Masako Ozaki

Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes.

Aims/hypothesisWe analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein.MethodsAutoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268–369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype.ResultsSeventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed.Conclusions/interpretationThese results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

CTLA4 gene polymorphism correlates with the mode of onset and presence of ICA512 Ab in Japanese Type 1 diabetes

Recently, the association of CTLA4 gene polymorphism with type 1 diabetes and AITD has been reported in several populations. CTLA4 was originally reported to regulate T-cell activity and T-B cognate interaction. To investigate the role of CTLA4 in autoimmune diseases, we examined the correlation between CTLA4 gene polymorphism and the clinical characteristics of Japanese patients with type 1 diabetes, including the mode of onset of diabetes and presence of islet-specific autoantibodies (GAD, ICA 512 Ab) in the serum. We studied 111 patients with type 1 diabetes and 445 normal subjects. CTLA4 exon 1 position 49 (A/G: codon 17: Thr/Ala) polymorphism was defined, employing PCR-RFLP. Sixty-three (57%) patients had AITD. The allele frequencies of G and A in both 111 patients (G: 65%; A: 35%) and 63 patients (G: 62%; A: 38%) were not significantly different from the control subjects (G: 63%; A: 37%). Serum samples of 69 patients were obtained within a year after onset and used for pancreas specific autoantibodies analysis. These samples were also used for further analysis between CTLA4 gene polymorphism and clinical characteristics. The allele frequencies of G and A in patients who presented with diabetic ketoacidosis (DK+) (G: 75%; A: 25%) were significantly different from those in DK- patients (G: 50%, A: 50%, P = 0.003). Allele and genotype analyses showed significant differences between DK+ patients and control subjects (P = 0.014, P = 0.046, respectively). Allele frequencies of G and A were not significant between patients who were positive and negative for GAD Ab, but significant for ICA 512 Ab (G: 83%, A:17% versus G: 59%, A: 41%: positive patients versus negative patients, P = 0.004). Our results showed a significant correlation between CTLA4 gene polymorphism and ICA 512 Ab. Our results also indicated that CTLA4 gene polymorphism is associated with the onset mode of Japanese type 1 diabetes and the presence of ICA512 Ab. Further analysis of this polymorphism is necessary to fully understand the pathogenesis and progression of type 1 diabetes.

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.

No deterioration in insulin sensitivity, but impairment of both pancreatic β-cell function and glucose sensitivity, in Japanese women with former gestational diabetes mellitus

To identify the primary pathogenic factors involved in the development of Type 2 diabetes mellitus (DM), we studied Japanese women with former gestational diabetes mellitus (GDM) who are at risk for the later development of Type 2 DM. We used the minimal model analysis derived from frequently sampled intravenous glucose tolerance test (FSIGT). The subjects consisted of eight non‐obese women with a history of GDM and eight non‐obese normal women as control subjects. The 75 g oral glucose tolerance test (75 g OGTT) performed within 6 months of delivery confirmed that all the subjects with former GDM had a normal glucose tolerance. Insulin sensitivity (SI) derived from the minimal model analysis was not different between the two groups. Glucose effectiveness at zero insulin (GEZI), reflecting tissue glucose sensitivity, was significantly lower in former GDM patients than in control subjects (1.18 ± 0.34 vs 2.26 ± 0.29 × 10−2 min−1, p < 0.05). The early phase insulin secretion found in FSIGT was markedly reduced to 56 % of that observed in control subjects (1250 ± 187.4 vs 2223 ± 304.3 pmol l−1 min, p < 0.01). Our results indicate that in former GDM patients, who are Japanese and non‐obese, impairment of the acute insulin response to glucose and a decrease in tissue glucose sensitivity rather than insulin sensitivity are the primary pathogenic factors involved. Copyright

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

Abstract: 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 ≤18 years, n= 60) was significantly higher than that in patients with adult‐onset diabetes (68.3 vs. 36.4%, P < 0.002). Ninety‐two percent of type 1 diabetic patients positive for IA‐2ic autoantibodies recognized the PTP domain of IA‐2, whereas 8% reacted with the JM region only. Among 60 patients with childhood‐onset type 1 diabetes, 2% recognized the JM region only, 48% bound the PTP domain of IA‐2 only, and 18% recognized both JM and PTP epitopes. Among 33 patients with adult‐onset diabetes, 9% recognized the IA‐2JM only, 18% bound the IA‐2PTP only, and 9% recognized both the IA‐2JM and the IA‐2PTP. IA‐2PTP autoantibodies were prevalent in patients with childhood‐onset type 1 diabetes. By contrast, the proportion of patients with the IA‐2JM autoantibody only in type 1 diabetes who were positive for IA‐2ic autoantibodies was significantly higher in adult‐onset than in childhood‐onset diabetes (P < 0.05). These results demonstrate that autoantibody recognition of the IA‐2 epitope is distinct in childhood‐onset and adult‐onset type 1 diabetes.

Humoral immune response to islet autoantigens in Japanese patients with type 1 diabetes.

In this study, we evaluated autoantibodies to IA‐2 (IA‐2As), glutamic acid decarboxylase 65 (GADAs), and islet cell antibodies (ICAs) in 233 patients with type 1 diabetes (M:F = 90:143, mean duration 4.0 ± 6.7 yr) as a cross‐sectional study. Of 233 patients with type 1 diabetes, IA‐2A was detected in 58% of patients with duration within 2 weeks, 61% of patients with duration <1 yr, 41% of patients with diabetes for 1–3 yr, 29% for 4–9 yr, and 21% for ≥10 yr. These prevalences were similar to those of ICA, while the prevalence of GADA was not influenced by duration of diabetes with positivity of 63–74%. Thus, as the duration of diabetes became longer, the frequency of GADA+/IA‐2A− patients increased and the frequency of GADA+/IA‐2A+ patients decreased. However, the frequency of GADA−/IA‐2A+ patients was not influenced by duration of diabetes. The prevalence of IA‐2A was significantly higher in abrupt‐onset group (68%, n= 79) compared to the slowly progressive group (23%, n= 22) in new‐onset patients (P= 0.0001). However, there was no difference in the IA‐2A frequency between these two groups (abrupt‐onset 26%, n= 53 vs. slowly progressive 24%, n= 21) in patients with long‐standing disease, suggesting that IA‐2A positivity might persist in patients with slowly progressive type 1 diabetes. These results emphasize the heterogeneity of humoral autoimmunity to protein tyrosine phosphatase–like molecules, but not to GAD, in patients with type 1 diabetes.

HEPATOCYTE NUCLEAR FACTOR-1α INHIBITS INSULIN PROMOTER FACTOR 1-DEPENDENT TRANSACTIVATION OF THE HUMAN INSULIN GENE

To investigate the regulational interaction of hepatocyte nuclear factor-1α (HNF-1α) and insulin promoter factor 1 (IPF1) on insulin gene expression, either or both of the expression vectors carrying each transcription factor were transiently transfected into HeLa cells, RINm5F cells and MIN6 cells together with the luciferase reporter construct driven by a human preproinsulin gene promoter (−1998 to +237) designated as pINS-1998/luc. IPF1-transfection into HeLa cells strongly stimulated the luciferase activity to 725 fold that of the basal level. In contrast, HNF-1α-transfection resulted in only a 6.7 fold increase. In co-transfection experiments, increasing the amount of HNF-1α resulted in an 84.5% and 74.4% decrease in IPF1-stimulated luciferase activity in HeLa and RINm5F cells, respectively. Deletion constructs designated as pINS-248/luc, pINS-213/luc and pINS-185/luc were transfected into RINm5F cells to determine the role of the A3 element and its 5′ flanking sequence in the inhibitory effect of HNF-1α. The results showed that the inhibiting effects of HNF-1α with pINS-213/luc and pINS-185/luc were significantly smaller than those with both pINS-1998/luc and pINS-248/luc. Transfection into MIN6 cells with pINS-1998/luc in the absence of IPF1 resulted in constitutional transactivation of the insulin gene, and this transactivation was abolished by the co-transfection with HNF-1α. The present data indicate that IPF1 rather than HNF-1α predominantly transactivates the insulin gene, and that HNF-1α inhibits IPF1-dependent insulin gene transactivation mediated through the 5′ flanking sequence of the A3 element. It is suggested that HNF-1α may be involved in insulin gene expression as a negative regulator.

Decrease in the insulin receptor protein level by anti-insulin receptor antibodies: roles of tyrosine kinase activity and receptor internalization

Abstract. To investigate the mechanism of severe impairment of insulin action in type B insulin resistance, we extracted IgG from the serum of a patient with type B insulin resistance (B-IgG) and analyzed the inhibiting effect of B-IgG not only on insulin signaling but also on IGF-I signaling in Chinese hamster ovary (CHO) cells expressing human insulin receptor or human IGF-I receptor. Preincubation with 1 mg/ml B-IgG prevented insulin-induced phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) but did not alter the IGF-I-induced phosphorylation of the IGF-I receptor and IRS-1. 125I-insulin binding was inhibited by 93% after preincubation with BigG at 37° C and was recovered up to 50% of the control value by acid washing. However, when cells were preincubated with B-IgG at 4° C, the insulin binding completely recovered the control value by acid washing. 125I-IGF-I binding was not altered by B-IgG preincubation. Immunoblot study revealed that the protein level of the insulin receptor was strongly decreased by preincubation with 1 mg/ml B-IgG at 37° C, but never at 4° C. The IRS-1 protein level did not change by B-IgG preincubation. In order to know the role of the insulin receptor internalization in the inhibiting effect of B-IgG, we employed CHO cells expressing mutant insulin receptors which do not undergo internalization (CHO-K1018R). B-IgG incubation of CHOK1018R at 37° C failed to decrease the protein level of the insulin receptor. The present data indicate that IgG from the diabetic patient with type B insulin resistance decreased insulin receptor protein level, probably due to the enhanced degradation rate of the insulin receptor, in which insulin receptor tyrosine kinase activity and internalization are required for this process. This effect of B-IgG was specific for the insulin receptor with no effect on either IGF-I receptor or IRS-1, as reflected by the IGF-I effectiveness on glycemic control in this patient.