Keiichi Koshinaka

Effect of exercise intensity and AICAR on isoform-specific expressions of murine skeletal muscle PGC-1α mRNA: a role of β2-adrenergic receptor activation

There are three isoforms of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA, which promotes mitochondrial biogenesis in skeletal muscles. Compared with PGC-1α-a mRNA, PGC-1α-b or PGC-1α-c mRNA is transcribed by a different exon 1 of the PGC-1α gene. In this study, effects of exercise intensity and 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) on isoform-specific expressions of PGC-1α were investigated. All isoforms were increased in proportion to exercise intensity of treadmill running (10-30 m/min for 30 min). Preinjection of β₂-adrenergic receptor (AR) antagonist (ICI 118551) inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs, but not the increase in PGC-1α-a mRNA, in response to high-intensity exercise. Although high-intensity exercise activated α2-AMP-activated protein kinase (α2-AMPK) in skeletal muscles, inactivation of α2-AMPK activity did not affect high-intensity exercise-induced mRNA expression of all PGC-1α isoforms, suggesting that activation of α2-AMPK is not mandatory for an increase in PGC-1α mRNA by high-intensity exercise. A single injection in mice of AICAR, an AMPK activator, increased mRNAs of all PGC-1α isoforms. AICAR increased blood catecholamine concentrations, and preinjection of β₂-AR antagonist inhibited the increase in PGC-1α-b and PGC-1α-c mRNAs but not the increase in PGC-1α-a mRNA. Direct exposure of epitrochlearis muscle to AICAR increased PGC-1α-a but not the -b isoform. These data indicate that exercise-induced PGC-1α expression was dependent on the intensity of exercise. Exercise or AICAR injection increased PGC-1α-b and PGC-1α-c mRNAs via β₂-AR activation, whereas high-intensity exercise increased PGC-1α-a expression by a multiple mechanism in which α2-AMPK is one of the signaling pathways.

Muscle contractile activity regulates Sirt3 protein expression in rat skeletal muscles

Sirt3, a member of the sirtuin family, is known to control cellular mitochondrial function. Furthermore, because sirtuins require NAD for their deacetylase activity, nicotinamide phosphoribosyltransferase (Nampt), which is a rate-limiting enzyme in the intracellular NAD biosynthetic pathway, influences their activity. We examined the effects of exercise training and normal postural contractile activity on Sirt3 and Nampt protein expression in rat skeletal muscles. Male rats were trained by treadmill running at 20 m/min, 60 min/day, 7 days/wk for 4 wk. This treadmill training program increased the Sirt3 protein expression in the soleus and plantaris muscles by 49% and 41%, respectively (P < 0.05). Moreover, a 4-wk voluntary wheel-running program also induced 66% and 95% increases in Sirt3 protein in the plantaris and triceps muscles of rats, respectively (P < 0.05). Treadmill-running and voluntary running training induced no significant changes in Nampt protein expression in skeletal muscles. In resting rats, the soleus muscle, which is recruited during normal postural activity, possessed the greatest expression levels of the Sirt3 and Nampt proteins, followed by the plantaris and triceps muscles. Furthermore, the Sirt3, but not Nampt, protein level was reduced in the soleus muscles from immobilized hindlimbs compared with that shown in the contralateral control muscle. These results demonstrated that 1) Sirt3 protein expression is upregulated by exercise training in skeletal muscles and 2) local postural contractile activity plays an important role in maintaining a high level of Sirt3 protein expression in postural muscle.

Characterization of obestatin in rat and human stomach and plasma, and its lack of acute effect on feeding behavior in rodents

Obestatin is a 23-amino acid peptide, initially isolated from rat stomach as an endogenous ligand for the orphan G-protein-coupled receptor. Obestatin is derived from proteolytic cleavage of a 117-amino acid precursor, preproghrelin. Ghrelin increases food intake, body weight, and gastric emptying, whereas obestatin has the opposite effects. In this study, we characterized obestatin in both rat and human stomach, and investigated the peptides effect on feeding behavior. Using reversed-phase high-performance liquid chromatography coupled with RIAs specific for rat and human obestatin, we detected a very small amount of obestatin, compared with ghrelin, in the gastric fundi. The ratios of obestatin to ghrelin are 0.0039 and 1.94% respectively in the rat and human gastric fundi. In humans, plasma obestatin accounted for 5.21% of the ghrelin concentration, whereas it was undetectable in rat plasma. Plasma ghrelin concentration decreased after a meal in normal subjects, whereas obestatin concentration did not change. When administered centrally or peripherally, obestatin did not suppress food intake in either free-feeding or fasted rodents. Administration of obestatin did not antagonize ghrelin-induced feeding. These findings indicate that obestatin is present at very low levels compared with ghrelin in both rat and human, and has no acute effect on feeding behavior.

Neuroendocrine regulatory peptide-2 regulates feeding behavior via the orexin system in the hypothalamus

Neuroendocrine regulatory peptide (NERP)-1 and NERP-2 are derived from distinct regions of VGF, a neurosecretory protein. Vgf(-/-) mice exhibit dwarfism and hypermetabolic rates, suggesting that VGF or VGF-derived peptides play important roles in energy metabolism. Here, we examined the role of NERPs in the central regulation of feeding and energy homeostasis. We attempted to identify NERPs expressing neurons in rats by immunohistochemistry. We studied the effects of intracerebroventricular (icv) administration of NERP-2 on feeding, body temperature, oxygen consumption, and locomotor activity in rats and mice. Intracerebroventricular administration of NERP-2, but not NERP-1 or a form of NERP-2 bearing a COOH-terminal glycine extension, increased food intake in rats. We investigated the downstream signal of NERP-2 on the basis of studies of NERP-2-induced feeding with neutralization of orexins, neuropeptide Y, or agouti-related protein. NERP-2 expression localized to the lateral hypothalamus (LH) and the dorsomedial perifornical hypothalamus in rats, colocalizing with orexins that activate feeding behavior and arousal. NERP-2 administration induced Fos protein, a marker of neuronal activation, in the orexin-immunoreactive neurons. Vgf mRNA levels were upregulated in the rat LH upon food deprivation. Intracerebroventricular administration of NERP-2 also increased body temperature, oxygen consumption, and locomotor activity in rats. Treatment with anti-NERP-2 IgG decreased food intake. NERP-2-induced bioactivities could be abrogated by administration of anti-orexins IgG or orexin receptor antagonists. NERP-2 did not induce food intake or locomotor activity in orexin-deficient mice. Our findings indicate that hypothalamic NERP-2 plays a role in the control of food intake and energy homeostasis via the orexin pathway. Thus, VGF serves as a precursor of multiple bioactive peptides exerting a diverse set of neuroendocrine functions.

Role of local muscle contractile activity in the exercise-induced increase in NR4A receptor mRNA expression

Exercise upregulates the expression of NR4A receptors, which are involved in regulation of glucose and fatty acid utilization genes in skeletal muscle. The aims of our study were 1) to determine the role of local contractile activity on NR4A mRNA expression in skeletal muscle during exercise; and 2) to elucidate the mechanisms underlying the induction of NR4A mRNA expression in response to muscle contractile activity. Rats were subjected to an acute 3-h low-intensity swimming or a 3-h low-intensity treadmill running as a model of endurance exercise. Low-intensity swimming increased NR4A1 and NR4A3 mRNA in triceps but not in soleus muscle. Conversely, low-intensity treadmill running increased NR4A1 and NR4A3 mRNA in soleus but not in triceps muscle. NR4A mRNA increased concomitantly with reduced postexercise muscle glycogen, suggesting that gene expression of NR4A receptors occurs in muscles recruited during exercise. Furthermore, in resting rats, an acute 1-h local electrical stimulation of a motor nerve to the tibialis anterior muscle caused increases in NR4A1 and NR4A3 mRNA relative to the contralateral control muscle of the same animals. On the other hand, after 6 h of hindlimb immobilization, NR4A1 and NR4A3 mRNA were reduced in immobilized soleus muscle relative to contralateral control muscle. In addition, both NR4A1 and NR4A3 mRNA in epitrochlearis muscle were increased after 6-h incubation with 0.5 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, which activates AMP-activated protein kinase. These results suggest that 1) local muscle contractile activity is required for increased expressions of NR4A1 and NR4A3 mRNA during exercise; and 2) muscle contractile activity-induced increases in NR4A1 and NR4A3 mRNA may be mediated by AMPK activation, at least in part.

Effect of acute high-intensity intermittent swimming on post-exercise insulin responsiveness in epitrochlearis muscle of fed rats

Maximally insulin-stimulated glucose uptake in skeletal muscle, ie, insulin responsiveness, is reduced in fed animals as compared with fasted animals; but acute prior endurance exercise improves insulin responsiveness in the muscles of fed rats. The effect of acute prior sprint interval exercise on insulin responsiveness in the muscles of fed animals has not been clarified, and we therefore compared the effect of short high-intensity swimming as a model of sprint interval exercise on insulin responsiveness in the muscles of fed rats with the effect of prolonged low-intensity swimming as a model of endurance exercise. The fed rats were subjected to an acute bout of high-intensity intermittent swimming (HIS) or low-intensity continuous swimming (LIS). The HIS rats swam for eight 20-second periods with a weight equal to 18% of their body weight. The LIS rats swam with no load for 3 hours. HIS increased (P < .05) the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) Thr(172) and that of its downstream target acetyl-CoA carboxylase (ACC) Ser(79) 12.6- and 3.1-fold, respectively, whereas LIS increased them 3.8- and 1.9-fold, respectively, immediately after exercise compared with rested muscle. HIS and LIS increased the insulin responsiveness of 2-deoxyglucose uptake measured 4 hours after exercise by 39% and 41%, respectively, compared with rested muscles. These results show that very short (160 seconds) HIS exercise with greater AMPK activation increases the responsiveness of glucose uptake to insulin in the muscles of fed rats to a similar level observed after prolonged (3 hours) LIS exercise with lower AMPK activation. Therefore, it is suggested that an acute bout of sprint interval exercise that activates AMPK to a sufficiently high level can increase post-exercise insulin responsiveness on muscle glucose uptake irrespective of very short exercise duration.

Therapeutic potential of ghrelin treatment for unloading-induced muscle atrophy in mice

Ghrelin is a growth hormone (GH) secretagogue secreted mainly from the stomach that functions in controlling muscle volume and energy homeostasis. We here studied the effects of ghrelin on unloading-induced muscle atrophy using a mouse model of hindlimb suspension (HS). Ghrelin administration during 2-week HS alleviated reductions of muscle mass in the fast-twitch fiber-rich plantaris muscle and the slow-twitch fiber-rich soleus muscle of the hindlimb. Ghrelin administration during a 5-day recovery period following 2-week HS enhanced food intake and facilitated recovery from atrophy in both muscles. Ghrelin administration normalized hypercorticosteronemia in these studies. Ghrelins anti-muscle atrophy effect was found even under pair-feeding condition, but not in mice given des-acyl ghrelin. Insulin-like growth factor (IGF)-1 mRNA expression was significantly reduced in the atrophied plantaris muscle compared with control muscles. A single ghrelin administration to HS mice acutely increased plasma GH and also amplified phosphorylation of signal transducer and activator of transcription (STAT) 5 and increased IGF-1 mRNA expression in the plantaris muscle, but not in the soleus muscle. This study demonstrated that ghrelin stimulated the GH-STAT5-IGF-1 axis in the locally atrophied plantaris muscle, and its administration alleviated muscle atrophy and facilitated recovery from muscle atrophy. Ghrelins effects represent a novel therapeutic paradigm for the treatment of unloading-induced muscle atrophy induced by factors such as bed rest, injury, and joint immobilization.

Elevation of muscle temperature stimulates muscle glucose uptake in vivo and in vitro.

The purpose of this study was to examine whether elevation of muscle temperature per se might be a stimulatory factor to increase muscle glucose uptake. Heat stimulation to rat hindlimbs increased glucose uptake measured in vivo in the extensor digitorum longus (EDL) and soleus muscles with a significant increase in muscle temperature. This thermal effect was observed again when glucose uptake was measured in vitro in both isolated muscles immediately after the heat stimulation in vivo. When heat stimulation was imposed on isolated EDL muscles, glucose uptake was facilitated in proportion to the increase in muscle temperature. The heat stimulation led to a significant amplification in the phosphorylation of AMP-activated protein kinase (AMPK) and Akt, and treatment with compound C, wortmannin, or LY294002 partially blocked the thermal effect on muscle glucose uptake. We provide evidence that elevation of muscle temperature per se can directly stimulate muscle glucose uptake and that this thermal effect is compound C-, wortmannin-, and LY294002-inhibitable.

Increased postexercise insulin sensitivity is accompanied by increased AS160 phosphorylation in slow-twitch soleus muscle.

A single bout of exercise can enhance insulin‐stimulated glucose uptake in both fast‐twitch (type II) and slow‐twitch (type I) skeletal muscle for several hours postexercise. Akt substrate of 160 kDa (AS160) is most distal insulin signaling proteins that have been proposed to contribute to the postexercise enhancement of insulin action in fast‐twitch muscle. In this study, we examined whether the postexercise increase in insulin action of glucose uptake in slow‐twitch muscle is accompanied by increased phosphorylation of AS160 and its paralog TBC1D1. Male Wistar rats (~1‐month‐old) were exercised on a treadmill for 180 min (9 m/min). Insulin (50 μU/mL)‐stimulated glucose uptake was increased at 2 h after cessation of exercise in soleus muscle composed of predominantly slow‐twitch fibers. This postexercise increase in insulin action of glucose uptake was accompanied by increased phosphorylation of AS160 (detected by phospho‐Thr642 and phospho‐Ser588 antibody). On the other hand, prior exercise did not increase phosphorylation of TBC1D1 (detected by phospho‐Thr590) at 2 h postexercise. These results suggest the possibility that an enhancement in AS160 phosphorylation but not TBC1D1 phosphorylation is involved with increased postexercise insulin action of glucose uptake in slow‐twitch muscle.

The effects of β-adrenergic stimulation and exercise on NR4A3 protein expression in rat skeletal muscle

Abstractβ-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.