Shigeo Uotani

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.

Glucose transporter gene expression in rat conceptus during high glucose culture

SummaryWe investigated the expression of glucose transporter genes and protein in embryo and yolk sac during organogenesis and the regulation of glucose transporters during culture in hyperglycaemic media. Erythrocyte-type glucose transporter (GLUT 1) and brain-type glucose transporter (GLUT 3) mRNA were expressed in embryo and yolk sac. The expression of GLUT-1 and GLUT-3 mRNA was abundant on day 9–11 and day 9–10 in the embryo, respectively, and day 9–14 and day 10–11 in the yolk sac, respectively. The levels of GLUT-1 protein in the embryo increased in parallel with the expression of GLUT-1 mRNA during the corresponding period. Immunohistochemical staining of GLUT-1 protein was found principally in the neuroepithelial cells surrounding the neural tube in the embryo on day 10 and appeared in the microvessels surrounding the neural tube after day 12.To test whether the expression of glucose transporter genes and protein was suppressed during hyperglycaemia, conceptuses were cultured in high glucose medium. The abundant expression of GLUT-1 protein was not decreased during culture in high glucose media for 24 h (day 9–10) and was only down-regulated by prolonged exposure to this media for 48 h (day 9–11). We have demonstrated the predominant expression of the high affinity glucose transporter (GLUT 1 and GLUT 3) genes and (GLUT 1) protein in embryo during the early period of organogenesis. The persistently abundant expression of glucose transporter during the critical period of neural tube formation (day 9–10) even in the presence of hyperglycaemia may explain one of the mechanisms of increased glucose flux into the neuroepithelium, which may lead to neural tube defects.

Anti-insulin receptor autoantibodies in a patient with type B insulin resistance and fasting hypoglycemia.

Abstract We studied a patient with systemic lupus erythematosus and type B insulin resistance who showed almost complete normalization of postprandial plasma glucose in 3 months and a transient coccurrence of fasting hypoglycemia from day 35 (i. e. the 35th day of hospitalization) to day 77. To determine the clinical relevance of the biological ability of anti-insulin receptor antibodies (anti-IRAb), we made multiple preparations of the patients dialyzed serum and IgG. Dialyzed serum prepared on day 1 showed 95% inhibition of insulin binding. The binding inhibition was, however, decreased parallel to the normalization of insulin sensitivity. For 2DG uptake, 6.2 μM IgG purified on 3 different days (day 7, 35 and 78, designated IgG-NOV, -JAN, and -FEB, respectively) stimulated 2DG uptake into CHO-hIR at 3.4-, 3.1-, and 1.5-fold, respectively. Phosphotyrosine immunoblotting revealed that apparent insulin receptor autophosphorylation was visible only with IgG-NOV, not with the IgG-JAN or -FEB. Mutation of tyrosine-960 or lysine-1018 of the insulin receptor failed to transduce the IgGs stimulatory effect. IgG-NOV was not able to stimulate the autophosphorylation of the human IGF-I receptor. In the present study, the insulin binding inhibitory activities of the dialyzed sera prepared at different time points were shown to be altered parallel to insulin sensitivity in vivo. Stimulatory activities of the patients IgG were, however, discordant for the occurrence of fasting hypoglycemia observed in vivo. Other pathogenic factors or mechanisms in addition to the insulin-like action of the anti-IRAb may be also required to fully understant the development of fasting hypoglycemia in type B insulin resistance.

Association of Interleukin-18 Gene Promoter Polymorphisms in Type 1 Diabetes and Autoimmune Thyroid Disease

Abstract: 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‐γ 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.

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 2850–320 g) were pretreated with intracerebroventricular injection of insulin, glucose, or saline, followed by leptin (7.5 μg) 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.

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.

TNF-α stimulates glucose uptake in L6 myoblasts

Abstract The mechanism of TNF-α to regulate glucose metabolism remains unclear. To further delineate the TNF-α signal transduction pathway mediating glucose metabolism, we utilized L6 rat myoblasts which contain the receptors for the insulin-like growth factor-I (IGF-I) and TNF-α, and the ability of both ligands to stimulate glucose uptake was compared. IGF-I (6.5 nM) maximally stimulated glucose uptake 7-fold after 24 h incubation, while 23 nM TNF-α maximally stimulated glucose uptake 3-fold only after 48 h incubation. IGF-I receptor β-subunit, insulin receptor substrate-1 (IRS-1), and mitogen-activated protein (MAP) kinase were all phosphorylated in response to 6.5 nM IGF-I after 10 min incubation. In contrast, the treatment with 23 nM TNF-α failed to phosphorylate either IGF-I receptor β-subunit or IRS-1 but did phosphorylate MAP kinase as much as IGF-I did. Despite a similar extent to which TNF-α induced MAP kinase phosphorylation as IGF-I did, TNF-α stimulated glucose uptake less compared to IGF-I. The results indicate that MAP kinase phosphorylation is not sufficient for glucose uptake in L6 myoblasts. TNF-α-elicited signal transduction to glucose uptake may utilize a different pathway from that seen with IGF-I.

Increased insulin responsiveness after CS-045 treatment in diabetes associated with Werner's syndrome

Werners syndrome is a rare inheritated disorder characterized by accelerated aging and is often accompanied by diabetes mellitus or impaired glucose tolerance. Previous reports suggest that insulin resistance is involved in the development of diabetes associated with Werners syndrome. In the present study, CS-045((+/-)-5-[4-(6-Hydroxy-2,5,7,8-tetramethylchroman-2-ylmet hoxy)benzyl] - 2,4-thiazolidinedione, a new oral hypoglycemic agent which reportedly reduces insulin resistance, was administered to 2 Werners syndrome patients. The patients were hospitalized for the duration of the study. During a pretreatment period lasting 8 weeks the patients received a controlled diet, however, their previous treatment was unchanged. Throughout the 4-week treatment period, each subjects blood glucose level was measured 7 times each day (07:30, 10:00, 11:30, 14:00, 17:30, 20:00, 22:00) for 1 week at 8, 4, and 1 week before treatment and at 2 and 4 weeks after treatment. To assess insulin action, the euglycemic glucose clamp technique was performed in these subjects at insulin infusion rates of 20, 120 and 400 mU/kg/min before and after 4 weeks of treatment. After 4 weeks of treatment with CS-045, the mean blood glucose level at each time point measured in this study was markedly lower compared to the corresponding pretreatment level.(ABSTRACT TRUNCATED AT 250 WORDS)

Epitope Analysis of GAD65 Autoantibodies in Japanese Patients with Autoimmune Diabetes

Abstract: Type 1 diabetes is an organ‐specific autoimmune disease characterized by T cell‐mediated destruction of pancreatic β 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.