Specific amino acids regulate Sestrin2 mRNA and protein levels in an ATF4-dependent manner in C2C12 myocytes.

BACKGROUND: Sestrin2 is a conserved protein in several species, and its expression is upregulated in cells under various environmental stresses. Sestrin2 content is involved in the function of mechanistic target of rapamycin complex 1 (mTORC1) in mouse embryonic fibroblasts and C2C12 cells. METHODS: C2C12 cells were treated with amino acid-free DMEM (AF-DMEM) for 5 h. The effects of the addition of specific amino acids to AF-DMEM on Sestrin2 mRNA and protein expression were examined using RT-qPCR and western blotting, respectively. The mechanism by which amino acids regulate Sestrin2 mRNA expression was examined using blocking and siRNA experiments. RESULTS: AF-DMEM increased the mRNA and protein levels of both Sestrin2 and activating transcription factor 4 (ATF4). The addition of a specific amino acid changed Sestrin2 mRNA and protein levels. The response pattern of Sestrin2 to specific amino acids was similar to that of ATF4. ATF4 siRNA reduced Sestrin2 mRNA levels. AF-DMEM increased eukaryotic initiation factor 2α (eIF2α) phosphorylation as early as 10 min after the treatment; however, ATF4 and Sestrin2 were increased 300 min after the treatment. Nuclear factor erythroid 2-related factor 2 and pancreatic and duodenal homeobox 1 siRNA did not affect ATF4 and Sestrin2 mRNA expression. CONCLUSIONS: Specific Amino acids regulate Sestrin2 levels in an ATF4-dependent manner in C2C12 cells. GENERAL SIGNIFICANCE: The results of the present study indicate that amino acids regulate levels of Sestrin2, which might cause phenotypic alterations, including mTORC1 activity, in C2C12 cells.

The involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1 activity in C2C12 and L6 myocytes.

OBJECTIVE: IGF-I and branched-chain amino acids have been reported to promote muscle hypertrophy via the stimulation of protein synthesis. Sestrin2, the function of which is regulated by leucine, has been reported to attenuate the activity of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) that stimulates protein synthesis. The objective of this study was to examine whether IGF-I modulates Sestrin2 abundance and to clarify the involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1. DESIGN: C2C12 and L6 myocytes were stimulated by leucine (1 mM) with or without pretreatment with IGF-I (100 ng/mL). Phosphorylation of p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), both of which are targets of the mTORC1, was examined by western blotting. Effects of Sestrin2 small interfering RNA (siRNA) on the actions of leucine and IGF-I were examined. Sestrin2 mRNA and protein levels were also determined after Sestrin2 siRNA. RESULTS: Leucine increased the phosphorylation of S6K and 4E-BP1 in a dose-dependent manner. Pretreatment with IGF-I for 5 h further increased the stimulatory effect of leucine on the phosphorylation of S6K and 4E-BP1 in C2C12 cells. IGF-I increased Sestrin2 protein and messenger RNA levels. Sestrin2 siRNA increased or tended to increase basal phosphorylation of 4E-BP1 and decreased the leucine-induced phosphorylation in C2C12 and L6 cells, in particular after IGF-I treatment, suggesting the involvement of Sestrin2 in the action of leucine and IGF-I. The net increase in leucine-induced 4E-BP1 phosphorylation appeared to be attenuated by Sestrin2 siRNA. Likewise, Sestrin2 siRNA attenuated leucine-induced S6K phosphorylation in L6 cells. However, Sestrin2 siRNA did not influence leucine-induced S6K phosphorylation in C2C12 cells. CONCLUSIONS: IGF-I and leucine cooperatively increased mTORC1 activity in C2C12 cells. IGF-I increased Sestrin2. Sestrin2 siRNA experiments showed that Sestrin2 was involved in the effect of leucine and IGF-I on mTORC1 activity in C2C12 and L6 cells, and suggested that increased Sestrin2 by IGF-I pretreatment might play a role in enhancing the effect of leucine on mTORC1.

Pathogenesis of Anti-PIT-1 Antibody Syndrome: PIT-1 Presentation by HLA Class I on Anterior Pituitary Cells.

CONTEXT: Anti-pituitary-specific transcriptional factor-1 (anti-PIT-1) antibody syndrome is characterized by acquired and specific deficiencies in growth hormone, prolactin, and thyroid-stimulating hormone. Although PIT-1-reactive cytotoxic T lymphocytes (CTLs) have been speculated to recognize anterior pituitary cells and to cause the injury in the pathogenesis of the syndrome, it remains unclear whether endogenous PIT-1 protein is processed through the proteolytic pathway and presented as an antigen on anterior pituitary cells. OBJECTIVE: To examine how PIT-1 protein is processed and whether its epitope is presented by major histocompatibility complex (MHC)/HLA class I on anterior pituitary cells. MATERIALS AND METHODS: Immunofluorescence staining and proximity ligation assay (PLA) were performed using anti-PIT-1 antibody and patients' sera on PIT-1-expressing cell line GH3 cells and human induced pluripotent stem cell (iPSC)-derived pituitary tissues. RESULTS: PIT-1 was colocalized with MHC class I molecules, calnexin, and GM130 in the cytosol. PLA results showed that PIT-1 epitope was presented by MHC/HLA class I molecules on the cell surface of GH3 cells and iPSC-derived pituitary cells. The number of PIT-1/HLA complexes on the cell surface of pituitary cells in the patient was comparable with that in the control subject. CONCLUSIONS: Our data indicate that PIT-1 protein is processed in the antigen presentation pathway and that its epitopes are presented by in MHC/HLA class I on anterior pituitary cells, supporting the hypothesis that PIT-1-reactive CTLs caused the cell-specific damage. It is also suggested that number of epitope presentation was not associated with the pathogenesis of anti-PIT-1 antibody syndrome.

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.

OBJECTIVE: Branched-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. DESIGN: After 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. RESULTS: GH 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. CONCLUSIONS: IGF-I increased LAT1 mRNA level in myocytes. However, the role of LAT1 in IGF-I-induced mTORC1 activation and cell functions remains unclear.

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-ß-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.

A novel thymoma-associated autoimmune disease: Anti-PIT-1 antibody syndrome.

Anti-PIT-1 antibody syndrome has recently been reported and characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with autoimmunity to a pituitary specific transcription factor PIT-1, which plays an essential role in GH-, PRL-, and TSH-producing cells. Although circulating anti-PIT-1 antibody and PIT-1-reactive cytotoxic T cells (CTLs) were detected in the patients, the pathophysiology and precise mechanisms for the autoimmunity remain unclarified. During the follow up, thymoma was diagnosed in all 3 cases with anti-PIT-1 antibody syndrome. Immunohistochemical analysis revealed that PIT-1 was strongly expressed in neoplastic cortical thymic epithelial cells. Importantly, after thymectomy, the titer of anti-PIT-1 antibody decreased and reactivity of CTLs toward PIT-1 diminished. These data strongly suggest that the aberrant expression of PIT-1 in the thymoma plays a causal role in the development of this syndrome. Thus, we define that this syndrome is a novel thymoma-associated autoimmune disease.

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.

Involvement of PIT-1-reactive cytotoxic T lymphocytes in anti-PIT-1 antibody syndrome.

CONTEXT: Anti-pituitary-specific transcriptional factor 1 (PIT-1) antibody syndrome is characterized by acquired growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiencies associated with circulating anti-PIT-1 antibodies. Although autoimmunity to PIT-1 has been suggested as a pathogenesis, the precise mechanism of the syndrome remains unclarified. OBJECTIVE: To elucidate the involvement of antibody- or cell-mediated immunity in anti-PIT-1 antibody syndrome. MATERIALS AND METHODS: To investigate a direct effect of anti-PIT-1 antibody on pituitary cells, cell proliferation, and cytotoxicity detection assays were performed using patient serum. Enzyme-linked immunospot (ELISpot) assay was performed to evaluate the involvement of PIT-1-reactive cytotoxic T lymphocytes (CTLs). An immunohistochemical analysis using anti-CD4 or anti-CD8 antibody was performed to examine tissue infiltration by CTLs. RESULTS: Patient serum did not exhibit any inhibitory effect on cell proliferation and secretion of GH and PRL in GH3 cells. In addition, complement-dependent cytotoxicity was not detected in patient serum on GH3 cells or primary pituitary cells. The ELISpot assay revealed the presence of CTLs that specifically reacted to the recombinant PIT-1 protein in the patient's peripheral lymphocytes. CD8(+) cell infiltrations, which is the characteristic of CTLs, were observed in the pituitary gland, adrenal gland, stomach, thyroid gland, liver, and pancreas of the patient with anti-PIT-1 antibody syndrome. CONCLUSIONS: These results suggest that the anti-PIT-1 antibody is not a cause but a marker of anti-PIT-1 antibody syndrome, in which CTLs play a pivotal role in the pathogenesis.

A PROP1-binding factor, AES cloned by yeast two-hybrid assay represses PROP1-induced Pit-1 gene expression.

PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1.

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.

Decreased serum chemerin levels in male Japanese patients with type 2 diabetes: sex dimorphism.

Chemerin, a recently discovered adipocytokine plays an important role in obesity and obesity-associated metabolic complications. However, the role of chemerin in the pathogenesis of type 2 diabetes mellitus (T2DM) has not fully been elucidated. We compared the serum chemerin levels and metabolic parameters between 88 control subjects, 86 patients with metabolic syndrome (MS), and 147 patients with T2DM in a Japanese population and further analyzed their correlation. Enzyme-linked immunosorbent assay was used to measure the serum chemerin levels. The chemerin levels were significantly higher in male than in female control subjects (p < 0.005), with significant decreases in patients with T2DM compared with those with MS and control subjects (164.9 ± 6.3 ng/mL vs. 209.8 ± 7.7 and 218.7 ± 7.3 ng/mL; p < 0.0001 vs. p < 0.0001, respectively) but no significant differences in female subjects. The multiple regression analysis revealed that the chemerin levels negatively correlated with the fasting glucose and HbA1c levels in total and male subjects. In the patients with T2DM, the chemerin levels negatively correlated with fasting glucose and high-density lipoprotein cholesterol but positively correlated with body mass index (BMI), and total cholesterol and triglyceride levels. The negative correlation between the chemerin and fasting glucose levels remained significant after adjustment for age, sex, and BMI in the total and male subjects and those with T2DM. These results suggest the role of chemerin in sex dimorphism and a potential link between chemerin levels and T2DM pathogenesis in a Japanese population.

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 (n = 228) 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.

Nonalcoholic fatty liver disease in adult hypopituitary patients with GH deficiency and the impact of GH replacement therapy.

BACKGROUND: Liver dysfunction in adult hypopituitary patients with GH deficiency (GHD) has been reported and an increased prevalence of nonalcoholic fatty liver disease (NAFLD) has been suggested. OBJECTIVE: The objective of the present study was to elucidate the pathophysiology of the liver in adult hypopituitary patients with GHD. PATIENTS AND METHODS: We recruited 69 consecutive Japanese adult hypopituitary patients with GHD and examined the prevalence of NAFLD by ultrasonography and nonalcoholic steatohepatitis (NASH) by liver biopsy. Patients had been given routine replacement therapy except for GH. We compared these patients with healthy age-, gender-, and BMI-matched controls. We further analyzed the effect of GH replacement therapy on liver function, inflammation and fibrotic markers, and histological changes. RESULTS: The prevalence of NAFLD in hypopituitary patients with GHD was significantly higher than in controls (77 vs 12%, P<0.001). Of 16 patients assessed by liver biopsy, 14 (21%) patients were diagnosed with NASH. GH replacement therapy significantly reduced serum liver enzyme concentrations in the patients and improved the histological changes in the liver concomitant with reduction in fibrotic marker concentrations in patients with NASH. CONCLUSIONS: Adult hypopituitary patients with GHD demonstrated a high NAFLD prevalence. The effect of GH replacement therapy suggests that the NAFLD is predominantly attributable to GHD.

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.

Chemerin regulates ß-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 ß-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 ß-cell function, was downregulated in chemerin-deficient islets and a chemerin-ablated ß-cell line and rescue of MafA expression restored GSIS, indicating that chemerin regulates ß-cell function via maintaining MafA expression. These results indicate that chemerin regulates ß-cell function and plays an important role in glucose homeostasis in a tissue-dependent manner.

Adult combined GH, prolactin, and TSH deficiency associated with circulating PIT-1 antibody in humans.

The pituitary-specific transcriptional factor-1 (PIT-1, also known as POU1F1), is an essential factor for multiple hormone-secreting cell types. A genetic defect in the PIT-1 gene results in congenital growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) deficiency. Here, we investigated 3 cases of adult-onset combined GH, PRL, and TSH deficiencies and found that the endocrinological phenotype in each was linked to autoimmunity directed against the PIT-1 protein. We detected anti-PIT-1 antibody along with various autoantibodies in the patients' sera. An ELISA-based screening revealed that this antibody was highly specific to the disease and absent in control subjects. Immunohistochemical analysis revealed that PIT-1-, GH-, PRL-, and TSH-positive cells were absent in the pituitary of patient 2, who also had a range of autoimmune endocrinopathies. These clinical manifestations were compatible with the definition of autoimmune polyendocrine syndrome (APS). However, the main manifestations of APS-I--hypoparathyroidism and Candida infection--were not observed and the pituitary abnormalities were obviously different from the hypophysitis associated with APS. These data suggest that these patients define a unique "anti-PIT-1 antibody syndrome," related to APS.

W194XProp1 and S156insTProp1, both of which have intact DNA-binding domain, show a different DNA-binding activity to the Prop1-binding element in human Pit-1 gene.

Prop1 activates POU1F1 (Pit-1) gene expression, which in turn stimulates GH, PRL, TSHbeta and GHRH receptor gene expressions. Therefore the patients with Prop1 mutation show GH, PRL, and TSH deficiency. The mutation of Prop1 is a major abnormality causing combined pituitary hormone deficiency (CPHD). However, DNA-binding and activating functions of mutant Prop1 have not been examined fully because Prop1-binding elements (PBEs) in human POU1F1 gene were not identified until 2008. The aim of this study is to test DNA-binding and transcriptional activities of two mutant Prop1s (W194XProp1 and S156insTProp1, both of them were found in the patients with CPHD) whose mutation is located in putative transactivating domain but not in DNA-binding domain. W194XProp1 showed a marked DNA-binding to PBE as well as a consensus element of paired-like transcription factors (PRDQ9). Activating function for POU1F1 reporter genes expression was lost or decreased in W194XProp1 but still preserved for PRDQ9 reporter gene. S156insTProp1 did not bind PBE but bound PRDQ9. Consistent with the result, S156insTProp1 did not stimulate POU1F1 reporter gene but stimulated PRDQ9 reporter gene. These results support the inference that W194XProp1 is unable to increase POU1F1 gene expression by the defect of transactivating domain and that S156insTProp1 is unable to increase due to the loss of DNA-binding activity. DNA-binding domain that has been assumed is not sufficient to provide full DNA-binding activity of Prop1 and transactivating domain of Prop1 is likely to affect DNA binding to PBE.

Branched-chain amino acids protect against dexamethasone-induced soleus muscle atrophy in rats.

We investigated the utility of branched-chain amino acids (BCAA) in dexamethasone-induced muscle atrophy. Dexamethasone (600 microg/kg, intraperitoneally) and/or BCAA (600 mg/kg, orally) were administered for 5 days in rats, and the effect of BCAA on dexamethasone-induced muscle atrophy was evaluated. Dexamethasone decreased total protein concentration of rat soleus muscles. Concomitant administration of BCAA reversed the decrease. Dexamethasone decreased mean cross-sectional area of soleus muscle fibers, which was reversed by BCAA. Dexamethasone increased atrogin-1 expression, which has been reported to play a pivotal role in muscle atrophy. The increased expression of atrogin-1 mRNA was significantly attenuated by BCAA. Furthermore, dexamethasone-induced conversion from microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, which is an indicator of autophagy, was blocked by BCAA. These findings suggest that BCAA decreased protein breakdown to prevent muscle atrophy. BCAA administration appears to be useful for prevention of steroid myopathy.

IGF-I stimulates reactive oxygen species (ROS) production and inhibits insulin-dependent glucose uptake via ROS in 3T3-L1 adipocytes.

OBJECTIVE: IGF-I is known to enhance insulin sensitivity in whole body mainly via the IGF-I receptors in muscles. However, the effect of IGF-I on the regulation of insulin sensitivity in the adipose tissue is yet unclear. Insulin sensitivity was found to be higher in the IGF-I receptor-deficient adipocytes than that in wild-type adipocytes, suggesting that IGF-I signaling induces insulin resistance in adipocytes. However, the underlying mechanism has not yet been elucidated. In addition, the effect of superphysiological levels of IGF-I, as is observed in patients with acromegaly, on insulin sensitivity remains unclear. DESIGN: To clarify the role of IGF-I on insulin sensitivity in adipocytes, we determined insulin-induced glucose uptake and IRS-1 status in 3T3-L1 adipocytes treated with IGF-I. Since reactive oxygen species (ROS) are causally related to insulin resistance, we investigated the effect of IGF-I on ROS production to elucidate the molecular mechanism underlying insulin resistance. RESULTS: Preincubation of the adipocytes with IGF-I attenuated insulin-dependent glucose uptake. Interestingly, we found that IGF-I significantly stimulated ROS production. Furthermore, preincubation of adipocytes with an antioxidant, N-acetyl-cysteine (NAC) restored the IGF-I-induced attenuation of insulin-dependent glucose uptake; this indicates that IGF-I induces insulin resistance via ROS. Serine phosphorylation of IRS-1 was strongly induced and the insulin-dependent tyrosine phosphorylation of IRS-1 was suppressed by preincubating the adipocytes with IGF-I. Further, NAC restored these changes induced by IGF-I on both serine and tyrosine phosphorylation of IRS-1. CONCLUSIONS: These data indicate that IGF-I inhibited insulin activity in the 3T3-L1 adipocytes via ROS production, which affects IRS-1 phosphorylation status.