Reiko Nakao

Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading.

ABSTRACT Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine608-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.

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.

Glucose Infusion Suppresses Surgery-induced Muscle Protein Breakdown by Inhibiting Ubiquitin-proteasome Pathway in Rats

Background:It appears to have been well established that after surgery, protein catabolism is accelerated and glucose infusion suppresses the catabolic reactions. However, in the early postoperative period, the effects of surgical stress and glucose infusion on muscle protein catabolism and the related mechanisms remain unclear. Methods:Rats undergoing laparotomy were infused with acetated Ringer’s solution (10 ml · kg–1 · h–1) without glucose (control) or containing 1% or 5% glucose. The infusion was continued for a further 4 h after the surgical treatment. The catabolic index, excretion of urinary nitrogen and 3-methylhistidine, and release of tyrosine and 3-methylhistidine from isolated muscle were determined. Furthermore, muscular mRNA expression of proteolytic-related genes (atrogin-1/MAFbx, muscle ring finger-1, &mgr;- and m-calpain, and cathepsin L and H) and phosphorylation of components of insulin signaling (forkhead box O3 and protein kinase B) were evaluated. Results:Surgery increased the catabolic index, and this increase was suppressed by glucose infusion (both 1% and 5%). In the control group, mRNA expression of atrogin-1/MAFbx and muscle ring finger-1 was increased, and they were suppressed in the two glucose groups. Furthermore, insulin signaling (phosphorylation of protein kinase B and forkhead box O3) in muscles was stimulated by glucose infusion. Conclusion:The present study indicates that glucose infusion, even at a relatively low rate, suppresses muscle protein breakdown in the early postoperative period. The mechanism of this effect is related to the suppression of the ubiquitin-proteasome pathway, accompanied by activation of insulin signaling.

Quercetin Prevents Unloading-Derived Disused Muscle Atrophy by Attenuating the Induction of Ubiquitin Ligases in Tail-Suspension Mice

The effects of quercetin (1) were investigated on disused muscle atrophy using mice that underwent tail suspension. Periodic injection of 1 into the gastrocnemius muscle suppressed muscle weight loss and ubiquitin ligase expression. Compound 1 reduced the enhancement of lipid peroxidation in the muscle. Injection of N-acetyl-l-cysteine, but not flavone (2), also prevented muscle weight loss and enhancement of lipid peroxidation. These findings demonstrate that 1 can prevent disused muscle atrophy by attenuating the expression of ubiquitin ligases and that such prevention originates from its antioxidant activity.

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

RANTES SECRETED FROM MACROPHAGES DISTURBS SKELETAL MUSCLE REGENERATION AFTER CARDIOTOXIN INJECTION IN Cbl-b-DEFICIENT MICE

Deficiency of the Cbl‐b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl‐b‐deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin‐injected tibialis anterior muscle of Cbl‐b‐deficient mice. Ablation of the Cbl‐b gene expression delayed regeneration of cardiotoxin‐induced skeletal muscle damage compared with wild‐type mice. CD8‐positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl‐b‐deficient mice, but there was dispersal of the same cells over that time‐frame in wild‐type mice. Infiltrating macrophages in Cbl‐b‐deficient mice showed strong expression of RANTES (regulated‐on‐activation, normal T cell expressed and secreted), a chemokine for CD8‐positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8‐positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl‐b is an important regulatory factor for cytotoxic T‐cell infiltration via RANTES production in macrophages. Muscle Nerve, 2011

Cathepsin C Propeptide Interacts with Intestinal Alkaline Phosphatase and Heat Shock Cognate Protein 70 in Human Caco-2 Cells

The oligomeric structure and the residual propeptide are distinct characteristics of cathepsin C from other members in the papain superfamily. In this study, we examined the physiological role of the cathepsin C propeptide. The stable overexpression of cathepsin C propeptide significantly decreased the activities of intestinal alkaline phosphatase (IAP) and sucrase in human Caco-2 intestinal epithelial cells, whereas it did not change the proliferation and cathepsin C activity. The overexpression of cathepsin C propeptide significantly decreased the amounts of IAP protein in differentiated Caco-2 cells, compared with the transfection of mock vector, whereas the amounts of IAP transcripts were not changed. Pulse-chase analysis confirmed that the reduction in IAP activity was due to an increase in IAP degradation, but not a decrease in IAP expression. For the mechanism of the enhanced IAP degradation, we identified proteins interacting with cathepsin C propeptide in Caco-2 cells by immunoprecipitation and mass spectrometry. Cathepsin C propeptide interacted with proteins with a molecular mass of approximately 70 kDa, including IAP and heat shock cognate protein 70. Our present results suggest that the propeptide of cathepsin C may stimulate the sorting to the lysosome, at least in part, contributing to the degradation of IAP in Caco-2 cells.