Time-of-day effects of consumption of fish oil-enriched sausages on serum lipid parameters and fatty acid composition in normolipidemic adults: A randomized, double-blind, placebo-controlled, and parallel-group pilot study.

OBJECTIVES: The body clock controls diurnal rhythms of nutrient digestion, absorption, and metabolism. Fish oil (FO) contains abundant ω-3 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), that are thought to lower triglyceride (TG) levels. This randomized, placebo-controlled, double-blind, parallel-group trial aimed to confirm the effects of the time of FO intake on TG in healthy Japanese adults. METHODS: Twenty healthy Japanese adults (age, 20-60 y) were assigned to either a group that consumed sausages enriched with FO (DHA 1010 mg; EPA 240 mg) in the morning and a placebo (DHA 40 mg; EPA 15 mg) in the evening (BF-FO) or another group that consumed FO-enriched sausages in the evening and the placebo in the morning (DN-FO). Serum lipid parameters, fatty acid (FA) composition, and messenger RNA expression of lipogenic genes in circulating blood cells were evaluated in fasting blood samples before, as well as after 4 and 8 wk of FO intake. RESULTS: Serum concentrations of TG and total saturated FA were significantly decreased in the BF-FO group, whereas those of ω-3 PUFA were significantly and identically increased in both groups. Serum concentrations of ω-6 PUFA were significantly decreased in the BF-FO but not the DN-FO group. Messenger RNA expression of the lipogenic genes ACLY, SCD, and FASN were similarly reduced in both groups. CONCLUSIONS: These findings suggested that the timing of FO intake affects both serum FA concentrations and TG metabolism in normolipidemic humans. The mechanisms of these effects of FO on lipid metabolism require further investigation.

Cacao polyphenols regulate the circadian clock gene expression and through glucagon-like peptide-1 secretion.

Energy metabolism and circadian rhythms are closely related together, i.e., the timing of nutrient intake affects metabolism under the regulation of circadian rhythms. Previously, we have reported that cacao liquor procyanidin (CLPr) promotes energy metabolism, resulting in preventing obesity and hyperglycemia. However, it is not unclear whether CLPr regulates clock gene expression. In this study, we investigated whether the administration timing of CLPr affected clock gene expression and found that CLPr regulated the circadian clock gene expression through the glucagon-like peptide-1 (GLP-1) signaling pathway. CLPr administration at Zeitgeber time 3 increased the expression level of Per family and Dbp in the liver. At the same administration timing, CLPr increased GLP-1 and insulin concentration in the plasma and phosphorylation of AMPK in the liver. It was noteworthy that an antagonist for GLP-1 receptor Exendin (9-39) canceled CLPr-increased expression of Per family and Dbp and phosphorylation of AMPK in the liver, in addition to insulin secretion. These results strongly suggest that CLPr-induced GLP-1 regulates the changes in clock gene expression in the liver through increased insulin. Thus, CLPr is a possible functional food material for prevention and/or amelioration of metabolic disorders through preventing circadian disruption through GLP-1 and AMPK pathways.

Chronically skipping breakfast impairs hippocampal memory-related gene expression and memory function accompanied by reduced wakefulness and body temperature in mice.

Acute or chronic effects of consuming or skipping breakfast on cognitive performance in humans are controversial. To evaluate the effects of chronically skipping breakfast (SB) on hippocampus-dependent long-term memory formation, we examined hippocampal gene expression and applied the novel object recognition test (NORT) after two weeks of repeated fasting for six hours from lights off to mimic SB in mice. We also examined the effects of SB on circadian rhythms of locomotor activity, food intake, core body temperature (CBT) and sleep-wake cycles. Skipping breakfast slightly but significantly decreased total daily food intake without affecting body weight gain. Locomotor activity and CBT significantly decreased during the fasting period under SB. The degree of fasting-dependent CBT reduction gradually increased and then became stabilized after four days of SB. Electroencephalographic data revealed that repeated SB significantly decreased the duration of wakefulness and increased that of rapid eye movement (REM) and of non-REM (NREM) sleep during the period of SB. Furthermore, total daily amounts of wakefulness and NREM sleep were significantly decreased and increased, respectively, under SB, suggesting that SB disrupts sleep homeostasis. Skipping breakfast significantly suppressed mRNA expression of the memory-related genes, Camk2a, Fkbp5, Gadd45b, Gria1, Sirt1 and Tet1 in the hippocampus. Recognition memory assessed by NORT was impaired by SB in accordance with the gene expression profiles. These findings suggested that chronic SB causes dysregulated CBT, sleep-wake cycles and hippocampal gene expression, which results in impaired long-term memory formation.

Cinnamic acid shortens the period of the circadian clock in mice.

Cinnamic acid (CA) derivatives have recently received focus due to their anticancer, antioxidant, and antidiabetic properties. The present study aimed to determine the effects of cinnamic acid on the circadian clock, which is a cell-autonomous endogenous system that generates circadian rhythms that govern the behavior and physiology of most organisms. Cinnamic acid significantly shortened the circadian period of PER2::LUC expression in neuronal cells that differentiated from neuronal progenitor cells derived from PER2::LUC mouse embryos. Cinnamic acid did not induce the transient mRNA expression of clock genes such as Per1 and Per2 in neuronal cells, but significantly shortened the half-life of PER2::LUC protein in neuronal cells incubated with actinomycin D, suggested that CA post-transcriptionally affects the molecular clock by decreasing Per2 mRNA stability. A continuous infusion of CA into mice via an Alzet osmotic pump under constant darkness significantly shortened the free-running period of wheel-running rhythms. These findings suggest that CA shortens the circadian period of the molecular clock in mammals.

Short-term feeding at the wrong time is sufficient to desynchronize peripheral clocks and induce obesity with hyperphagia, physical inactivity and metabolic disorders in mice.

BACKGROUND: The circadian clock regulates various physiological and behavioral rhythms such as feeding and locomotor activity. Feeding at unusual times of the day (inactive phase) is thought to be associated with obesity and metabolic disorders in experimental animals and in humans. OBJECTIVE: The present study aimed to determine the underlying mechanisms through which time-of-day-dependent feeding influences metabolic homeostasis. METHODS: We compared food consumption, wheel-running activity, core body temperature, hormonal and metabolic variables in blood, lipid accumulation in the liver, circadian expression of clock and metabolic genes in peripheral tissues, and body weight gain between mice fed only during the sleep phase (DF, daytime feeding) and those fed only during the active phase (NF, nighttime feeding). All mice were fed with the same high-fat high-sucrose diet throughout the experiment. To the best of our knowledge, this is the first study to examine the metabolic effects of time-imposed restricted feeding (RF) in mice with free access to a running wheel. RESULTS: After one week of RF, DF mice gained more weight and developed hyperphagia, higher feed efficiency and more adiposity than NF mice. The daily amount of running on the wheel was rapidly and obviously reduced by DF, which might have been the result of time-of-day-dependent hypothermia. The amount of daily food consumption and hypothalamic mRNA expression of orexigenic neuropeptide Y and agouti-related protein were significantly higher in DF, than in NF mice, although levels of plasma leptin that fluctuate in an RF-dependent circadian manner, were significantly higher in DF mice. These findings suggested that the DF induced leptin resistance. The circadian phases of plasma insulin and ghrelin were synchronized to RF, although the corticosterone phase was unaffected. Peak levels of plasma insulin were remarkably higher in DF mice, although HOMA-IR was identical between the two groups. Significantly more free fatty acids, triglycerides and cholesterol accumulated in the livers of DF, than NF mice, which resulted from the increased expression of lipogenic genes such as Scd1, Acaca, and Fasn. Temporal expression of circadian clock genes became synchronized to RF in the liver but not in skeletal muscle, suggesting that uncoupling metabolic rhythms between the liver and skeletal muscle also contribute to DF-induced adiposity. CONCLUSION: Feeding at an unusual time of day (inactive phase) desynchronizes peripheral clocks and causes obesity and metabolic disorders by inducing leptin resistance, hyperphagia, physical inactivity, hepatic fat accumulation and adiposity.

Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/µL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods.

Quercetin suppresses immune cell accumulation and improves mitochondrial gene expression in adipose tissue of diet-induced obese mice.

SCOPE: To examine the effect of dietary quercetin on the function of epididymal adipose tissue (EAT) in Western diet-induced obese mice. METHODS AND RESULTS: C57BL/6J mice were fed a control diet; a Western diet high in fat, cholesterol, and sucrose; or the same Western diet containing 0.05% quercetin for 18 weeks. Supplementation with quercetin suppressed the increase in the number of macrophages, the decrease in the ratio of CD4(+) to CD8(+) T cells in EAT, and the elevation of plasma leptin and tumor necrosis factor α levels in mice fed the Western diet. Comprehensive gene expression analysis revealed that quercetin suppressed gene expression associated with the accumulation and activation of immune cells, including macrophages and lymphocytes in EAT. It also improved the expression of the oxidative stress-sensitive transcription factor NFκB, NADPH oxidases, and antioxidant enzymes. Quercetin markedly increased gene expression associated with mitochondrial oxidative phosphorylation and mitochondrial DNA content. CONCLUSION: Quercetin most likely universally suppresses the accumulation and activation of immune cells, including antiinflammatory cells, whereas it specifically increased gene expression associated with mitochondrial oxidative phosphorylation. Suppression of oxidative stress and NFκB activity likely contributed to the prevention of the accumulation and activation of immune cells and resulting chronic inflammation.

High glucose increases the expression of proinflammatory cytokines and secretion of TNFα and ß-hexosaminidase in human mast cells.

Epidemiological studies demonstrated that obesity, which is a high-risk factor for development of hyperglycemia-associated metabolic syndromes, is associated with prevalence/incidence of allergic diseases. To elucidate the underlying mechanisms of the relationship between hyperglycemia and allergy, we examined the effect of high glucose on the activation of human mast cell lines, HMC-1 and LAD2. HMC-1 and LAD2 cells were cultured in low (5.5 mM) and high (25 mM)-glucose Dulbecco's modified Eagle's medium (DMEM). High-glucose medium increased the intracellular reactive oxygen species levels in HMC-1 and LAD2 cells after 2 days of incubation; in HMC-1 cells, the expression levels of tumor necrosis factor (TNF) α, interleukin (IL)-1ß, IL-6, and IL-13 were increased significantly. The ß-hexosaminidase release rates were not significantly different between LAD2 cells cultured in both media; however, the intracellular and extracellular activities of ß-hexosaminidase in cells were significantly higher in high-glucose than in low-glucose media. High glucose increased the secretion of TNFα by unstimulated HMC-1 cells and IgE crosslinking-stimulated LAD2 cells. High glucose increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs), which regulate the expression of TNFα and other inflammatory cytokines, in both HMC-1 and LAD2 cells. Thus, high glucose increased the expression of proinflammatory and proallergic cytokines, the secretion of TNFα, and ß-hexosaminidase activity in human mast cells. Our result suggests that hyperglycemia promotes the activation of human mast cells associated with allergy and inflammation under unstimulated and stimulated conditions.

Caffeine lengthens circadian rhythms in mice.

Although caffeine alters sleep in many animals, whether or not it affects mammalian circadian clocks remains unknown. Here, we found that incubating cultured mammalian cell lines, human osteosarcoma U2OS cells and mouse fibroblast NIH3T3 cells, with caffeine lengthened the period of circadian rhythms. Adding caffeine to ex vivo cultures also lengthened the circadian period in mouse liver explants from Per2::Luciferase reporter gene knockin mice, and caused a phase delay in brain slices containing the suprachiasmatic nucleus (SCN), where the central circadian clock in mammals is located. Furthermore, chronic caffeine consumption ad libitum for a week delayed the phase of the mouse liver clock in vivo under 12 h light-dark conditions and lengthened the period of circadian locomotor rhythms in mice under constant darkness. Our results showed that caffeine alters circadian clocks in mammalian cells in vitro and in the mouse ex vivo and in vivo.

Chronic dietary intake of quercetin alleviates hepatic fat accumulation associated with consumption of a Western-style diet in C57/BL6J mice.

SCOPE: To determine the effect of consumption of a quercetin-rich diet on obesity and dysregulated hepatic gene expression. METHODS AND RESULTS: C56BL/6J mice were fed for 20 wk on AIN93G (control) or a Western diet high in fat, cholesterol and sucrose, both with or without 0.05% quercetin. Triglyceride levels in plasma, thiobarbituric acid-reactive substances (oxidative stress marker) and glutathione levels and peroxisome proliferator-activated receptor α expression in livers of mice fed with the Western diet were all improved after 8 wk feeding with quercetin. After 20 wk, further reductions of visceral and liver fat accumulation and improved hyperglycemia, hyperinsulinemia, dyslipidemia and plasma adiponectin and TNFα levels in these mice fed with quercetin were observed. The expression of hepatic genes related to steatosis, such as peroxisome proliferator-activated receptor γ and sterol regulatory element-binding protein-1c was also normalized by quercetin. In mice fed with the control diet, quercetin did not affect body weight but reduces the plasma TNFα and hepatic thiobarbituric acid-reactive substance levels. CONCLUSION: In mice fed with a Western diet, chronic dietary intake of quercetin reduces liver fat accumulation and improves systemic parameters related to metabolic syndrome, probably mainly through decreasing oxidative stress and reducing PPARα expression, and the subsequent reduced expression in the liver of genes related to steatosis.

Evaluation of the skin irritation using a DNA microarray on a reconstructed human epidermal model.

To avoid the need to use animals to test the skin irritancy potential of chemicals and cosmetics, it is important to establish an in vitro method based on the reconstructed human epidermal model. To evaluate skin irritancy efficiently and sensitively, we determined the gene expression induced by a topically-applied mild irritant sodium dodecyl sulfate (SDS) in a reconstructed human epidermal model LabCyte EPI-MODEL (LabCyte) using a DNA microarray carrying genes that were related to inflammation, immunity, stress and housekeeping. The expression and secretion of IL-1alpha in reconstructed human epidermal culture is known to be induced by irritation. We detected the induction of IL-1alpha expression and its secretion into the cell culture medium by treatment with 0.075% SDS for 18 h in LabCyte culture using DNA microarray, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and ELISA. DNA microarray analysis indicated that the expression of 10 of the 205 genes carried on the DNA microarray was significantly induced in a LabCyte culture by 0.05% or 0.075% SDS irritation for 18 h. RT-PCR analysis confirmed that SDS treatment significantly induced the expressions of interleukin-1 receptor antagonist (IL-1RN), FOS-like antigen 1 (FOSL1), heat shock 70 kDa protein 1A (HSPA1) and myeloid differentiation primary response gene (88) (MYD88), as well as the known marker genes for irritation IL-1beta and IL-8 in a LabCyte culture. Our results showed that a DNA microarray is a useful tool for efficiently evaluating mild skin irritation using a reconstructed human epidermal model.

Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells.

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.