Combined resistant dextrin and low-dose Mg oxide administration increases short-chain fatty acid and lactic acid production by gut microbiota.

The consumption of resistant dextrin improves constipation, while its fermentation and degradation by the intestinal microbiota produce short-chain fatty acids (SCFA) and lactic acid, which have beneficial effects on host metabolism and immunity. Mg oxide (MgO) is an important mineral that is used to treat constipation. Therefore, resistant dextrin and MgO are often administered together to improve constipation. However, limited information is available regarding the effect of this combination on SCFA and lactic acid production. Crl:CD1(ICR) mice were fed a Mg-free diet with 5% resistant dextrin, followed by oral administration of MgO. We collected the cecum contents and measured SCFA and lactic acid levels. Additionally, the human subjects received resistant dextrin and Mg supplements as part of their habitual diet. The results of this study demonstrate that intestinal microbiota cannot promote SCFA and lactic acid production in the absence of Mg. In a mouse model, low doses of MgO promoted the production of SCFA and lactic acid, whereas high doses decreased their production. In humans, the combined consumption of resistant dextrin and Mg supplements increased the production of SCFA and lactic acid. The production of SCFA and lactic acid from dietary fiber may be augmented by the presence of MgO.

Mid-Point of the Active Phase Is Better to Achieve the Natriuretic Effect of Acute Salt Load in Mice.

Excess sodium intake and insufficient potassium intake are a prominent global issue because of their influence on high blood pressure. Supplementation of potassium induces kaliuresis and natriuresis, which partially explains its antihypertensive effect. Balancing of minerals takes place in the kidney and is controlled by the circadian clock; in fact, various renal functions exhibit circadian rhythms. In our previous research, higher intake of potassium at lunch time was negatively associated with blood pressure, suggesting the importance of timing for sodium and potassium intake. However, the effects of intake timing on urinary excretion remain unclear. In this study, we investigated the effect of 24 h urinary sodium and potassium excretion after acute sodium and potassium load with different timings in mice. Compared to other timings, the middle of the active phase resulted in higher urinary sodium and potassium excretion. In Clock mutant mice, in which the circadian clock is genetically disrupted, urinary excretion differences from intake timings were not observed. Restricted feeding during the inactive phase reversed the excretion timing difference, suggesting that a feeding-induced signal may cause this timing difference. Our results indicate that salt intake timing is important for urinary sodium and potassium excretion and provide new perspectives regarding hypertension prevention.

Effect of circadian clock and claudin regulations on inulin-induced calcium absorption in the mouse intestinal tract.

Dietary calcium supplementation has been shown to be an effective adjunct therapy in an inflammatory bowel disease model. Soluble dietary fiber reduces intestinal pH and is known to enhance calcium absorption. Although many circadian clock regulations of nutrient absorption in the intestinal tract have been reported, the effects of clock regulation on calcium absorption have yet to be understood. In this study, we investigated the timing of efficient calcium intake by measuring urinary calcium excretion in mice. The diurnal variations in channel-forming tight junctions (claudins) were detected in both the jejunum and ileum. Following 2 days of feeding with a Ca2+-free diet, Ca2+-containing diets with or without soluble fiber (inulin) were fed at specific timings, and urine was subsequently examined every 4 hr. There was an evident increase in urinary calcium concentration when the inulin diet was fed at the beginning of the resting period. The Claudin 2 (Cldn2) expression level also showed a significant day-night change, which seemed to be a mechanism for the increased calcium excretion after inulin intake. This diurnal rhythm and enhanced Cldn2 expression were abolished by disruption of the suprachiasmatic nucleus, the central clock in the hypothalamus. This study suggests that intestinal calcium absorption might be modulated by the circadian clock and that the intake of inulin is more effective at the beginning of the resting period in mice.

Effects of the timing of acute mulberry leaf extract intake on postprandial glucose metabolism in healthy adults: a randomised, placebo-controlled, double-blind study.

BACKGROUND/OBJECTIVES: Glucose tolerance is controlled by the internal clock and is worse in the evening. From a chrononutrition perspective, diabetes prevention requires evaluating the antidiabetic effects of the timing of functional ingredients and nutrient intake. The purpose of this study was to investigate the timing effects of acute mulberry leaf extract (MLE) intake on postprandial glucose levels in young adults. SUBJECTS/METHODS: Twelve young adults underwent four trials. Blood samples were collected in a fasting state and at 30, 60, 120, and 180 min after eating a mixed meal. The study had a randomised, placebo-controlled, double-blind trial design involving: (1) morning placebo trial (08:00 h; MP trial), (2) evening placebo trial (18:00 h; EP trial), (3) morning MLE trial (08:00 h; MM trial), and (4) evening MLE trial (18:00 h; EM trial). RESULTS: The incremental area under the blood glucose curve (iAUC) in the EM trials was significantly lower than that in the EP trials (P = 0.010). The postprandial glucose concentrations 120 min after the meal were significantly lower in the EM trials than those in the EP trials (P = 0.006). The postprandial insulin concentrations at 120 min were significantly lower in the MM trials than those in the MP trials (P = 0.034). Moreover, the postprandial insulin concentrations 180 min after the meal were significantly lower in the EM trials than those in the EP trials (P = 0.034). CONCLUSIONS: MLE intake in the evening, but not in the morning, was effective in improving glucose tolerance. TRIAL REGISTRATION: Clinical trial reference: UMIN 000045301; website of trial registry: https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000051340 .

Association of Japanese Breakfast Intake with Macro- and Micronutrients and Morning Chronotype.

(1) Background: Dietary intake may have a remarkable effect on sleep because skipping breakfast and having a late dinner affects many sleep parameters. Breakfast is the most important meal of the day for children and adults to maintain morning chronotype. We examine whether breakfast style is associated with nutrient intake and sleep factors. (2) Methods: This cross-sectional analysis, with a large sample size of 2671 (766 men and 1805 women aged 20-60 years after data brush-up), was based on data obtained from an online survey. Correlation analysis was performed using Spearman's rank correlation test. The Kruskal-Wallis's test followed by post hoc Dunn's multiple comparison test was used to evaluate the interaction between sleep factors and breakfast categories. Multiple regression analyses were performed to identify variables associated with multiple confounding factors. Dietary data were analyzed using approximately one-month average dietary records from the application. The basic characteristics of the participants (age, sex, and BMI) and other lifestyle-related factors (sleep and physical activity) were obtained accordingly. Sleep parameters including the timing of weekday sleep onset, weekday wake-up, weekend (free day) sleep onset, weekend wake-up, sleep, and midpoints of sleep phase were calculated for each participant. We categorized participants' breakfast types into five groups: (1) Japanese meal, where breakfast may contain Japanese ingredients such as rice; (2) Western meal, where breakfast may contain bread; (3) alternating eating patterns of Japanese and Western meals; (4) cereals and supplements, where breakfast may contain cereals or supplements and energy bars; and (5) skipped breakfast (no breakfast). (3) Results: The midpoint values of the sleep phase on weekends adjusted for sleep debt on work days (MSFsc) related to chronotype were higher in women, suggesting that they may prefer eveningness. Participants with obesity, young age, and low physical activity preferred eveningness with longer sleep durations. Intake of Japanese-style breakfast was significantly associated with early wake-up time on both weekdays and weekends. Cereal-style breakfast intake was significantly associated with late wake-up on both weekdays and weekends. Intake of macronutrients such as protein, fat, carbohydrate, and sodium at breakfast time was positively and strongly associated with the intake of Japanese breakfast, whereas macronutrients were negatively associated with the intake of cereal breakfast. Among micronutrients, vitamin K was positively correlated with Japanese breakfast and negatively correlated with cereal breakfast; (4) Conclusions: Japanese-style breakfast is associated not only with morning preference but also with high intake of macro- and micronutrients.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway.

CONTEXT: The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampo, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. OBJECTIVE: We examine effective crude drugs, which cover at least one or two of Kampo, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. MATERIALS AND METHODS: We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 µg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. RESULTS: We found that Polygalae Radix (ED50: 24.01 µg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. DISCUSSION AND CONCLUSIONS: Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

Effects of Timing of Acute and Consecutive Catechin Ingestion on Postprandial Glucose Metabolism in Mice and Humans.

We examined the effects of the timing of acute and consecutive epigallocatechin gallate (EGCG) and catechin-rich green tea ingestion on postprandial glucose in mice and human adults. In mouse experiments, we compared the effects of EGCG administration early (morning) and late (evening) in the active period on postprandial glucose. In human experiments, participants were randomly assigned to the morning-placebo (MP, n = 10), morning-green tea (MGT, n = 10), evening-placebo (EP, n = 9), and evening-green tea (EGT, n = 9) groups, and consumed either catechin-rich green tea or a placebo beverage for 1 week. At baseline and after 1 week, participants consumed their designated beverages with breakfast (MP and MGT) or supper (EP and EGT). Venous blood samples were collected in the fasted state and 30, 60, 120, and 180 min after each meal. Consecutive administration of EGCG in the evening, but not in the morning, reduced postprandial glucose at 30 (p = 0.006) and 60 (p = 0.037) min in the evening trials in mice. In humans, ingestion of catechin-rich green tea in the evening decreased postprandial glucose (three-factor analysis of variance, p < 0.05). Thus, catechin intake in the evening more effectively suppressed elevation of postprandial glucose.

Effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in healthy men.

Green tea polyphenols, particularly catechins, decrease fasting and postprandial glucose. However, no studies have compared the timing of green tea ingestion on glucose metabolism and changes in catechin concentrations. Here, we examined the effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in young men. Seventeen healthy young men completed four trials involving blood collection in a fasting state and at 30, 60, 120, and 180 min after meal consumption in a random order: 1) morning placebo trial (09:00 h; MP trial), 2) evening placebo trial (17:00 h; EP trial), 3) morning catechin-rich green tea trial (09:00 h; MGT trial), and 4) evening catechin-rich green tea trial (17:00 h; EGT trial). The concentrations of glucose at 120 min (P=.031) and 180 min (P=.013) after meal intake were significantly higher in the MGT trials than in the MP trials. Additionally, the concentration of glucose was significantly lower in EGT trials than in the EP trials at 60 min (P=.014). Moreover, the concentrations of glucose-dependent insulinotropic polypeptide were significantly lower in the green tea trials than in the placebo trials at 30 min (morning: P=.010, evening: P=.006) and 60 min (morning: P=.001, evening: P=.006) after meal intake in both the morning and evening trials. Our study demonstrated that acute ingestion of catechin-rich green tea in the evening reduced postprandial plasma glucose concentrations.

Glucagon and/or IGF-1 Production Regulates Resetting of the Liver Circadian Clock in Response to a Protein or Amino Acid-only Diet.

The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment.

[Chrono-nutrition and n-3 polyunsaturated fatty acid].

Circadian clock system has been widely maintained in many spices from prokaryote to mammals. "Circadian" means "approximately day" in Latin, thus circadian rhythm means about 24 hour rhythms. The earth revolves once every 24 hours, and our circadian system has been developed for adjusting to this 24 hour cycles, to get sun light information for getting their foods or for alive in birds or mammals. We have two different circadian systems so-called main oscillator located in the suprachiasmatic nucleus (SCN) of the hypothalamus, and local oscillator located in the various peripheral organ tissues such as liver, kidney and skeletal muscle. The SCN is directly entrained by light-dark information through retinal-hypothalamic tract, and then organizes local clock in peripheral tissues via many pathways including neural and hormonal functions. On the other hand, peripheral local clocks are entrained by feeding, exercise and stress stimuli through several cell signaling. Foods (protein, carbohydrate, and lipid) are important regulator of circadian clocks in peripheral tissues. Thus, controlling the timing of food consumption and food composition, a concept known as chrononutrition, is one area of active research to aid recovery from many physiological dysfunctions. In this review, we focus on molecular mechanisms of entrainment and the relationships between circadian clock systems and n-3 polyunsaturated fatty acid. We concentrate on experimental data obtained from cells or animals and humans and discuss how these findings translate into clinical research, and we highlight the latest developments in chrononutritional studies.

Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue.

Microbiota-derived short-chain fatty acids (SCFAs) and organic acids produced by the fermentation of non-digestible fibre can communicate from the microbiome to host tissues and modulate homeostasis in mammals. The microbiome has circadian rhythmicity and helps the host circadian clock function. We investigated the effect of SCFA or fibre-containing diets on circadian clock phase adjustment in mouse peripheral tissues (liver, kidney, and submandibular gland). Initially, caecal SCFA concentrations, particularly acetate and butyrate, induced significant day-night differences at high concentrations during the active period, which were correlated with lower caecal pH. By monitoring luciferase activity correlated with the clock gene Period2 in vivo, we found that oral administration of mixed SCFA (acetate, butyrate, and propionate) and an organic acid (lactate), or single administration of each SCFA or lactate for three days, caused phase changes in the peripheral clocks with stimulation timing dependency. However, this effect was not detected in cultured fibroblasts or cultured liver slices with SCFA applied to the culture medium, suggesting SCFA-induced indirect modulation of circadian clocks in vivo. Finally, cellobiose-containing diets facilitated SCFA production and refeeding-induced peripheral clock entrainment. SCFA oral gavage and prebiotic supplementation can facilitate peripheral clock adjustment, suggesting prebiotics as novel therapeutic candidates for misalignment.

Polyporus and Bupleuri radix effectively alter peripheral circadian clock phase acutely in male mice.

In mammals, daily physiological events are precisely regulated by an internal circadian clock system. An important function of this system is to readjust the phase of the clock daily. In Japan, traditional herb medicines, so-called crude drugs (Shoyaku), are widely used for many diseases, and some are reported to affect circadian clock impairment, suggesting that some of them might have an ability to modify clock gene expression rhythms. Therefore, from selected 40 crude drugs, finding candidates that control the circadian clock phases was the first purpose of this study. As there are several crude drugs used for liver- and/or kidney-related diseases, the second aim of the present study was to find some crude drugs affecting liver/kidney circadian clock in vivo. To assess phase changes in the daily circadian rhythm, bioluminescence from the core clock gene product Period 2 was continuously monitored in mouse embryonic fibroblasts in vitro and in some peripheral tissues (kidney, liver, and submandibular gland) of PERIOD2::LUCIFERASE knock-in mice in vivo. In our screening, Polyporus and Bupleuri radix were found to be good candidates to effectively manipulate the peripheral circadian clock phase acutely, with stimulation time-of-day dependency in vitro as well as in vivo. Interestingly, Polyporus and Bupleuri radix are traditional herb medicines use for treating edema and promoting diuresis, and for chronic hepatitis, respectively. These crude drugs may be therefore good modulators of the circadian peripheral clocks including liver and kidney, and circadian clock genes become new molecular targets for these crude drugs.

Fish Oil Accelerates Diet-Induced Entrainment of the Mouse Peripheral Clock via GPR120.

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock.

Time-restricted feeding of rapidly digested starches causes stronger entrainment of the liver clock in PER2::LUCIFERASE knock-in mice.

Restricting feeding to daytime can entrain circadian clocks in peripheral organs of rodents, and nutrients that rapidly increase the blood glucose level are suitable for inducing entrainment. However, dietetic issues, for example, whether or not the diet comprises heated food, have not been fully explored. We therefore hypothesized that rapidly digested starch causes stronger entrainment than slowly digested starch. The entrainment ability of the liver clock in PER2::LUCIFERASE knock-in mice, blood glucose levels, insulin levels, and acute changes in liver clock gene expression were compared between a ß-starch (native)-substituted AIN-93M standard diet and an α-starch (gelatinized)-substituted diet. ß-Corn and ß-rice starch induced larger phase delays of the liver clock, larger blood glucose increases, and higher Per2 gene expression in the liver compared with ß-potato starch. Starch granule size, as examined by electron microscopy, was larger for ß-potato starch than for ß-corn or ß-rice starch. After heating, we obtained gelatinized α-potato, α-corn, and α-rice starch, which showed destruction of the crystal structure and a high level of gelatinization. No difference in the increase of blood glucose or insulin levels was observed between ß-corn and α-corn starch, or between ß-rice and α-rice starch. In contrast, α-potato starch caused higher levels of glucose and insulin compared with ß-potato starch. An α-potato starch-substituted diet induced larger phase delays of the liver clock than did ß-potato starch. Therefore, rapidly digested starch is appropriate for peripheral clock entrainment. Dietetic issues (heated vs unheated) are important when applying basic mouse data to humans.

Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).

Prednisolone causes anxiety- and depression-like behaviors and altered expression of apoptotic genes in mice hippocampus.

Glucocorticoids are known to cause psychiatric disorders including depression. Prednisolone (PSL) is one of the most widely used synthetic glucocorticoids to treat various medical diseases; however, little is known about PSL-induced behavioral changes and its molecular basis in the brain. Growing evidence has implicated that hippocampal remodeling or damage play a role in the pathogenic effect of glucocorticoids. In this study, mice were administered PSL (50 or 100mg/kg) or vehicle for 6 or 7 days and subjected to a series of behavioral tests, i.e., open field, elevated plus maze, prepulse inhibition, forced swim, and tail suspension tests. Hippocampal tissues were subject to microarray analysis using the GeneChip Mouse Genome 430 2.0 Array (Affymetrix) containing 45,101 probes of transcripts. Increased anxiety- and depression-like behaviors assessed with open field, elevated plus maze, and tail suspension tests were observed. Microarray analysis detected 108 transcripts with a fold change of >2.0 or <0.5 in which many cell-death-related genes were found. The microarray data was validated by quantitative reverse transcriptase-polymerase chain reaction analysis. Our results demonstrated that PSL causes anxiety- and depression-like behaviors, and suggest that altered gene expressions related to hippocampal remodeling or damage are involved in the effect of PSL on such behaviors.

Neural regulation of the hepatic circadian rhythm.

A microarray analysis experiment has revealed that there are many genes, including so-called clock genes, expressing a circadian rhythm in the liver. The clock genes mentioned above are expressed not only in the suprachiasmatic nucleus (SCN) of the hypothalamus, where the master clock exists, but also in other brain regions and various peripheral tissues. In the liver, clock genes are abundantly expressed and show a clear circadian rhythm. Thus, clock genes seem to play a critical role in the molecular clockworks of both the SCN and the liver. Although oscillation of clock genes in the liver is controlled under the circadian clock mechanism in the SCN, we do not know the resetting signals on liver clock function. Over the past few years, use of the pseudorabies virus, a transsynaptic tract tracer, has allowed us to map neural connections between the SCN and peripheral tissues in several physiological systems. Communication between the SCN and peripheral tissues occurs through autonomic nervous systems involving the sympathetic and parasympathetic neurons. This review mainly describes both anatomical and physiological experiments to reveal the sympathetic control over liver clock function. Although further study is necessary to produce the precise mechanism underlying neural control of liver clock systems, evolution of this mechanism will help our understanding of liver clock functions such as drug metabolism and energy metabolism.