7he tcrture of six varieties of cooked rice with different atnylose contents was meusured with a Texturometer. Masticatory behavior of the rice was ernluuted using electromyography (EMG) of masticatory muscles. Differences in tnasticution patterns among the seven subjects examined were greater than between rice varieties. Masticatory behavior was more related to the adhesiveness and stickiness cf rice as measured using a Texturometer than to the hardness. The number of chews and masticatory time, total duration of mastication and total muscle activities were highly correlated with each other, and were greater in cooked rice with a high arnylose content, which showed low adhesiveness and stickiness values using a Texturometer. Cooked rice with a high amylose content was masticated with high masseter muscle activities. The ratio of jaw-opening muscle activity to the preceding jaw-closing muscle activity was low in high-amylose varieties, which corresponds to the ratio of stickiness to hardness using a Texturometer. Between-.sample differences in mastication parameters were more apparent in the 'Correspondent: Dr. KCION Kohyama, Food

Key pointsr Epac (exchange protein directly activated by cyclic AMP (cAMP)), a PKA-independent cAMP sensor, plays important roles in multiple cellular processes, but its role in the pathogenesis of skeletal muscle hypertrophy and myosin heavy chain (MHC) transition is poorly understood.r Chronic stimulation of β 2 -adrenoceptor (β 2 -AR) with clenbuterol (CB), a selective β 2 -AR agonist, induced masseter muscle hypertrophy in wild-type (WT) mice, but not in Epac1-null mice (Epac1KO), even if slow-to-fast MHC isoform transition was similarly induced by CB treatment in both WT and Epac1KO.r Disruption of Epac1 inhibited development of masseter muscle hypertrophy concomitantly with decreased phosphorylation of Akt and its downstream molecules 70 kDa ribosomal S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1, and also, in parallel, glycogen synthase kinase-3β.r Disruption of Epac1 decreased histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy.r We conclude that Epac1 induces β 2 -AR-mediated masseter muscle hypertrophy without influencing slow-to-fast MHC isoform transition, probably via activation of Akt and its downstream molecules and increase of CaMKII-mediated HDAC4 phosphorylation.Abstract The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β 2 -AR and that in the cardiac muscle is β 1 -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 β 2 -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 β 2 -AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.

In skeletal muscle, the major isoform of β-adrenergic receptor (β-AR) is β 2 -AR and the minor isoform is β 1 -AR, which is opposite to the situation in cardiac muscle. Despite extensive studies in cardiac muscle, the physiological roles of the β-AR subtypes in skeletal muscle are not fully understood. Therefore, in this work, we compared the effects of chronic β 1 - or β 2 -AR activation with a specific β 1 -AR agonist, dobutamine (DOB), or a specific β 2 -AR agonist, clenbuterol (CB), on masseter and cardiac muscles in mice. In cardiac muscle, chronic β 1 -AR stimulation induced cardiac hypertrophy, fibrosis and myocyte apoptosis, whereas chronic β 2 -AR stimulation induced cardiac hypertrophy without histological abnormalities. In masseter muscle, however, chronic β 1 -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 β 2 -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 β 1 -AR pathway is deleterious and the β 2 -AR is protective in masseter muscle. These data should be helpful in developing pharmacological approaches for the treatment of skeletal muscle wasting and weakness.

BackgroundGlucocorticoid 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 β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition.MethodologyWe 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 ConclusionMasseter 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.

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Abstract. Chronic administration of clenbuterol (CB), a lipophilic b 2 -adrenoceptor (b 2 -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 b 2 -AR stimulation, we compared these effects of CB with those of salbutamol (SB), a hydrophilic b 2 -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 b 2 -AR stimulation, but the increase of daily duty time was mediated through the CNS.

Abstract.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.

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