The effects of cold water immersion after rugby training on muscle power and biochemical markers.

During rugby game, or intensive rugby training there are many high intensity explosive exercises and eccentric muscle contractions, therefore adequate recovery is very important to rugby players. In the present study we have tested the effects of cold water immersion (CWI) after game-simulated (80 min.) rugby training on muscle power recovery and blood markers of muscle damage. Twenty well-trained collegiate male rugby players (age: 20.3 ± 0.6 years old, body height: 1.74 ± 0.05 m, body weight: 85.4 ± 2.0 kg, body fat: 18.2 ± 1.4 %) volunteered for this study. This study was conducted as a cross-over design; i.e., the subjects were randomly assigned either to CWI (n = 10) or passive rest condition (n = 10) for the 1(st) trial and 1 week later the subjects were switched conditions for the 2(nd) trial. After the simulated rugby training, including tackles and body contacts, muscle functional ability and blood markers of muscle damage were tested immediately, after CWI or passive rest, and again 24 hours later. Statistical analysis of all muscle functional tests (10 m dash, counter movement jump, reaction time, side steps) except for 10 seconds maximal pedaling power and blood makers of muscle damage (aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and creatinine) revealed significant main effects for time (p < 0.05). However, no statistically significant interactions were found in any of the muscle functional tests and blood markers between groups and time courses. Our results suggest that a rugby game induces muscle damage and reduces muscle function. However, CWI has no significant restorative effect after an 80-minute rugby game in terms of muscle damage. Key PointsCold water immersion study for the recovery of rugby playersMuscle strength and muscle power were mainly evaluated as well as muscle enzymes of muscle break downSubjects were highly trained rugby players with control group.

Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway.

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis within the individual cell. Recent reports have suggested that leptin, an adipocyte-secreted hormone, phosphorylates AMPK in skeletal muscle directly. However, little is known about the interaction between leptin signaling and AMPK activation. Here, we report that the leptin-induced phosphorylation of AMPK was detected in Huh7 cells expressing long form leptin receptor (OBRb) as well as short form leptin receptor (OBRa). In addition, we demonstrate that AMPK activation does not require the phosphorylation of either Tyr-985 or Tyr-1138 within the OBRb and may occur via a STAT3-independent signaling pathway. We also show that Huh7 cells expressing OBRb and SOCS3 (inhibitor of JAK2) resulted in a marked reduction of AMPK activation in response to leptin. These findings suggest that the activation of JAK2, but not STAT3, may play a critical role in leptin-induced AMPK activation in Huh7 cells.

Rapid inhibition of leptin signaling by glucocorticoids in vitro and in vivo.

Elevated secretion of glucocorticoids (GCs) or hypersensitivity to GCs has a permissive effect on the development of obesity and leads to abnormalities of body fat distribution. Recent studies demonstrated GCs act as antagonists of leptin in rodents. However, little is known about the interaction between GCs and leptin signaling. In the present study, we investigated the effects of GCs on leptin action in vitro and in vivo. GCs rapidly inhibited the leptin-induced STAT3 phosphorylation in a dose- and time-dependent manner, as assayed by Western blotting using anti-phosphospecific-STAT3 in human hepatoma cell lines (Huh7) transiently expressing long form leptin receptor. GCs also inhibited the leptin-induced JAK2 tyrosine phosphorylation but unaltered the specific binding of (125)I-leptin to the cells. Parallel experiments, however, demonstrated that the inhibitory effects of GCs were not observed in either IL-6- or LIF-induced STAT3 phosphorylation. Furthermore, we examined the feeding behavior and hypothalamic leptin signaling following intracerebroventricular (icv) infusion of GCs prior to icv leptin infusion in Sprague-Dawley rats. The food intake after 24 h of icv leptin injection increased 3-fold in GCs-treated animals. In addition, central infusion of GCs resulted in a marked reduction of hypothalamic STAT3 phosphorylation in response to icv infusion of leptin. To clarify the molecular mechanism by which GCs rapidly reduce leptin-induced JAK/STAT signaling, we examined the intracellular signal transduction pathway potentially mediated by GCs. PD98059, a specific MEK inhibitor, blocked the inhibitory effects of GCs on leptin-induced JAK/STAT activation in Huh7 cells. These results suggest GCs antagonize leptin action by a rapid inhibition of the leptin-induced JAK/STAT pathway partly via MAPK cascade.

Association between IL-18 gene promoter polymorphisms and CTLA-4 gene 49A/G polymorphism in Japanese patients with type 1 diabetes.

Interleukin-18 (IL-18) is a potent proinflammatory cytokine which is strongly associated with the development of diabetes in NOD mice. To test the putative involvement of IL-18 gene polymorphism in predisposition to human type 1 diabetes, the SNPs at position -607 (C/A) and -137 (G/C) in the promoter region of IL-18 gene were analyzed by sequence-specific PCR in 116 patients with type 1 diabetes and 114 normal controls. A linkage disequilibrium found only three of the four possible haplotypes defined by these SNPs. The distribution of the IL-18 gene genotypes at position -607 was significantly different between patients with type 1 diabetes and normal controls (P=0.023). Furthermore, there was a significant increase in haplotype 1 (-607C/-137G) in the patients compared with controls (P=0.006). The association study of the susceptible CTLA-4 genotype (GG at nucleotide position 49 in exon 1) or HLA-DR4-DQB1*0401 and type 1 diabetes showed that the predisposing IL-18 gene haplotype modulates the risk on CTLA-4 GG genotype, but not on HLA-DR4-DQB1*0401 haplotype. Among subjects carrying the CTLA-4 GG genotype, the frequency of IL-18 haplotype 1 in patients with type 1 diabetes was significantly higher than that in controls (91% vs. 71%, P=0.012). However, IL-18 haplotype 1 was not frequent in patients who do not exhibit the CTLA-4 high-risk genotype. These results suggest that the IL-18 gene polymorphism is associated with a type 1 diabetes susceptibility, and there might be a gene-gene interaction between IL-18 gene with susceptible CTLA-4 gene.

Interleukin-10 gene promoter region polymorphisms in patients with type 1 diabetes and autoimmune thyroid disease.

Type 1 diabetes is a heterogeneous autoimmune disease and is frequently associated with other organ-specific autoimmune diseases, including autoimmune thyroid disease (AITD). Type 1 diabetic patients with AITD are known to show distinct clinical and immunological features from patients without AITD. This study investigated whether interleukin-10 (IL-10) gene promoter region polymorphisms are associated with susceptibility to type 1 diabetes and AITD. The frequency of -1082G/A, -819C/T, and -592C/A polymorphisms was analyzed in 54 type 1 diabetic patients with AITD, 74 type 1 diabetic patients without AITD, 124 nondiabetic patients with AITD, and 107 healthy subjects in a case-control study. No significant differences on the allele and genotype frequencies of three polymorphisms were found not only in type 1 diabetic patients with AITD compared with normal controls, but also between nondiabetic patients with AITD and healthy controls. The distribution of IL-10 gene haplotypes was also similar between both patient groups and normal controls. These results suggest that IL-10 gene promoter region polymorphisms are not associated with genetic susceptibility to type 1 diabetes and AITD.

Association of interleukin-18 gene promoter polymorphisms in type 1 diabetes and autoimmune thyroid disease.

Type 1 diabetes is a heterogeneous autoimmune disease and is often associated with other organ-specific autoimmune diseases, including autoimmune thyroid disease (AITD). IL-18 is a potent proinflammatory cytokine capable of inducing IFN-gamma production that is associated with the development of type 1 diabetes and AITD. The gene for IL-18 is located near Idd2 and has been reported to be associated with a susceptibility to type 1 diabetes. To test the putative involvement of IL-18 gene polymorphism in predisposition to type 1 diabetes and AITD, we conducted a case-control study in Japanese population. The SNPs at position -607 (C/A) and -137 (G/C) in the promoter region of the IL-18 gene were analyzed by sequence-specific PCR in 74 nondiabetic patients with AITD, 47 type 1 diabetic patients with AITD, and 114 normal controls. There was no significant increase in the genotype and allele frequencies not only in nondiabetic patients with AITD compared with normal controls, but also in type 1 diabetic patients with AITD compared with normal controls. The distribution of IL-18 gene haplotypes was also similar between both patient groups and normal controls. These results suggest that polymorphisms of the IL-18 gene are not associated with a susceptibility to AITD and type 1 diabetes coexistent with AITD in Japanese population.

Epitope analysis of GAD65 autoantibodies in Japanese patients with autoimmune diabetes.

Type 1 diabetes is an organ-specific autoimmune disease characterized by T cell-mediated destruction of pancreatic beta cells. In Japanese population, the incidence of type 1 diabetes in children is very low compared to European countries. However, there are more patients with type 1 diabetes in adults, including latent autoimmune diabetes in adults (LADA). The circulating autoantibodies to multiple islet autoantigens including GAD, insulin, and IA-2 are the important immunological features of type 1 diabetes. The prevalences of anti-islet autoantibodies in patients with Japanese type 1 diabetes are 60-70% for GAD autoantibodies, 45-50% for insulin autoantibodies (IAA), and 60-65% for IA-2 autoantibodies at disease onset, which are similar to those reported in Caucasian patients. With combinatorial analysis of these autoantibodies, 90% of patients express at least one of these autoantibodies and are classified as type 1A diabetes. Although the majority of patients with type 1 diabetes are young, lean, and ketosis-prone, there are a number of patients with type 1 diabetes initially diagnosed as having type 2 diabetes at disease onset called LADA. These patients with LADA often progress toward an insulin-deficient state within several years after diagnosis. High levels of GAD autoantibodies have a high predictive value for future insulin deficiency in LADA. Further, epitope analysis of GAD65 autoantibodies may be helpful to predict future insulin dependency in LADA patients. In conclusion, Japanese patients with type 1 diabetes are clinically heterogeneous and the determination of immunological features are helpful to clarify the characteristics of the Japanese type 1 diabetic syndrome.

Intracerebroventricular administration of insulin and glucose inhibits the anorectic action of leptin in rats.

Obese individuals with glucose intolerance present with high serum levels of glucose, insulin, and leptin. These substances are potent inhibitors of feeding in the brain. Obese subjects still present with over-feeding despite elevation of the above factors. To elucidate the mechanism of this paradox, the effects of insulin and glucose on the anorectic action of leptin in the hypothalamus were examined. Adult male Sprague-Dawley rats (weighing 285-320 g) were pretreated with intracerebroventricular injection of insulin, glucose, or saline, followed by leptin (7.5 microg) or phosphate-buffered saline (PBS) injection into the third cerebral ventricle (icv). The cumulative food intakes were measured 24 hr after leptin icv. The tyrosine phosphorylation of signal transducer and activator transcription factor 3 (STAT3) in the hypothalamus was determined by Western blotting. In rats pretreated with saline and stimulated with leptin (saline/LEPTIN group), food intake diminished to about 50% of that of the saline/PBS group (P < 0.005). Food intake in the insulin/LEPTIN group was significantly higher compared with the saline/LEPTIN group (P < 0.005) and reached the level seen in the saline/PBS group. Similar data were obtained in glucose pretreatment experiments. Insulin and glucose icv resulted in reduction of leptin-induced STAT3 tyrosine phosphorylation compared with saline. Infusion of insulin and glucose icv did not alter peripheral blood glucose levels in all groups. High insulin or glucose levels in the brain could result in leptin resistance as manifested by food intake, which is probably due to the attenuation of STAT3 phosphorylation downstream the leptin receptor.

Stromal-cell derived factor-1 chemokine gene variant is associated with type 1 diabetes age at onset in Japanese population.

Stromal-cell derived factor-1 (SDF-1) is a powerful chemokine that upregulates T-cell migration and activation. The gene for SDF-1 is located near type 1 diabetes susceptibility locus IDDM10, suggesting a contribution by SDF-1 to the induction of diabetes. Recently the role of SDF-1 gene polymorphism in the clinical presentation of type 1 diabetes in French population has been reported. To test the putative involvement of SDF-1 gene polymorphism in predisposition to or clinical heterogeneity of type 1 diabetes in Japanese population, we conducted the case-control study. The SDF1-3'A variant (801 G to A in the 3'-untranslated region) was determined by the polymerase chain reaction-restriction fragment length polymorphism technique in 184 patients with abrupt-onset type 1 diabetes and 106 healthy control subjects. No significant difference in allele and genotype frequencies of SDF1-3'A variant was found between type 1 diabetic patients and healthy controls. However, the SDF1-3'A variant was strongly associated with early-onset diabetes in a recessive model (AA versus AG + GG, p = 0.017). The mean age-at-onset in patients carrying SDF1-3'AA genotype was significantly younger than that in patients with SDF1-3' AG or GG genotype (p = 0.028). The frequencies of SDF1-3' A variant were significantly increased in HLA-DR4/9 patients compared with non-DR4/9 patients (p = 0.008). These results suggest that the SDF-1 gene polymorphism is associated with the age-at-onset of type 1 diabetes in Japanese population.

Insulin attenuates leptin-induced STAT3 tyrosine-phosphorylation in a hepatoma cell line.

Leptin, the 16 kDa protein product of the ob gene, is secreted by adipocytes. The long form leptin receptor (ObRb) is expressed at high levels in the hypothalamus, and regulates appetite and energy expenditure. The fact that serum concentration of leptin is correlated with body mass index (BMI) suggests reduced sensitivity to leptin. Even though hyperinsulinemia and hyperleptinemia could coexist in obese humans, little is known about the interaction of insulin and leptin. In this study, we examined the effect of insulin on leptin signaling using Huh 7 cells transiently transfected with ObRb cDNA. Insulin inhibits leptin-induced STAT3 phosphorylation in a time- and dose-dependent manner without affecting Janus tyrosine kinases (JAKs) JAK2 phosphorylation. Okadaic acid prevents the inhibitory effect of insulin on leptin-induced STAT3 activation.

Vanadate enhances leptin-induced activation of JAK/STAT pathway in CHO cells.

Leptin, the product of the ob gene, is an adipocyte-derived hormone that plays a key role in the control of food intake and energy expenditure. Leptin acts through receptors that belong to a member of the class I cytokine receptor family. It has been demonstrated that the SH2 domain-containing tyrosine phosphatase 2 (SHP-2) negatively regulates STAT3-mediated transcriptional activation through long form leptin receptor (OBRb). Vanadate has been shown to be a potent and selective inhibitor of PTPase activity in vitro. In this study, we have demonstrated that vanadate increases leptin-induced JAK2 and STAT3 phosphorylation in CHO cells expressing OBRb. The increased leptin-dependent luciferase activity of SOCS3 gene was also seen in vanadate-treated cell. Furthermore, vanadate reversed the inhibitory effects of SOCS3 on leptin-induced STAT3 phosphorylation. The present findings suggest that PTP inhibitors including vanadate and vanadate-derived compounds could be used as a therapeutic agent in the treatment of obesity.

Ethanol inhibits leptin-induced STAT3 activation in Huh7 cells.

Leptin, an adipocyte-derived hormone, regulates food intake and energy expenditure in the hypothalamus via its receptor, member of the class I cytokine receptor family. Leptin resistance has been observed in rodents and in humans. However, the mechanisms could not be explained in most cases of human obesity, except for rare cases with mutations in the leptin receptor. Recent reports demonstrated that ethanol inhibited the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activated by some members of the class I cytokine receptor family. In this study, we examined the effects of ethanol on the leptin-induced JAK/STAT signaling pathway using human hepatoma cell lines transiently expressing long form of the leptin receptor. A 30 min pretreatment with ethanol dose-dependently inhibited the leptin-induced STAT3 phosphorylation. Furthermore, to determine the time course of ethanol inhibitory effects, the cells were incubated in 10 mM ethanol for various times. Partial inhibition of leptin-induced STAT3 activation was seen after 1 min of treatment with ethanol and completely inhibited after 30 min pretreatment. SB 202190, a p38 mitogen-activated protein kinase (MAPK) inhibitor, partly prevented this inhibition by ethanol of leptin-induced STAT3 activation. These findings suggest that ethanol time- and dose-dependently inhibits the leptin action, in part via p38 MAPK.

Genetic association between interleukin-10 gene promoter region polymorphisms and type 1 diabetes age-at-onset.

This study investigated whether interleukin-10 (IL-10) gene promoter region polymorphisms are associated with susceptibility to or clinical presentation of type 1 diabetes. The frequency of -1082G/A, -819C/T, and -592C/A polymorphisms was analyzed in 128 Japanese patients with type 1 diabetes and in 107 healthy control subjects in a case-controlled study. The allelic and haplotypic frequencies of the IL-10 gene promoter region polymorphisms were similar in patients with type 1 diabetes and in control subjects. However, the -819T and -592A allele were associated with adult-onset (>18 years) of the disease (p = 0.037). Furthermore, the frequency of ATA haplotype was increased in adult-onset patients than that in early-onset patients (< or =18 years; p = 0.037). Among the genotypes comprising ATA haplotype, the frequency of ATA/ATA was significantly higher in adult-onset patients than in early-onset patients (p = 0.004). These results suggest that the IL-10 gene promoter polymorphisms are associated with the age-at-onset in Japanese patients with type 1 diabetes.

Distinct IA-2 autoantibody epitope recognition between childhood-onset and adult-onset type 1 diabetes.

Different autoimmune mechanisms may be involved in childhood- and adult-onset type 1 diabetes. Our aim was to explore the differences in IA-2 autoantibody epitope recognition between childhood- and adult-onset type 1 diabetes. Therefore, in vitro synthesized radiolabeled IA-2ic (amino acid 601-979), IA-2JM (amino acid 557-629), and IA-2PTP (amino acid 630-979) were used to analyze the IA-2 autoantibody epitope specificities in 93 patients with new-onset type 1 diabetes. Among 93 patients with type 1 diabetes the prevalences of autoantibodies to GAD, IA-2ic, and insulin were 69.9%, 58.1%, and 45.2%, respectively. The prevalence of IA-2ic autoantibodies in patients with childhood-onset type 1 diabetes (aged