Hiromasa Ikegami

Association of Trp64Arg mutation of the β3-adrenergic-receptor with NIDDM and body weight gain

SummaryA possible pathogenic mutation in the Β3-adrenergic-receptor gene (Trp64Arg) has been reported to be associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and clinical features of the insulin resistance syndrome in Pima Indian, Finnish and French subjects. Since marked heterogeneity has been reported in the association of mutations of candidate genes with NIDDM between Japanese and other ethnic groups, we investigated the association of Trp64Arg with NIDDM in Japanese subjects. The allele frequency of the mutation (Arg) was slightly, but not significantly, higher in NIDDM than in control subjects (70 out of 342 alleles [20.5%] vs 40 out of 248 [16.1%], respectively, p>0.2). When our data were combined with those of Pima Indian and Finnish subjects, however, the Arg/Arg genotype was significantly associated with NIDDM as compared with the other two genotypes (p<0.005, relative risk [RR] 2.13, 95% confidence interval [CI] 1.28–3.55). The Arg allele was also associated with NIDDM (p<0.05, RR 1.27, 95% CI 1.06–1.52). Japanese subjects homozygous for the mutation had a significantly higher body mass index (mean ± SD∶25.5±3.9 kg/ m2) than heterozygotes (22.6±4.1, p<0.05) and normal homozygotes (22.8±3.8, p<0.05). NIDDM patients homozygous for the mutation tended to have an earlier age of onset of NIDDM than those with other genotypes. These data suggest that the Trp64Arg mutation not only contributes to weight gain and age-at-onset of NIDDM but is also associated with susceptibility to NIDDM.

The NSY mouse: a new animal model of spontaneous NIDDM with moderate obesity

SummaryThe NSY (Nagoya-Shibata-Yasuda) mouse was established as an inbred strain of mouse with spontaneous development of diabetes mellitus, by selective breeding for glucose intolerance from outbred Jcl∶ICR mice. NSY mice spontaneously develop diabetes mellitus in an age-dependent manner. The cumulative incidence of diabetes is 98% in males and 31% in females at 48 weeks of age. Neither severe obesity nor extreme hyperinsulinaemia is observed at any age in these mice. Glucose-stimulated insulin secretion was markedly impaired in NSY mice after 24 weeks of age. In contrast, fasting plasma insulin level was higher in male NSY mice than that in male C3H/He mice (545±73 vs 350±40 pmol/l, p<0.05, at 36 weeks of age). Pancreatic insulin content was higher in male NSY mice than that in male C3H/He mice (76±8 vs 52±5 ng/mg wet weight, p<0.05, at 36 weeks of age). Morphologically, no abnormal findings, such as hypertrophy or inflammatory changes in the pancreatic islets, were observed in NSY mice at any age. These data suggest that functional changes of insulin secretion in response to glucose from pancreatic beta cells may contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM) in the NSY mouse. Although insulin sensitivity was not measured, fasting hyperinsulinaemia in NSY mice suggests that insulin resistance may also contribute to the pathogenesis of NIDDM. Since these findings are similar to the pathophysiologic features of human NIDDM patients, the NSY mouse is considered to be useful for investigating the pathogenesis and genetic predisposition to NIDDM.

Role of Mitogen-Activated Protein Kinase Pathway in Prostaglandin F2α-Induced Rat Puerperal Uterine Contraction

In this study, prostaglandin (PG) F2α was found to activate mitogen-activated protein (MAP) kinase and MAP kinase kinase (MEK) in cultured rat puerperal uterine myometrial cells. PGF2α stimulation also led to an increase in phosphorylation of raf-1, son of sevenless (SOS), and Shc. Furthermore, we examined the mechanism by which PGF2α induced MAP kinase phosphorylation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the β-adrenergic receptor kinase 1 (βARK1), which specifically blocks signaling mediated by the βγ subunits of G proteins, blocked the PGF2α-induced activation of MAP kinase. Ritodrine (1 μm), which is known to relax uterine muscle contraction, attenuated PGF2α-induced tyrosine phosphorylation of MAP kinase. Moreover, to examine the role of MAP kinase pathway in uterine contraction, an inhibitor of MEK activity, PD098059, was used. Although MEK inhibitor had no effect on PGF2α-induced calcium mobilization, th...

A novel microsatellite polymorphism in the human OB gene : a highly polymorphic marker for linkage analysis

Summary The mouse ob gene and its human homologue OB have recently been cloned. The mutations in the ob gene are known to be associated with extreme obesity. The relationship between the human OB gene and disease, however, is largely unknown due to the lack of suitable markers within or adjacent to the OB gene. To obtain informative markers, we searched for simple tandem repeat polymorphisms in the genomic sequence of the human OB gene and identified a novel tetranucleotide repeat in the 3′ flanking region. Fifteen alleles were detected in this marker with a heterozygosity of 0.85 and polymorphism information content of 0.83, indicating a highly informative nature of this marker. Two-point linkage mapping in two Centre Etude Polymorphisme Humaine (CEPH) reference families suggested that this marker is located in the interval between D7S514 and D7S530, the same interval where the OB gene is located (recombination fractions with D7S514 and D7S530 were 0.026 and 0.034, respectively). Although allele frequency distributions of this marker did not differ between 84 control subjects and 69 NIDDM patients, there was a tendency to higher body weight in control subjects with class I/class I genotype than in those without this genotype (68.8 ± 11.1 vs 60.8 ± 10.3 kg, p = 0.05). The highly polymorphic nature of this marker and its location in the OB gene makes this marker useful for linkage studies of the OB gene with a number of phenotypes, such as obesity, non-insulin-dependent diabetes mellitus, hypertension and the insulin resistance syndrome. [Diabetologia (1996) 36: 1398–1401]

A mutation in the glucagon receptor gene (Gly40Ser) : heterogeneity in the association with diabetes mellitus

SummaryA possible pathogenic mutation in the glucagon receptor gene causing a Gly to Ser change at codon 40 (Gly40Ser) was reported to be associated and linked with non-insulin-dependent diabetes mellitus (NIDDM), in France and Sardinia, Since the frequency of the mutation (Gly40Ser), about 5% in the French population of familial NIDDM and 8% in randomly chosen diabetic patients in Sardinia, was much higher than that of any of the previously reported mutations in candidate genes, it is important to clarify whether the contribution of this mutation to NIDDM is universal. In this study, we investigated the association of this mutation with diabetes mellitus in a large number of Japanese diabetic patients (383 NIDDM and 53 insulin-dependent diabetic patients) by polymerase chain reaction-restriction fragment length polymorphism analysis. None of the Japanese diabetic patients showed Gly40Ser mutation and the association of this mutation with NIDDM was significantly different (p<4·10−5 vs French, p<3·10−6 vs Sardinian by Fishers exact test). The results not only indicate that the mutation plays little, if any, role in susceptibility to diabetes in Japan, but also indicate the genetic heterogeneity in NIDDM and further emphasize the importance of studies on genetic susceptibility to NIDDM and other complex traits in different ethnic groups.

Class I HLA is associated with age-at-onset of IDDM, while class II HLA confers susceptibility to IDDM.

Dear Sir, We thank Dr. Torffvit for his query regarding the efficacy of different forms of insulin in the rat. Our own study has not specifically addressed this issue. However, over the last 10 years, we have used different doses of both bovine and human ultra~ lente insulins in rats with both shortand long-term experimental diabetes. We were unable to locate a direct comparison of bovine and human insulin in rats although this issue has been addressed in a range of human studies. These studies have suggested that human ultralente insulin, although similar in potency to bovine insulin, has a shorter duration of action in man [1, 2]. In our previous animal studies, we used heat-treated bovine Ultralente insulin and as noted by Dr. Torffvit, we achieved blood glucose levels of less than 22 mmol/1 with a dose of 2 IU/day [3]. However, as indicated previously, there was a heterogeneous response among the diabetic rats. In our more recent studies, using human ultralente insulin, the dose of 2 IU/ day was not associated with blood glucose levels greater than 20 mmol/1 [4]. However, the possibility that human insulin is ineffective in rats is unlikely since in a recent study using human insulin administered via silastic implants, we were able to achieve euglycaemia [5]. Another factor which needs to be considered relates to the severity of the diabetes that is induced by streptozotocin. This relates not only to the susceptibility of the individual rat strain to streptozotocin but also to the dose and mode of administration of streptozotocin [6]. In our studies, streptozotocin was given at a dose of about 45-60 mg/kg whereas Dr. Torffvit gave a

Association of plasma fibrinogen level and blood pressure with diabetic retinopathy, and renal complications associated with proliferative diabetic retinopathy, in Type 2 diabetes mellitus

Aim To clarify the association of several clinical parameters, including plasma fibrinogen level, with diabetic retinopathy in patients with Type 2 diabetes mellitus (DM).

Interleukin-6 stimulates cell proliferation of rat pituitary clonal cell lines in vitro

We investigated the effect of recombinant human IL-6 (rhlL-6) on cell proliferation using the MtT/E rat pituitary tumor cell line, which was recently established by Inoue et al. This cell line expresses the homeodomain protein Pit-1/GHF 1 and does not produce any significant amount of pituitary hormones, but retains its tumorigenicity by back-transplantation into rats, resulting in production of prolactin. MtT/E cells were seeded into Falcon 24-well plates at a density of 2×104 cells/well in a cultured medium, containing 10% horse serum and 2.5% fetal bovine, with test drug. After four-days (12 days for the time-course study) incubations, the cells were counted using a hemocytometer. Incubation for 4 days with rhlL-6 caused concentration-dependent stimulation of MtT/E cell growth and [3H]-thymidine incorporation into MtT/E cells. Addition of 20 ng/ml rhlL-6 to the culture medium stimulated MtT/E cell growth in a time-dependent manner, withdrawal of rhlL-6 from the culture medium reduced MtT/E cell growth, and re-addition of rhlL-6 to the culture medium again stimulated MtT/E cell growth. Among the cytokines tested, granulocyte colony-stimulating factor (rh G-CSF) also showed a slight but significant mitogenic activity on the MtT/E cells. Analysis of 125|-rhlL-6 binding to the MtT/E cells indicated a dissociation constant of 0.953×10−9 mmol/l and the presence of 968 binding sites per cell. These results confirm the previous finding that IL-6 exerts mitogenic activity on pituitary tumor cells and also support the hypothsis that IL-6 may be involved in the control of cell growth and proliferation in the pituitary.

Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.

Allelic variation in class I K gene as candidate for a second component of MHC-linked susceptibility to Type 1 diabetes in non-obese diabetic mice

Aims/hypothesisRecent studies have revealed that MHC-linked susceptibility to Type 1 diabetes is determined by multiple components. In the non-obese diabetic (NOD) mouse, a second component (Idd16) has been mapped to a region adjacent to, but distinct from Idd1 in the class II region. In this study, we investigated the class I K gene as a candidate gene for Idd16.MethodsWe determined the genomic sequences of the class I K gene as well as the reactivity of K molecules with monoclonal antibodies in the NOD mouse, the Cataract Shionogi (CTS) mouse, and the NOD.CTS-H-2 congenic strain, which possesses a resistance allele to Type 1 diabetes at the Idd16 on the NOD genetic background genes.ResultsWhile the K sequence of the NOD mouse was identical to that of Kd type, ten nucleotide substitutions were identified in the CTS mouse compared with the NOD mouse. Of these, three were in exon 4, giving two amino acid substitutions, which were identical to those seen in KK type. These characteristics were retained in the NOD.CTS-H-2 congenic strain, which had a lower incidence and delayed onset of Type 1 diabetes owing to a resistance allele at Idd16. Lymphocytes from NOD.CTS-H2 congenic mice reacted with anti-Kd and anti-Kk monoclonal antibodies, reflecting the unique sequence of the K gene. The nucleotide sequence of the K gene in the non-obese non-diabetic (NON) mouse was also unique, consisting of a combination of Kk- and Kb-like sequences.Conclusions/interpretationThese data suggest that H2-K is unique in CTS and NON mice, and that allelic variation of the class I K gene may be responsible for Idd16.