Yuki Yasumoto

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.

Wheat Alkylresorcinols Suppress High-Fat, High-Sucrose Diet-Induced Obesity and Glucose Intolerance by Increasing Insulin Sensitivity and Cholesterol Excretion in Male Mice

BACKGROUND Epidemiologic studies have shown that the consumption of whole grains can reduce the risk of type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. However, the underlying mechanisms remain a matter of debate. OBJECTIVE We aimed to determine the effects of wheat bran-derived alkylresorcinols on diet-induced metabolic disorders in mice. METHODS We fed C57BL/6J mice a normal refined diet or a high-fat, high-sucrose diet [29.1% fat, 20.7% protein, 34.0% carbohydrates containing 20.0% sucrose (w/w)] alone (FS) or containing 0.4% (wt:wt) alkylresorcinols (FS-AR) for 10 wk. RESULTS The alkylresorcinols suppressed FS-induced increases in body weight by 31.0% as well as FS-induced hepatic triglyceride accumulation (means ± SEMs: 29.6 ± 3.18 and 19.8 ± 2.42 mg/g tissue in the FS and FS-AR groups, respectively), without affecting energy intake. We measured circadian changes in blood metabolic hormones and found that FS-induced hyperinsulinemia (5.1 and 2.1 μg/L at night in the FS and FS-AR groups, respectively) and hyperleptinemia (21.6 and 10.8 μg/L at night in the FS and FS-AR groups, respectively) were suppressed by alkylresorcinols. Glucose and insulin tolerance tests showed that alkylresorcinols significantly reduced fasting blood glucose concentrations (190 ± 3.62 and 160 ± 8.98 mg/dL in the FS and FS-AR groups, respectively) and suppressed glucose intolerance as well as insulin resistance induced by the FS diet. Furthermore, alkylresorcinols significantly increased insulin-stimulated hepatic serine/threonine protein kinase B phosphorylation compared to the FS diet (+81.3% and +57.4% for Ser473 and Thr308, respectively). On the other hand, pyruvate and starch tolerance tests suggested that alkylresorcinols did not affect gluconeogenesis and carbohydrate digestion, respectively. Alkylresorcinols significantly increased fecal cholesterol excretion by 39.6% and reduced blood cholesterol concentrations by 30.4%, while upregulating the expression of hepatic cholesterol synthetic genes such as sterol regulatory element binding protein 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1). CONCLUSIONS These findings suggest that wheat alkylresorcinols increase glucose tolerance and insulin sensitivity by suppressing hepatic lipid accumulation and intestinal cholesterol absorption, which subsequently suppresses diet-induced obesity in mice.

Free Access to a Running-Wheel Advances the Phase of Behavioral and Physiological Circadian Rhythms and Peripheral Molecular Clocks in Mice

Behavioral and physiological circadian rhythms are controlled by endogenous oscillators in animals. Voluntary wheel-running in rodents is thought to be an appropriate model of aerobic exercise in humans. We evaluated the effects of chronic voluntary exercise on the circadian system by analyzing temporal profiles of feeding, core body temperature, plasma hormone concentrations and peripheral expression of clock and clock-controlled genes in mice housed under sedentary (SED) conditions or given free access to a running-wheel (RW) for four weeks. Voluntary wheel-running activity advanced the circadian phases of increases in body temperature, food intake and corticosterone secretion in the mice. The circadian expression of clock and clock-controlled genes was tissue- and gene-specifically affected in the RW mice. The temporal expression of E-box-dependent circadian clock genes such as Per1, Per2, Nr1d1 and Dbp were slightly, but significantly phase-advanced in the liver and white adipose tissue, but not in brown adipose tissue and skeletal muscle. Peak levels of Per1, Per2 and Nr1d1 expression were significantly increased in the skeletal muscle of RW mice. The circadian phase and levels of hepatic mRNA expression of the clock-controlled genes that are involved in cholesterol and fatty acid metabolism significantly differed between SED and RW mice. These findings indicated that endogenous clock-governed voluntary wheel-running activity provides feedback to the central circadian clock that systemically governs behavioral and physiological rhythms.

Atypical expression of circadian clock genes in denervated mouse skeletal muscle.

The central circadian clock in the suprachiasmatic nucleus of the hypothalamus synchronizes peripheral clocks through neural and humoral signals in most mammalian tissues. Here, we analyzed the effects of unilateral sciatic denervation on the expression of circadian clock- and clock-controlled genes in the gastrocnemius muscles of mice twice per day on days 0, 3, 7, 9, 11 and 14 after denervation and six times on each of days 7 and 28 after denervation to assess the regulation mechanism of the circadian clock in skeletal muscle. Sciatic denervation did not affect systemic circadian rhythms since core body temperature (Day 7), corticosterone secretion (Days 7 and 28), and hepatic clock gene expression remained intact (Days 7 and 28). Expression levels of most circadian clock-related genes such as Arntl, Per1, Rora, Nr1d1 and Dbp were reduced in accordance with the extent of muscle atrophy, although circadian Per2 expression was significantly augmented (Day 28). Cosinor analysis revealed that the circadian expression of Arntl (Days 7 and 28) and Dbp (Day 28) was phase advanced in denervated muscle. The mRNA expression of Clock was significantly increased in denervated muscle on Day 3 when the severe atrophy was absent, and it was not affected by atrophic progression for 28 days. Sciatic denervation did not affect the expression of these genes in the contralateral muscle (Days 7 and 28), suggesting that humoral changes were not involved in denervation-induced muscle clock disruption. We then analyzed genome-wide gene expression using microarrays to determine the effects of disrupting the molecular clock in muscle on circadian rhythms at Day 7. Among 478 circadian genes, 313 lost rhythmicity in the denervated muscles. These denervation-sensitive genes included the lipid metabolism-related genes, Nrip1, Bbs1, Ptgis, Acot1, Scd2, Hpgd, Insig1, Dhcr24, Ldlr and Mboat1. Our findings revealed that sciatic denervation disrupts the circadian expression of clock and clock-controlled genes either directly or indirectly via muscle atrophy in the gastrocnemius muscles of mice in a gene-specific manner.

Impact of denervation-induced muscle atrophy on housekeeping gene expression in mice.

Introduction: Immobilization induced by experimental denervation leads to rapid and progressive alterations in structural and biochemical properties of skeletal muscle. Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) is a popular method of elucidating the molecular mechanisms involved in muscle atrophy. Identification of suitable reference genes that are not affected by experimental conditions is a critical step in accurate normalization of real‐time RT‐PCR. Methods: We investigated the impact of denervation‐induced muscle atrophy for 2 weeks on the expression of common housekeeping genes. Results: Denervation differentially affected the expression levels of these genes. RefFinder software identified TATA box binding protein (Tbp) as the most stable gene and showed that the stability of glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh) and hypoxanthine guanine phosphoribosyl transferase (Hprt) genes was low, even though they are widely used for normalization. Conclusions: The appropriate reference gene for normalization of genes of interest in denervated muscle is Tbp. Muscle Nerve 51: 276–281, 2015

Disrupted light-dark cycle abolishes circadian expression of peripheral clock genes without inducing behavioral arrhythmicity in mice.

The environmental light-dark (LD) cycle entrains the central circadian clock located in the suprachiasmatic nucleus (SCN) of mammals. The present study examined the effects of disrupted LD cycles on peripheral clocks in mice housed under a normal 12 h light-12 h dark cycle (LD 12:12) or an ultradian LD 3:3 cycle. Drinking behavior seemed to be free-running with a long period (26.03 h) under ultradian LD 3:3 cycles, in addition to light-induced direct suppression (masking effect). Core body temperature completely lost robust circadian rhythm and acquired a 6-h rhythm with a low amplitude under LD 3:3. Robust circadian expression of Per1, Per2, Clock and Bmal1 mRNAs was similarly flattened to intermediate levels in the liver, heart and white adipose tissue under LD 3:3. Robust circadian expression of Rev-erbα mRNA was completely damped in these tissues. Circadian expression of Dbp, a clock-controlled gene, was also disrupted in these tissues from mice housed under LD 3:3. The aberrant LD cycle seemed to induce the loss of circadian gene expression at the level of transcription, because rhythmic pre-mRNA expression of these genes was also abolished under LD 3:3. In addition to the direct effect of the aberrant LD cycle, abolished systemic time cues such as those of plasma corticosterone and body temperature might be involved in the disrupted expression of these circadian genes under LD 3:3. Our findings suggest that disrupted environmental LD cycles abolish the normal oscillation of peripheral clocks and induce internal desynchrony in mammals.

Circadian fluctuations in circulating plasminogen activator inhibitor-1 are independent of feeding cycles in mice

ABSTRACT To evaluate the involvement of the day-night feeding cycle in the circadian regulation of circulating plasminogen activator inhibitor-1 (PAI-1) concentrations, mice were fed with a diet for eight hours during either daytime (DF) or nighttime (NF) for one week. The reversed feeding cycle did not affect the circadian phases of plasma PAI-1 levels as well as the nocturnal wheel-running activity, although the phase of Pai-1 mRNA expression was significantly advanced for 8.6 hours in the livers of DF, compared with NF mice. The day-night feeding cycle is not a critical Zeitgeber for circadian rhythm of circulating PAI-1.

Molecular Clock Regulates Daily α1–2-Fucosylation of the Neural Cell Adhesion Molecule (NCAM) within Mouse Secondary Olfactory Neurons

Background: Mammalian olfaction has circadian rhythm, and glycosylation plays critical roles in the olfactory system. Results: α1–2-Fucosylation increases during the nighttime in axons of secondary olfactory neurons in WT but not in Clock mutant mice. Conclusion: Rhythmic α1–2-fucosylation governed by clock genes is a potential mechanism of circadian olfaction. Significance: Glycosylation in the central nervous system is circadian. The circadian clock regulates various behavioral and physiological rhythms in mammals. Circadian changes in olfactory functions such as neuronal firing in the olfactory bulb (OB) and olfactory sensitivity have recently been identified, although the underlying molecular mechanisms remain unknown. We analyzed the temporal profiles of glycan structures in the mouse OB using a high-density microarray that includes 96 lectins, because glycoconjugates play important roles in the nervous system such as neurite outgrowth and synaptogenesis. Sixteen lectin signals significantly fluctuated in the OB, and the intensity of all three that had high affinity for α1–2-fucose (α1–2Fuc) glycan in the microarray was higher during the nighttime. Histochemical analysis revealed that α1–2Fuc glycan is located in a diurnal manner in the lateral olfactory tract that comprises axon bundles of secondary olfactory neurons. The amount of α1–2Fuc glycan associated with the major target glycoprotein neural cell adhesion molecule (NCAM) varied in a diurnal fashion, although the mRNA and protein expression of Ncam1 did not. The mRNA and protein expression of Fut1, a α1–2-specific fucosyltransferase gene, was diurnal in the OB. Daily fluctuation of the α1–2Fuc glycan was obviously damped in homozygous Clock mutant mice with disrupted diurnal Fut1 expression, suggesting that the molecular clock governs rhythmic α1–2-fucosylation in secondary olfactory neurons. These findings suggest the possibility that the molecular clock is involved in the diurnal regulation of olfaction via α1–2-fucosylation in the olfactory system.

Dosing schedule-dependent attenuation of dexamethasone-induced muscle atrophy in mice

Many inflammatory and autoimmune diseases are treated using synthetic glucocorticoids. However, excessive glucocorticoid can often cause unpredictable effects including muscle atrophy. Endogenous glucocorticoid levels robustly fluctuate in a circadian manner and peak just before the onset of the active phase in both humans and nocturnal rodents. The present study determines whether muscle atrophy induced by exogenous glucocorticoid can be avoided by optimizing dosing times. We administered single daily doses of the glucocorticoid analog dexamethasone (Dex) to mice for 10 days at the times of day corresponding to peak (early night) or trough (early morning) endogenous glucocorticoid levels. Administration at the acrophase of endogenous glucocorticoids significantly attenuated Dex-induced wasting of the gastrocnemius (Ga) and tibialis anterior (TA) muscles that comprise mostly fast-twitch muscle fibers. Real-time RT-PCR revealed that the Dex-induced mRNA expression of genes encoding the atrophy-related ubiquitin ligases Muscle Atrophy F-box (Fbxo32, also known as MAFbx/Atrogin-1) and Muscle RING finger 1 (Trim63, also known as MuRF1) in the Ga and TA muscles was significantly attenuated by Dex when administered during the early night. Dex negligibly affected the weight of the soleus (So) muscle that mostly comprises slow-twitch muscle fibers, but significantly and similarly decreased the weight of the spleen at both dosing times. These results suggest that glucocorticoid-induced muscle atrophy can be attenuated by optimizing the dosing schedule.