there have been no reports on the second-meal effect of snacking on popular snacks, such as potato chips (PC), roasted sweet potato (SP), and black beans (BB), or on the interval between snacking and dinner. METHOD: The present study was an open-label randomized crossover trial of five study groups (PC, SP, BB, FG, and no snack) regarding the second-meal effects with different intervals between snacks and dinner. The subjects consumed prescribed meals for lunch and dinner at 12:00 and 19:00, and a snack fixed at 838 kJ (= 200 kcal) at 15:00 or 17:00. RESULTS: When the participants snacked at 15:00, the postprandial glucose elevation at dinner was suppressed in the FG and SP groups, and the area under the curve (AUC) was also low. When they snacked at 17:00, the postprandial glucose elevation was suppressed in all the groups. The AUCs for PC, FG, and SP were lower than those for no snacking. On the other hand, carbohydrate intake increased with snacking, but the total AUC of snacks and dinner did not differ in any of the groups. The duration of hyperglycemia decreased with snack intake, as did the glucose amplitude. CONCLUSION: We believe that the intake of carbohydrates and soluble fiber in snacks is an important factor in the second-meal effect at dinner. These results will contribute to the development of snacking and research into the second-meal effect.

The differential effect of two cereal foods on gut environment: a randomized, controlled, double-blind, parallel-group study.

Background and aims: Cereal-based foods such as fruit granola (FG) and corn flakes (CF) form part of a fiber-rich diet. Dietary fiber has a good effect on human health. However, changes in gut microbiota and intestinal immunity have not been investigated. We conducted a randomized, double-blind, placebo-controlled trial to investigate the effects of FG and CF intake on gut microbiota, metabolome, and the immune system. Methods: Subjects continuously consume CF or FG for 4 weeks. Stool samples, and questionnaires on defecation were collected before, 2 weeks after, and 4 weeks after intake. Gut microbiota was analyzed using 16S rRNA gene amplicon sequencing. Fecal metabolomes were analyzed using GC/MS and CE-TOF/MS. Fecal IgA was analyzed using ELISA. Results: The defecation frequency after cereal based food intake was improved. The different cereal-based foods had different effects on gut microbiome. The increase in intestinal IgA levels was positively correlated with the relative abundance of Dialister and the Lachnospiraceae ND3007 group in CF and FG group, respectively. SCFAs showed a positive correlation with Prevotella 9 in the FG group. Conclusion: This study showed that the supplement in dietary fiber contained in CF and FG improves bowel movements. CF and FG each had different effects on gut microbes, metabolites and different relationships between fecal IgA or SCFAs and gut microbiota.

A Potato Peel Extract Stimulates Type I Collagen Synthesis via Akt and ERK Signaling in Normal Human Dermal Fibroblasts.

The ability of dermal fibroblasts to synthesize collagen decreases with ages. The integrity of collagen fibers severely decreases in aged skin, causing its characteristic morphological changes such as wrinkles and sagging. To prevent and improve skin aging, the stimulation of collagen synthesis in dermal fibroblasts is important. Potato peels contain many biofunctional compounds, but not much is known about their effects on human skin physiology. To characterize the potential effects of a potato peel extract (PPE) against skin aging, we examined its effects on the synthesis of type I collagen by normal human dermal fibroblasts (NHDFs). Treatment with the PPE significantly increased the expression of type I collagen mRNA in NHDFs and their secretion of type I collagen. To elucidate the mechanism involved, we examined the signaling pathway controlled by transforming growth factor-ß (TGF-ß), which regulates the synthesis of type I collagen. Treatment of NHDFs with the PPE significantly increased the expression of TGF-ß receptor mRNA. TGF-ß signaling involves Smad-dependent and Smad-independent pathways, like phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). The PPE did not activate Smad, but significantly activated Akt and ERK. These results demonstrate that the PPE activates PI3K/Akt and MAPK/ERK signals via TGF-ß receptors, which stimulate the synthesis of type I collagen in NHDFs. These results suggest that the PPE could be a novel and effective antiaging material.

Age-dependent changes in vancomycin-induced nephrotoxicity in mice.

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic that is commonly used to eradicate methicillin-resistant gram-positive cocci, despite its nephrotoxic side effects. Elderly people are particularly susceptible to developing VCM-induced nephrotoxicity. However, the precise mechanism by which VCM induces nephrotoxicity in elderly people is not completely understood. Therefore, we investigated VCM-induced nephrotoxicity in mice of different ages. VCM was injected intraperitoneally into mice at 1, 3, 6, 12, and 24 months of age at a dosage of 400 mg/kg body weight for 3 and 14 days. Twenty-four hours after the last injection, we examined plasma creatinine levels and histopathological alterations in the kidneys. VCM administration increased plasma creatinine levels, and these values gradually increased to higher levels with aging. The histological examination revealed renal tubular degeneration, such as brush-border atrophy, apoptosis/necrosis of the tubular epithelium, and epithelial desquamation, that gradually became more severe with aging. Furthermore, immunohistochemical staining with anti-CD10 and anti-single-stranded DNA antibodies revealed damaged renal proximal tubules with marked dilatation, as well as numerous apoptotic cells, and these features increased in severity in 12- and 24-month-old mice receiving VCM. Based on these results, aged mice were highly susceptible to kidney damage induced by VCM administration. In addition, proximal tubular epithelial cells likely underwent apoptosis after the administration of VCM. This report is the first to document VCM-induced nephrotoxicity in mice of different ages. Thus, this mouse model could be useful for understanding the mechanisms of VCM-induced nephrotoxicity in the elderly.

Time-Dependent Alterations of Vancomycin-Induced Nephrotoxicity in Mice.

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic that is commonly used against methicillin-resistant, Gram-positive cocci despite the nephrotoxic side effects. VCM-induced nephrotoxicity has been reported in 5-28% of recipient patients. Therefore, renal failure induced by VCM has become an important clinical problem. However, the exceedingly complex mechanism of VCM-induced nephrotoxicity is not fully understood. Therefore, this study was designed to clarify time-dependent alterations of VCM-induced nephrotoxicity in mice as a step toward decreasing the risks of kidney injury associated with VCM therapy. VCM was injected intraperitoneally into mice at a dose of 400 mg/kg body weight at 24-h intervals for 3, 5, 7, and 14 d. At 24 h after the last injection, we examined histopathological alterations of the kidney as well as blood biochemistry. VCM administration resulted in a decrease of body weight and increase of kidney weight. Histological examination revealed renal damage such as dilated proximal tubules with occasional casts and interstitial fibrosis in VCM-treated mice. Furthermore, immunohistochemical staining with anti-CD10 and anti-single-stranded DNA antibodies highlighted damaged renal proximal tubules with marked dilatation as well as numerous apoptotic cells as early as day 4 of VCM-treatment. The severity of symptoms progressed until day 15. These results suggest that VCM-induced renal damage and incipient renal failure begin soon after the start of treatment and progressively worsen. This is the first report describing the time-dependence of VCM-induced nephrotoxicity in mice and depicting a model that clarifies the mechanisms of this tissue damage.

Transcriptome Analysis of Skin from SMP30/GNL Knockout Mice Reveals the Effect of Ascorbic Acid Deficiency on Skin and Hair.

BACKGROUND/AIM: Senescence marker protein-30/gluconolactonase knockout mice (SMP-30/GNL-KO) are a very useful model for clarifying the involvement of vitamin C (VC) in aging-related diseases. In this study, the effects of VC deficiency on skin and hair growth were investigated using SMP-30/GNL-KO mice by RNA sequencing. MATERIALS AND METHODS: SMP-30/GNL-KO mice were given water containing 1.5 g/l VC until up to 8 weeks after birth to maintain a VC concentration in their organs and plasma equivalent to that in wild-type mice. The mice were then divided into two groups: a VC(+) group, where VC was administered, and a VC(-) group, where VC was not administered. Skin samples were collected at 4 and 8 weeks after the treatment. RNA was extracted from each skin sample, followed by cDNA synthesis and RNA-seq. In addition, hair growth was compared between the VC(-) and VC(+) groups after shaving. Skin samples were collected from the shaved area for histological examination by hematoxylin & eosin (HE) staining. RESULTS: RNA-seq revealed that there were 1,736 (FDR<0.001) differentially expressed genes in the VC(-) and VC(+) groups. From the functional analysis of the differentially expressed genes in the VC(-) and VC(+) groups, predicted functionalities including cell death and cytotoxicity increased in the VC(+) group. Furthermore, it was predicted that the difference in hair growth between the VC(-) and VC(+) groups was caused by the expression of genes including keratin-related genes and the Sonic hedgehog gene. It was confirmed that hair growth was significantly promoted; hair growth from hair papilla cells was also confirmed by HE staining of the shaved backs of SMP-30/GNL-KO mice in the VC(+) group. CONCLUSION: RNA-seq of the skin from VC-deficient mice showed the effects of VC deficiency on the expression of genes involved in cell growth and the hair cycle. Visual inspection suggested that changes in the expression of the genes are involved in delaying hair growth in the VC(-) group. Further research on the relationship among VC deficiency, the hair cycle, and skin cell growth may contribute to research on hair restoration and skin aging.

Induction of peptidylarginine deiminase 2 and 3 by dibutyryl cAMP via cAMP-PKA signaling in human astrocytoma U-251MG cells.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.

Mass spectrometric identification of citrullination sites and immunohistochemical detection of citrullinated glial fibrillary acidic protein in Alzheimer's disease brains.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.

Age-associated decrease of senescence marker protein-30/gluconolactonase in individual mouse liver cells: Immunohistochemistry and immunofluorescence.

AIM: Senescence marker protein-30 (SMP30)/gluconolactonase (GNL) is an age-associated protein in that its presence decreases with aging. Here, we used immunohistochemical analysis to investigate the changes of SMP30/GNL in individual cells of the liver from progressively aged mice. METHODS: Male C57BL/6 strain mice at 1, 3, 6, 12, 24 and 30 months-of-age were the source of hepatic cells used to detect SMP30/GNL. Liver sections from these mice were subjected to immunohistochemical staining with anti-SMP30/GNL antibody. For immunofluorescent staining, primary cultured hepatocytes from mice at various ages were stained with SMP30/GNL and albumin. RESULTS: In liver cells from mice of all ages, SMP30/GNL staining appeared in some but not all parenchymal cells, and localized in both the nuclei and cytoplasm. Moreover, SMP30/GNL-positive staining of parenchymal cells was present only around central vein areas, but not at sites of portal veins. Furthermore, the number of SMP30/GNL-positive cells increased as mice aged from 1 to 12 months, then decreased from the 12th to 24th month. Results were similar in primary cultured hepatocytes from mice of various ages. CONCLUSIONS: SMP30/GNL-positive cells localized mainly around the central veins in the livers of mice and decreased numerically with aging, although there was no age-related change in counts of albumin-positive cells. SMP30/GNL protein occupied the nuclei and cytoplasm. Therefore, nuclear SMP30/GNL protein might be a regulatory factor specific for genes whose expression governs transcription and the aging process.

Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis.

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30) is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA) biosynthesis. SMP30 also participates in Ca(2+) efflux by activating the calmodulin-dependent Ca(2+)-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO) mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15-24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1) higher plasma levels of triglyceride and aspartate aminotransferase; (2) severe accumulation of hepatic triglyceride and total cholesterol; (3) higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4) decreased mRNA and protein levels of Apolipoprotein B (ApoB) in livers - ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion.