Genzo Iguchi

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.

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.

The prevalence of IgG4-related hypophysitis in 170 consecutive patients with hypopituitarism and/or central diabetes insipidus and review of the literature

OBJECTIVE The prevalence and clinical characteristics of IgG4-related hypophysitis remain unclear due to the limited number of case reports. Therefore, in this study, we screened consecutive outpatients with hypopituitarism and/or diabetes insipidus (DI) to estimate its prevalence. METHODS A total of 170 consecutive outpatients with hypopituitarism and/or central DI were screened at Kobe University Hospital for detecting IgG4-related hypophysitis by pituitary magnetic resonance imaging, measuring serum IgG4 concentrations, assessing the involvement of other organs, and carrying out an immunohistochemical analysis to detect IgG4-positive cell infiltration. RESULTS Among the screened cases, 116 cases were excluded due to diagnosis of other causes such as tumors and congenital abnormalities. Additionally, 22 cases with isolated ACTH deficiency were analyzed and were found not to meet the criteria of IgG4-related hypophysitis. The remaining 32 cases were screened and seven were diagnosed with IgG4-related hypophysitis, of which three cases were diagnosed by analyzing pituitary specimens. IgG4-related hypophysitis was detected in 30% (seven of 23 patients) of hypophysitis cases and 4% of all hypopituitarism/DI cases. The mean age at the onset of IgG4-related hypophysitis was 61.8±8.8 years, and the serum IgG4 concentration was 191.1±78.3 mg/dl (normal values 5-105 mg/dl and values in IgG4-related disease (RD) ≥135 mg/dl). Pituitary gland and/or stalk swelling was observed in six patients, and an empty sella was observed in one patient. Multiple co-existing organ involvement was observed in four of the seven patients prior to the onset of IgG4-related hypophysitis. CONCLUSION These data suggest that the prevalence of IgG4-related hypophysitis has been underestimated. We should also consider the possibility of the development of hypopituitarism/DI caused by IgG4-related hypophysitis during the clinical course of other IgG4-RDs.

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 μg/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. Muscle Nerve, 2010

Cloning and Characterization of the 5′-Flanking Region of the Human Growth Hormone-releasing Hormone Receptor Gene

We cloned the 5′-flanking region of the human growth hormone-releasing hormone receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 kilobases upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site. The 5′-end of the longest product of 5′-rapid amplification of cDNA ends was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-1-binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated that 5′-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in nonpituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed that the regions from −310 to −130 and from −130 to −120 were important for the GHRH-R gene expression in GH3 cells, although the latter contributed less to the gene expression. In BeWo cells co-transfected with Pit-1 expression vector, the region from −310 to −130 was essential for the Pit-1-dependent expression of GHRH-R gene. The region from −310 to −120 has two putative Pit-1-binding sites, P1 and P2, located from −129 to −123 and from −171 to −160, respectively. Both mobility shift assay and DNase-I footprint analysis showed that P2 had much higher Pit-1 binding affinity than P1. Mutation of P2 decreased GHRH-R gene expression in GH3 cells. These findings were consistent with the results that the region from −310 to −130 is an important element for Pit-1-dependent expression of GHRH-R gene.

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.

SIRT1 regulates adaptive response of the growth hormone--insulin-like growth factor-I axis under fasting conditions in liver

Adaptation under fasting conditions is critical for survival in animals. Sirtuin 1 (SIRT1), a protein deacetylase, plays an essential role in adaptive metabolic and endocrine responses under fasting conditions by modifying the acetylation status of various proteins. Fasting induces growth hormone (GH) resistance in the liver, leading to decreased serum insulin-like growth factor-I (IGF-I) levels as an endocrine adaptation for malnutrition; however, the underlying mechanisms of this action remain to be fully elucidated. Here we report that in vivo knockdown of SIRT1 in the liver restored the fasting-induced decrease in serum IGF-I levels and enhanced the GH-dependent increase in IGF-I levels, indicating that SIRT1 negatively regulates GH-dependent IGF-I production in the liver. In vitro analysis using hepatocytes demonstrated that SIRT1 suppresses GH-dependent IGF-I expression, accompanied by decreased tyrosine phosphorylation on signal transducer and activator of transcription (STAT) 5. GST pull-down assays revealed that SIRT1 interacts directly with STAT5. When the lysine residues adjacent to the SH2 domain of STAT5 were mutated, STAT5 acetylation decreased concomitant with a decrease in its transcriptional activity. Knockdown of SIRT1 enhanced the acetylation and GH-induced tyrosine phosphorylation of STAT5, as well as the GH-induced interaction of the GH receptor with STAT5. These data indicate that SIRT1 negatively regulates GH-induced STAT5 phosphorylation and IGF-I production via deacetylation of STAT5 in the liver. In addition, our findings explain the underlying mechanisms of GH resistance under fasting conditions, which is a known element of endocrine adaptation during fasting.

The role of circulating ghrelin in growth hormone (GH) secretion in freely moving male rats

To examine the physiological significance of plasma ghrelin in generating pulsatile growth hormone (GH) secretion in rats, plasma GH and ghrelin levels were determined in freely moving male rats. Plasma GH was pulsatilely secreted as reported previously. Plasma ghrelin levels were measured by both N-RIA recognizing the active form of ghrelin and C-RIA determining total amount of ghrelin. Mean +/- SE plasma ghrelin levels determined by N-RIA and C-RIA were 21.6 +/- 8.5 and 315.5 +/- 67.5 pM, respectively, during peak periods when plasma GH levels were greater than 100 ng / ml. During trough periods when plasma GH levels were less than 10 ng / ml, they were 16.5 +/- 4.5 and 342.1 +/- 29.8 pM, respectively. There were no significant differences in plasma ghrelin levels between two periods. Next, effect of a GH secretagogue antagonist, [D-Lys-3]-GHRP-6, on plasma GH profiles was examined. There were no significant differences in both peak GH levels and area under the curves of GH (AUCs) between [D-Lys-3]-GHRP-6-treated and control rats. These findings suggest circulating ghrelin in peripheral blood does not play a role in generating pulsatile GH secretion in freely moving male rats.

GH-independent IGF-I action is essential to prevent the development of nonalcoholic steatohepatitis in a GH-deficient rat model

The progression to nonalcoholic steatohepatitis (NASH) from simple steatosis is associated with the mitochondrial dysfunction, enhanced oxidative stress, and inflammation. Recently, it has been reported that the prevalence of NAFLD (nonalcoholic fatty liver disease)/NASH is increased in patients with adult growth hormone deficiency (AGHD), suggesting that the deficiencies in GH and insulin-like growth factor (IGF-I) are involved in the development of NAFLD/NASH; however, the precise underlying mechanism remains to be elucidated. To clarify the mechanisms and the specific contribution of GH and IGF-I in these conditions, we examined the liver of a GH-deficient rat model, spontaneous dwarf rat (SDR) and the effect of GH and IGF-I administration. SDR showed steatosis and fibrosis in the liver in line with the phenotype observed in AGHD. Serum AST and ALT levels and triglyceride content in the liver were significantly increased in the SDR compared with the control. Intriguingly, the mitochondrial morphology in the SDR hepatocyte was impaired and the area was significantly decreased. Furthermore, oxidative stress in the SDR liver was enhanced. These changes were improved not only by GH but also by IGF-I administration, suggesting that GH-independent IGF-I action plays an essential role in the liver. In conclusion, we demonstrated that GH-deficient rat exhibits NASH and IGF-I plays an essential role to prevent the development of NASH. The improved mitochondrial function and reduced oxidative stress may contribute the effect of IGF-I in the liver.