Yasuhiko Okimura

Chemerin regulates β-cell function in mice

Although various function of chemerin have been suggested, its physiological role remains to be elucidated. Here we show that chemerin-deficient mice are glucose intolerant irrespective of exhibiting reduced macrophage accumulation in adipose tissue. The glucose intolerance was mainly due to increased hepatic glucose production and impaired insulin secretion. Chemerin and its receptor ChemR23 were expressed in β-cell. Studies using isolated islets and perfused pancreas revealed impaired glucose-dependent insulin secretion (GSIS) in chemerin-deficient mice. Conversely, chemerin transgenic mice revealed enhanced GSIS and improved glucose tolerance. Expression of MafA, a pivotal transcriptional factor for β-cell function, was downregulated in chemerin-deficient islets and a chemerin-ablated β-cell line and rescue of MafA expression restored GSIS, indicating that chemerin regulates β-cell function via maintaining MafA expression. These results indicate that chemerin regulates β-cell function and plays an important role in glucose homeostasis in a tissue-dependent manner.

Reactive oxygen species play an essential role in Igf-i signaling and Igf-i-induced myocyte hypertrophy in C2c12 myocytes.

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.

Branched-chain amino acids reduce hindlimb suspension-induced muscle atrophy and protein levels of atrogin-1 and MuRF1 in rats.

Atrogin-1 and MuRF1, muscle-specific ubiquitin ligases, and autophagy play a role in protein degradation in muscles. We hypothesized that branched-chain amino acids (BCAAs) may decrease atrogin-1, MuRF1, and autophagy, and may have a protective effect on disuse muscle atrophy. To test this hypothesis, we selected hindlimb suspension (HS)-induced muscle atrophy as a model of disuse muscle atrophy because it is an established model to investigate the effects of decreased muscle activity. Sprague-Dawley male rats were assigned to 4 groups: control, HS (14 days), oral BCAA administration (600 mg/[kg day], 22.9% L-isoleucine, 45.8% L-leucine, and 27.6% L-valine), and HS and BCAA administration. After 14 days of the treatment, muscle weights and protein concentrations, cross-sectional area (CSA) of the muscle fibers, atrogin-1 and MuRF1 proteins, and microtubule-associated protein 1 light chain 3 II/I (ratio of LC3 II/I) were measured. Hindlimb suspension significantly reduced soleus muscle weight and CSA of the muscle fibers. Branched-chain amino acid administration partly but significantly reversed the HS-induced decrease in CSA. Hindlimb suspension increased atrogin-1 and MuRF1 proteins, which play a pivotal role in various muscle atrophies. Branched-chain amino acid attenuated the increase in atrogin-1 and MuRF1 in soleus muscles. Hindlimb suspension significantly increased the ratio of LC3 II/I, an indicator of autophagy, whereas BCAA did not attenuate the increase in the ratio of LC3 II/I. These results indicate the possibility that BCAA inhibits HS-induced muscle atrophy, at least in part, via the inhibition of the ubiquitin-proteasome pathway. Oral BCAA administration appears to have the potential to prevent disuse muscle atrophy.

Development and Validation of a New Questionnaire Assessing Quality of Life in Adults with Hypopituitarism: Adult Hypopituitarism Questionnaire (AHQ)

Objective To develop and validate the Adult Hypopituitarism Questionnaire (AHQ) as a disease-specific, self-administered questionnaire for evaluation of quality of life (QOL) in adult patients with hypopituitarism. Methods We developed and validated this new questionnaire, using a standardized procedure which included item development, pilot-testing and psychometric validation. Of the patients who participated in psychometric validation, those whose clinical conditions were judged to be stable were asked to answer the survey questionnaire twice, in order to assess test-retest reliability. Results Content validity of the initial questionnaire was evaluated via two pilot tests. After these tests, we made minor revisions and finalized the initial version of the questionnaire. The questionnaire was constructed with two domains, one psycho-social and the other physical. For psychometric assessment, analyses were performed on the responses of 192 adult patients with various types of hypopituitarism. The intraclass correlations of the respective domains were 0.91 and 0.95, and the Cronbach’s alpha coefficients were 0.96 and 0.95, indicating adequate test-retest reliability and internal consistency for each domain. For known-group validity, patients with hypopituitarism due to hypothalamic disorder showed significantly lower scores in 11 out of 13 sub-domains compared to those who had hypopituitarism due to pituitary disorder. Regarding construct validity, the domain structure was found to be almost the same as that initially hypothesized. Exploratory factor analysis (nu200a=u200a228) demonstrated that each domain consisted of six and seven sub-domains. Conclusion The AHQ showed good reliability and validity for evaluating QOL in adult patients with hypopituitarism.

Dexamethasone and BCAA Failed to Modulate Muscle Mass and mTOR Signaling in GH-Deficient Rats

Branched-chain amino acids (BCAAs) and IGF-I, the secretion of which is stimulated by growth hormone (GH), prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex)-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs). Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA) of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR) mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex’s action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.

Glucose-mediated inactivation of AMP-activated protein kinase reduces the levels of L-type amino acid transporter 1 mRNA in C2C12 cells

Branched chain amino acids (BCAAs) have protective effects against muscle atrophy. Although plasma BCAA concentrations are higher in patients with diabetes than in healthy subjects, diabetes is related to sarcopenia. We hypothesized that high glucose concentration reduces the quantity of BCAA transporters, and consequently, the effects of BCAAs are diminished despite their high levels. We examined whether glucose reduces the expression of L-type amino acid transporter 1 (LAT1), which transports neutral amino acids, including BCAA, in C2C12 myocytes. Glucose reduced LAT1 mRNA level by 80% in the C2C12 cells, compared with that in the glucose-free control cells. Regarding LAT1-related transporters, glucose also reduced the level of sodium-dependent neutral amino acid transporter 2 mRNA, but not that of 4F2 heavy chain. Although fructose reduced LAT1 mRNA levels, 2-deoxyglucose exhibited low effectiveness in reducing LAT1 mRNA level; galactose and mannitol had no effect. These results suggest a relationship between ATP produced during glycolysis and LAT1 mRNA levels. In fact, the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin reduced LAT1 mRNA levels in the absence of glucose, whereas the AMPK activator 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside increased LAT1 mRNA levels even in the presence of glucose. Consistent with these findings, glucose reduced the levels of phospho-AMPKα (Thr172) compared with that in the glucose-free control. These findings indicate that glucose inactivates AMPK, leading to a reduction in LAT1 mRNA levels in the C2C12 cells. This glucose-induced reduction in LAT1 expression may explain the unresponsiveness to BCAA in the patients with diabetes.

Growth hormone and Insulin-like growth factor-I (IGF-I) modulate the expression of L-type amino acid transporters in the muscles of spontaneous dwarf rats and L6 and C2C12 myocytes

OBJECTIVEnBranched-chain amino acids (BCAAs) have been reported to inhibit several types of muscle atrophy via the activation of the mechanistic target of rapamycin complex 1 (mTORC1). However, we previously found that BCAA did not activate mTORC1 in growth hormone (GH)-deficient spontaneous dwarf rats (SDRs), and that GH restored the stimulatory effect of BCAAs toward the mTORC1. The objective of this study was to determine whether GH or Insulin-like growth factor-I (IGF-I) stimulated the expression of L-type amino acid transporters (LATs) that delivered BCAAs, and whether LATs were involved in the mTORC1 activation.nnnDESIGNnAfter the continuous administration of GH, cross-sectional areas (CSAs) of muscle fibers and LAT mRNA levels in the skeletal muscles of SDRs were compared to those from the SDRs that received normal saline. The effect of GH and IGF-I on LAT mRNA levels were determined in L6 and C2C12 myocytes. The effects of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), a blocker for LATs, and LAT1 siRNA on mTORC1 activation and cell functions were examined in C2C12 cells.nnnRESULTSnGH increased LAT1 and LAT4 mRNA levels in accordance with the increase in CSAs of muscle fibers in SDRs. IGF-I, and not GH, increased LAT1 mRNA levels in cultured L6 myocytes. IGF-I also increased LAT1 mRNA level in another myocyte line, C2C12. Furthermore, IGF-I reduced LAT3 and LAT4 mRNA levels in both cell lines. GH reduced LAT3 and LAT4 mRNA levels in L6 cells. BCH decreased basal C2C12 cell proliferation and reduced IGF-I-induced phosphorylation of 4E-BP1 and S6K, both of which are mTORC1 targets, but LAT1 siRNA did not affect the phosphorylation. This suggests that BCH may exert its effect via other pathway than LAT1.nnnCONCLUSIONSnIGF-I increased LAT1 mRNA level in myocytes. However, the role of LAT1 in IGF-I-induced mTORC1 activation and cell functions remains unclear.

Branched Chain Amino Acids and Muscle Atrophy Protection

A variety of diseases and conditions result in muscle atrophy. Muscle atrophy causes a decrease of mobility, increased susceptibility to injuries and reduced Quality of Life (QOL). The various types of muscle atrophy are due to increased protein breakdown, decreased protein synthesis or both. Muscle mass is maintained by the balance between muscle protein synthesis and degradation.

Lentiviral and Moloney Retroviral Expression of Green Fluorescent Protein in Somatotrophs In Vivo

Previous studies have shown that the locus control region (LCR) and the promoter of the growth hormone (GH) gene can control the expression of GH. Therefore, lenti- and retro-viral vectors with these elements might be useful to monitor the activation of the GH gene and the development of newborn somatotrophs. To test this, we first constructed a lentiviral vector, which expresses green fluorescent protein (GFP) under the control of these elements, and injected them into rat pituitaries in situ and in vivo. The lentiviral vector expressed GFP specifically in the anterior lobe, and nearly all GFP-positive cells were anti-GH immunoreactive. The GFP expression was upregulated by the administration of growth hormone releasing hormone and an IGF-1 receptor blocker. Furthermore, the social isolation stress, which was shown to decrease the GH secretion, decreased the GFP expression. Second, we injected the retroviral vector into neonatal rat pituitaries in vivo. At 30 days postinjection (DPI), almost all GFP-positive cells were anti-GH positive and anti-prolactin negative as the lentiviral expression. However, GFP was transiently expressed by developing lactotrophs at 8 and 16 DPI, suggesting that our vector lacks an element(s) which suppresses the expression. Meanwhile, the retrovirally labeled cells tended to cluster with the cells of same type. An analysis of cell numbers in each cluster revealed some features of cell proliferation. These viral vectors are shown to be useful tools to monitor the activation of the GH gene and the development of somatotrophs.