Gender differences in eating behavior and masticatory performance: An analysis of the Three-Factor-Eating Questionnaire and its association with body mass index in healthy subjects.

OBJECTIVES: The Three-Factor-Eating Questionnaire (TFEQ) is an established instrument to assess eating behavior in terms of dietary restraint, disinhibition and hunger. METHODS: The aims of this study were to examine (1) the correlation between eating behavior and body mass index (BMI), (2) the correlation between eating behavior and masticatory performance in terms of bite size and eating speed, and (3) the effects of gender on these correlations in 56 healthy subjects (33 males [21.9 ± 2.8 years old] and 23 females [21.7 ± 2.2 years old]). RESULTS: We found a significant correlation between restraint and BMI only in females and between hunger and BMI only in males. However, disinhibition and BMI were significantly correlated in both males and females. We also found a significant correlation between bite size and hunger only in males and between eating speed and disinhibition in both males and females. CONCLUSIONS: These findings underline the importance of gender-specific counselling and behavioral treatment of obesity.

Role of ß-adrenergic signaling in masseter muscle.

In skeletal muscle, the major isoform of ß-adrenergic receptor (ß-AR) is ß2-AR and the minor isoform is ß1-AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the ß-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic ß1- or ß2-AR activation with a specific ß1-AR agonist, dobutamine (DOB), or a specific ß2-AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic ß1-AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic ß2-AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic ß1-AR stimulation did not induce muscle hypertrophy, but did induce fibrosis and apoptosis concomitantly with increased levels of p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), calmodulin kinase II (Thr-286) and mammalian target of rapamycin (mTOR) (Ser-2481) phosphorylation. On the other hand, chronic ß2-AR stimulation in masseter muscle induced muscle hypertrophy without histological abnormalities, as in the case of cardiac muscle, concomitantly with phosphorylation of Akt (Ser-473) and mTOR (Ser-2448) and increased expression of microtubule-associated protein light chain 3-II, an autophagosome marker. These results suggest that the ß1-AR pathway is deleterious and the ß2-AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness.

Effects of chronic Porphyromonas gingivalis lipopolysaccharide infusion on skeletal muscles in mice.

Periodontitis, which is caused by various oral organisms, predominantly affects adults, and is one of the main causes of tooth loss, as well as leading to progression of numerous systemic diseases. However, its relationship to sarcopenia (aging-associated degenerative loss of skeletal muscle mass and function) remains unclear. The aim of this study was to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on skeletal muscle in mice, and to establish the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) for 4 weeks. This treatment significantly decreased the weight of fast-twitch skeletal muscles (masseter and tibialis anterior muscles), but not that of slow-twitch skeletal muscle (soleus muscle). The area of fibrosis was significantly increased in masseter muscle, but remained unchanged in the other two muscles. The number of apoptotic myocytes was significantly increased (approximately eightfold) in masseter muscle. These data suggest that persistent subclinical exposure to PG-LPS might reduce the size of fast-twitch skeletal muscle, but not slow-twitch skeletal muscle. Masseter muscle appears to be especially susceptible to the adverse effects of PG-LPS, because muscle remodeling (muscle fibrosis and myocyte apoptosis) was induced solely in masseter muscle. Thus, periodontitis might be one of the major causes of oral sarcopenia.

Relationship between bite size per mouthful and dental arch size in healthy subjects.

Although multiple factors influence food bite size, the relationship between food bite size per mouthful and mandible or tongue size remains poorly understood. Here, we examined the correlations between food bite size and the lower dental arch size (an indicator of tongue size) in human subjects with good oral and general health, using fish sausage and bread as test foods. Notably, bite size of both foods was significantly positively correlated with the lower dental arch size, whereas masticatory performance (measured in terms of glucose extraction from a gummy jelly) showed no dependence on bite size. Further, bite size was significantly positively correlated with the body mass index. Our findings suggest that larger bite size is associated with larger tongue size, which might be a contributory factor to obesity.

Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in ß-arrestin 2-Akt signaling and dopaminergic behaviors.

The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-ß-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and ß-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with ß-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a ß-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-ß-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in ß-arrestin-dependent, G protein-independent signaling.

Cardiac overexpression of Epac1 in transgenic mice rescues lipopolysaccharide-induced cardiac dysfunction and inhibits Jak-STAT pathway.

Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the ß-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.

Role of phosphodiesterase 4 expression in the Epac1 signaling-dependent skeletal muscle hypertrophic action of clenbuterol.

Clenbuterol (CB), a selective ß2-adrenergic receptor (AR) agonist, induces muscle hypertrophy and counteracts muscle atrophy. However, it is paradoxically less effective in slow-twitch muscle than in fast-twitch muscle, though slow-twitch muscle has a greater density of ß-AR We recently demonstrated that Epac1 (exchange protein activated by cyclic AMP [cAMP]1) plays a pivotal role in ß2-AR-mediated masseter muscle hypertrophy through activation of the Akt and calmodulin kinase II (CaMKII)/histone deacetylase 4 (HDAC4) signaling pathways. Here, we investigated the role of Epac1 in the differential hypertrophic effect of CB using tibialis anterior muscle (TA; typical fast-twitch muscle) and soleus muscle (SOL; typical slow-twitch muscle) of wild-type (WT) and Epac1-null mice (Epac1KO). The TA mass to tibial length (TL) ratio was similar in WT and Epac1KO at baseline and was significantly increased after CB infusion in WT, but not in Epac1KO The SOL mass to TL ratio was also similar in WT and Epac1KO at baseline, but CB-induced hypertrophy was suppressed in both mice. In order to understand the mechanism involved, we measured the protein expression levels of ß-AR signaling-related molecules, and found that phosphodiesterase 4 (PDE4) expression was 12-fold greater in SOL than in TA These results are consistent with the idea that increased PDE4-mediated cAMP hydrolysis occurs in SOL compared to TA, resulting in a reduced cAMP concentration that is insufficient to activate Epac1 and its downstream Akt and CaMKII/HDAC4 hypertrophic signaling pathways in SOL of WT This scenario can account for the differential effects of CB on fast- and slow-twitch muscles.

Effects of food diameter on bite size per mouthful and chewing behavior.

Obesity is well known to be associated with a wide variety of illnesses, and is an increasing problem not only in developed countries but also in developing countries. It is well known that large bite size contributes to excess energy intake and obesity, whereas an increased number of chews before swallowing the food bolus is associated with suppression of obesity. However, the effect of food diameter on bite size per mouthful and on chewing behavior remains poorly understood. Here, we examined the effects of food diameter on bite size and chewing behavior using a masticatory counter during the mastication of stick-type biscuits having the same length (10 cm) and ingredients, but with four different diameters (3.0, 3.5, 4.0, and 8.0 mm). Bite length and bite weight per mouthful were similar among the 3.0, 3.5, and 4.0 mm groups. However, bite length in the 8.0 mm group was significantly smaller, whereas bite weight was significantly greater than in the 3.0/3.5 mm groups. Further, the number of chews gradually increased, whereas the number of chews per bite weight gradually decreased, with an increase of biscuit diameter. These results indicate that a smaller biscuit diameter is associated with a smaller bite weight per mouthful and a greater number of chews per bite weight. This is the first report to quantity the effect of food diameter on bite weight per mouthful and on chewing behavior; these results should be helpful in the design of effective, safe, and low-cost behavioral modification therapy to combat obesity.

Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

BACKGROUND: Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle ß2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. METHODOLOGY: We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. RESULTS AND CONCLUSION: Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy.

Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by ß2-adrenoceptor stimulation.

The predominant isoform of ß-adrenoceptor (ß-AR) in skeletal muscle is ß2-AR and that in the cardiac muscle is ß1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic ß2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in ß2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.

Effects of protein kinase a on the phosphorylation status and transverse stiffness of cardiac myofibrils.

Stimulation of ß-adrenergic receptors in cardiac myocytes activates cyclic AMP-dependent protein kinase A (PKA). PKA-mediated phosphorylation of myofibrils decreases their longitudinal stiffness, but its effect on transverse stiffness is not fully understood. We thus examined the effects of PKA treatment on the transverse stiffness of cardiac myofibrils by atomic force microscopy and determined the phosphorylation levels of myofibril components by SDS-PAGE. Transverse stiffness was significantly decreased by PKA treatment concomitantly with increased phosphorylation of troponin I, myosin-binding protein C, and titin (also called connectin). Subsequent treatment with protein phosphatase 1 abrogated these PKA-mediated effects.

Effects of chronic Akt/mTOR inhibition by rapamycin on mechanical overload-induced hypertrophy and myosin heavy chain transition in masseter muscle.

To examine the effects of the Akt/mammalian target of rapamycin (mTOR) pathway on masseter muscle hypertrophy and myosin heavy chain (MHC) transition in response to mechanical overload, we analyzed the effects of bite-opening (BO) on the hypertrophy and MHC composition of masseter muscle of BO-rats treated or not treated with rapamycin (RAPA), a selective mTOR inhibitor. The masseter muscle weight in BO-rats was significantly greater than that in controls, and this increase was attenuated by RAPA treatment. Expression of slow-twitch MHC isoforms was significantly increased in BO-rats with/without RAPA treatment, compared with controls, but the magnitude of the increase was much smaller in RAPA-treated BO-rats. Phosphorylation of p44/42 MAPK (ERK1/2), which preserves fast-twitch MHC isoforms in skeletal muscle, was significantly decreased in BO-rats, but the decrease was abrogated by RAPA treatment. Calcineurin signaling is known to be important for masseter muscle hypertrophy and fast-to-slow MHC isoform transition, but expression of known calcineurin activity modulators was unaffected by RAPA treatment. Taken together, these results indicate that the Akt/mTOR pathway is involved in both development of masseter muscle hypertrophy and fast-to-slow MHC isoform transition in response to mechanical overload with inhibition of the ERK1/2 pathway and operates independently of the calcineurin pathway.

Role of masseter muscle ß2-adrenergic signaling in regulation of muscle activity, myosin heavy chain transition, and hypertrophy.

Chronic administration of clenbuterol (CB), a lipophilic ß2-adrenoceptor (ß2-AR) agonist, induces skeletal muscle hypertrophy and slow-to-fast fiber-type transitions in mammalian species, but the mechanism and pathophysiological roles of these changes have not been explored. Here, we examined the effects of CB not only on masseter muscle mass, fiber diameter, and myosin heavy chain (MHC) composition, but also on daily muscle activity, a factor influencing muscle phenotype, by means of electromyogram analysis in rats. MHC transition towards faster isoforms was induced by 2-week CB treatment. In addition, daily duty time was increased at 1 day, 1 week, and 2 weeks after the start of CB treatment and its increase was greater at high activity level (6-fold) than at low activity level (2-fold). In order to examine whether these effects of CB were mediated through muscle or CNS ß2-AR stimulation, we compared these effects of CB with those of salbutamol (SB), a hydrophilic ß2-AR agonist. SB treatment induced masseter hypertrophy and MHC transition, like CB, but did not increase daily activity. These results suggest that CB-mediated slow-to-fast MHC transition with hypertrophy was induced through direct muscle ß2-AR stimulation, but the increase of daily duty time was mediated through the CNS.

Pharmacological stimulation of type 5 adenylyl cyclase stabilizes heart rate under both microgravity and hypergravity induced by parabolic flight.

We previously demonstrated that type 5 adenylyl cyclase (AC5) functions in autonomic regulation in the heart. Based on that work, we hypothesized that pharmacological modulation of AC5 activity could regulate the autonomic control of the heart rate under micro- and hypergravity. To test this hypothesis, we selected the approach of activating AC5 activity in mice with a selective AC5 activator (NKH477) or inhibitor (vidarabine) and examining heart rate variability during parabolic flight. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater under micro- and hypergravity in the vidarabine group, while there were no significant changes in the NKH477 group, suggesting that autonomic regulation was unstable in the vidarabine group. The ratio of low frequency and high frequency (HF) in heart rate variability analysis, a marker of sympathetic activity, became significantly decreased under micro- and hypergravity in the NKH477 group, while there was no such decrease in the vidarabine group. Normalized HF, a marker of parasympathetic activity, became significantly greater under micro- and hypergravity in the NKH477 group. In contrast, there was no such increase in the vidarabine group. This study is the first to indicate that pharmacological modulation of AC5 activity under micro- and hypergravity could be useful to regulate the autonomic control of the heart rate.