Saori Yamamoto

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.

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

Ketogenic diet and fasting induce the expression of cold-inducible RNA-binding protein with time-dependent hypothermia in the mouse liver

Cold‐inducible RNA‐binding protein (CIRBP) induced by cold stress modulates the molecular circadian clock in vitro. The present study examines the effect of a ketogenic diet (KD) and fasting on Cirbp expression in the mouse liver. Chronic KD administration induced time‐dependent Cirbp expression with hypothermia in mice. The circadian expression of clock genes such as Bmal1 and Clock was phase‐advanced and augmented in the liver of mice fed with a KD. Transient food deprivation also induced time‐dependent Cirbp expression with hypothermia in mice. These findings suggest that hypothermia is involved in the increased expression of Cirbp under ketogenic or fasting conditions.

Dietary heat-killed Lactobacillus brevis SBC8803 promotes voluntary wheel-running and affects sleep rhythms in mice

AIMS We previously reported that heat-killed Lactobacillus brevis SBC8803 enhances appetite via changes in autonomic neurotransmission. Here we assessed whether a diet supplemented with heat-killed SBC8803 affects circadian locomotor rhythmicity and sleep architecture. MAIN METHODS AND KEY FINDINGS Daily total activity gradually increased in mice over 4 weeks and supplementation with heat-killed SBC8803 significantly intensified the increase, which reached saturation at 25 days. Electroencephalography revealed that SBC8803 supplementation significantly reduced the total amount of time spent in non-rapid eye movement (NREM) sleep and increased the amount of time spent being awake during the latter half of the nighttime, but tended to increase the total amount of time spent in NREM sleep during the daytime. Dietary supplementation with SBC8803 can extend the duration of activity during the nighttime and of sleep during the daytime. Daily voluntary wheel-running and sleep rhythmicity become intensified when heat-killed SBC8803 is added to the diet. SIGNIFICANCE Dietary heat-killed SBC8803 can modulate circadian locomotion and sleep rhythms, which might benefit individuals with circadian rhythms that have been disrupted by stress or ageing.

Effects of intraduodenal injection of Lactobacillus brevis SBC8803 on autonomic neurotransmission and appetite in rodents

Lactobacilli provide several health benefits to mammals, including humans. We previously observed that in rats, intraduodenal injection of Lactobacillus johnsonii La1 elevated efferent gastric vagal nerve activity (efferent-GVNA), while Lactobacillus paracasei ST11 suppressed efferent-GVNA, and thereby increased or decreased food intake. To determine the function of Lactobacillus brevis (SBC8803), its effect on food intake was examined by providing food containing heat-killed SBC8803 to mice. We observed that administration of SBC8803 elevated food intake. Because the afferent intestinal vagal nerve (IVN) is hypothesized to be involved in efferent-GVNA changes, we examined the effect of intraduodenal administration of heat-killed SBC8803 on efferent-GVNA and afferent-IVN activity (IVNA) in rats. In this study, we found that intraduodenal administration of heat-killed SBC8803 increased both efferent-GVNA and afferent-IVNA in rats. Moreover, IV administration of the serotonin 3 receptor antagonist granisetron eliminated the effects of SBC8803 on efferent-GVNA and afferent-IVNA. These findings suggest that heat-killed SBC8803 enhances appetite by elevating digestion and absorption abilities via changes in autonomic neurotransmission that might be mediated by the serotonin 3 receptor.

Disruption of behavioral circadian rhythms induced by psychophysiological stress affects plasma free amino acid profiles without affecting peripheral clock gene expression in mice.

Disordered circadian rhythms are associated with various psychiatric conditions and metabolic diseases. We recently established a mouse model of a psychophysiological stress-induced chronic sleep disorder (CSD) characterized by reduced amplitude of circadian wheel-running activity and sleep-wake cycles, sleep fragmentation and hyperphagia. Here, we evaluate day-night fluctuations in plasma concentrations of free amino acids (FAA), appetite hormones and prolactin as well as the hepatic expression of circadian clock-related genes in mice with CSD (CSD mice). Nocturnal increases in wheel-running activity and circadian rhythms of plasma prolactin concentrations were significantly disrupted in CSD mice. Hyperphagia with a decreased leptin/ghrelin ratio was found in CSD mice. Day-night fluctuations in plasma FAA contents were severely disrupted without affecting total FAA levels in CSD mice. Nocturnal increases in branched-chain amino acids such as Ile, Leu, and Val were further augmented in CSD mice, while daytime increases in Gly, Ala, Ser, Thr, Lys, Arg, His, Tyr, Met, Cys, Glu, and Asn were significantly attenuated. Importantly, the circadian expression of hepatic clock genes was completely unaffected in CSD mice. These findings suggest that circadian clock gene expression does not always reflect disordered behavior and sleep rhythms and that plasma FFA profiles could serve as a potential biomarker of circadian rhythm disorders.

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.

Engineering neoglycoproteins with multiple O-glycans using repetitive pentapeptide glycosylation units

Controlled protein remodeling with O-linked glycans has been limited by our incomplete understanding of the process of glycosylation. Here we describe a secretable fibroblast growth factor (FGF) with multiple mucin-type O-glycans produced by introducing a minimum pentapeptide glycosylation unit in a decarepeat format at its N- or C-terminus. Expressed in Chinese hamster ovary cells, chemical and biochemical analyses of the resultant proteins (Nm10-FGF and Cm10-FGF, respectively) demonstrated that all O-glycosylation units were glycosylated and the dominant structure was sialylated Gal[β1–3]GalNAc. This indicates that minimum O-glycosylation unit in multirepeat format serves as a remarkably efficient acceptor in CHO cells. The Nm10-FGF and Cm10-FGF proteins maintained the mitogenic activity to vascular endothelial cells. In addition, intact Cm10-FGF and its desialylated form interacted with several lectins in the same way as mucin-type glycoproteins. The intact Cm10-FGF with multiple sialylated O-glycans exhibited a longer lifetime in circulating blood, whereas the Cm10-FGF with desialylated O-glycans exhibited a shorter lifetime than the deglycosylated form of Cm10-FGF. Our approach would thus appear to be highly effective for engineering neoglycoproteins, the characteristics of which are determined by their multiple mucin-type O-glycans.