Abstract Aims/Introduction This study aimed to identify risk factors that contribute to the progression of slowly‐progressive type 1 diabetes by evaluating the positive predictive value (PPV) of factors associated with the progression to an insulin‐dependent state. Materials and Methods We selected 60 slowly‐progressive type 1 diabetes patients who tested positive for glutamic acid decarboxylase autoantibodies (GADA) at diagnosis from the Japanese Type 1 Diabetes Database Study. GADA levels in these patients were concurrently measured using both radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) techniques. Results Compared with the non‐progressor group (fasting C‐peptide [F‐CPR] levels maintained ≥0.6 ng/mL), the progressor group showed a younger age at diagnosis, lower body mass index (BMI), lower F‐CPR levels and a higher prevalence of insulinoma‐associated antigen‐2 autoantibodies (IA‐2A). The PPV of RIA‐GADA increased from 56.3 to 70.0% in the high titer group (≥10 U/mL), and further increased to 76.9, 84.2, 81.0 and 75.0% when combined with specific thresholds for age at diagnosis <47 years, BMI <22.6 kg/m², F‐CPR <1.41 ng/mL and IA‐2A positivity, respectively. In contrast, the PPV of ELISA‐GADA (71.8%) remained the same at 73.1% in the high titer group (≥180 U/mL), but increased to 81.8, 82.4 and 79.0% when evaluated in conjunction with age at diagnosis, BMI and F‐CPR level, respectively. Conclusions Our findings show that, unlike RIA‐GADA, ELISA‐GADA shows no association between GADA titers and the risk of progression to an insulin‐dependent state. The PPV improves when age at diagnosis, BMI and F‐CPR levels are considered in combination.

Abstract Context Glucose tolerance worsens after distal pancreatectomy (DP); however, the long-term incidence and factors affecting interindividual variation in this worsening are unclear. Objective To investigate the changes in diabetes-related traits before and after DP and to clarify the incidence of diabetes and its predictors. Methods Among 493 registered patients, 117 underwent DP. Among these, 56 patients without diabetes before surgery were included in the study. Glucose and endocrine function were prospectively assessed using a 75-g oral glucose tolerance test preoperatively, 1 month after DP, and every 6 months thereafter for up to 36 months. Pancreatic volumetry was performed using multidetector row computed tomography before and after surgery. Results Insulin secretion decreased and blood glucose levels worsened after DP. Residual pancreatic volume was significantly associated with the reserve capacity of insulin secretion but not with blood glucose levels or the development of diabetes. Among 56 patients, 33 developed diabetes mellitus. The cumulative incidence of diabetes at 36 months after DP was 74.1%. Multivariate Cox regression analysis showed that impaired glucose tolerance as a preoperative factor as well as a decreased insulinogenic index and impaired glucose tolerance at 1 month postoperatively were identified as risk factors for diabetes following DP. Conclusion Impaired glucose tolerance and reduced early-phase insulin response to glucose are involved in the development of new-onset diabetes after DP; the latter is an additional factor in the development of diabetes and becomes apparent when pancreatic beta cell mass is reduced after DP.

Abstract Continuous glucose monitoring (CGM) values obtained from CGM systems using the same sensor but with different internal algorithms (the first- and third-generation FreeStyle Libre (1st-gen-libre and 3rd-gen-libre, respectively)) were compared. We used 19,819 paired and simultaneously measured CGM values of 13 patients with diabetes. The average CGM value was significantly higher (P < 0.0001) and the time below range (CGM value < 70 mg/dL) was significantly lower (P < 0.0001) with the 3rd-gen-libre than with the 1st-gen-libre. There was a significant correlation (P < 0.0001) between the CGM values of the 3rd-gen-libre (y-axis, mg/dL) and 1st-gen-libre (x-axis, mg/dL) using the following formula: y = 0.9728x + 10.024. On assessing the association between glycated hemoglobin (HbA1c (%), y-axis) and the average CGM values (x-axis, mg/dL) by applying the obtained equation to previously reported 1st-gen-libre data and converting it to 3rd-gen-libre data, we obtained the equation y = 0.02628x + 3.233, indicating that the glucose management indicator reported in the West may be underestimated compared with the laboratory-measured HbA1c in the Japanese population. Glucose values from the same sensor were found to be significantly different between readers with different algorithms, and the calculation of CGM-related indices may need to be individualized for each device.

Abstract The diagnostic criteria for slowly progressive type 1 diabetes (slowly progressive insulin‐dependent diabetes mellitus; SPIDDM) have been revised by the Committee on Type 1 Diabetes of the Japan Diabetes Society. All of the following three criteria must be met for ‘a definitive diagnosis of SPIDDM’: (1) presence of anti‐islet autoantibodies at some point in time during the disease course; (2) absence of ketosis or ketoacidosis at the diagnosis of diabetes with no requirement for insulin treatment to correct hyperglycemia immediately after diagnosis in principle; and (3) gradual decrease of insulin secretion over time, with insulin treatment required at more than 3 months after diagnosis, and the presence of severe endogenous insulin deficiency (fasting serum C‐peptide immunoreactivity <0.6 ng/mL) at the last observed point in time. When a patient fulfills only (1) and (2), but not (3), he/she is diagnosed with ‘SPIDDM (probable)’ because the diabetes is non‐insulin‐dependent type.

Abstract We previously reported that four hyperglycemia loci are located on three chromosomes in the Nagoya-Shibata-Yasuda (NSY) mouse model, commonly used to study type 2 diabetes. However, we did not search for hyperglycemia loci across all chromosomes. In this study, we performed quantitative trait loci (QTLs) mapping of longitudinal phenotypes from crosses between NSY (hyperglycemic) and C3H (normoglycemic) mice. We identified four new QTLs for hyperglycemia, namely Nidd5nsy, Nidd6nsy, Nidd1c3h, and Nidd2c3h, on Chromosome 1, 4, 10, and 13, respectively. These QTLs were associated with hyperglycemia in young mice and had attenuated effects in older mice. Nidd5nsy and Nidd6nsy were hyperglycemic with NSY alleles, and Nidd1c3h and Nidd2c3h were hyperglycemic with C3H alleles. We further bred Nidd5nsy congenic mice and demonstrated that Nidd5nsy has a strong effect on hyperglycemia when young, accompanied by insulin resistance and visceral fat accumulation. These results showed that the effects of individual QTLs strengthened or weakened with age, and that the sum of the effects of QTLs captured the age-related deterioration of glucose tolerance in individuals. Our results support the importance of longitudinal phenotypes in the genetic analysis of polygenic traits and have implications for the genetic basis and pathogenesis of type 2 diabetes in humans.

Abstract Context The glucose tolerance of patients changes considerably from before to after pancreaticoduodenectomy wherein approximately half of the pancreas is resected. Objective The aim of this prospective study was to investigate the incidence of and risk factors for diabetes after pancreaticoduodenectomy. Methods This study is a part of an ongoing prospective study, the Kindai Prospective Study on Metabolism and Endocrinology after Pancreatectomy (KIP-MEP) study. Of the 457 patients enrolled to date, 96 patients without diabetes who underwent pancreaticoduodenectomy were investigated in this study. Preoperatively, 1 month post-pancreaticoduodenectomy, and every 6 months thereafter, the glucose metabolism and endocrine function were evaluated using the 75 g oral glucose tolerance test. Various other metabolic, endocrine, and exocrine indices were also examined over a period of up to 36 months. Results Of the 96 patients analyzed in this study, 33 were newly diagnosed with diabetes. The cumulative diabetes incidence at 36 months following pancreaticoduodenectomy was 53.8%. The preoperative insulinogenic index and ΔC-peptide in the glucagon stimulation test were significantly lower in the progressors to diabetes than in the nonprogressors. Multivariate Cox regression analysis demonstrated that the insulinogenic index was the only significant risk factor for new-onset diabetes. Conclusion The majority of patients developed new-onset diabetes after pancreaticoduodenectomy, and a low value of the insulinogenic index was suggested to be a risk factor for diabetes. Preoperative assessment for the prediction of the onset of diabetes serves as useful information for patients and is important for postoperative glycemic control and diabetes management in patients who require pancreaticoduodenectomy.

The targets for continuous glucose monitoring (CGM)-derived metrics were recently set; however, studies on CGM data over a long period with stable glycemic control are limited. We analyzed 194,279 CGM values obtained from 19 adult Japanese patients with type 1 diabetes. CGM data obtained during stable glycemic control over four months were analyzed. CGM-related metrics of different durations “within 120, 90, 60, 30, and 7 days” were calculated from baseline. Time in range (TIR; glucose 70–180 mg/dL), time above range (TAR; glucose ≥ 181 mg/dL), and average glucose levels, but not time below range (TBR; glucose ≤ 69 mg/dL), strongly correlated with glycated hemoglobin (HbA1c) values (P < 0.0001). TBR correlated with glucose coefficient of variation (CV) (P < 0.01). Fasting serum C-peptide levels negatively correlated with glucose CV (P < 0.01). HbA1c of approximately 7% corresponded to TIR of 74% and TAR of 20%. The shorter the CGM period, the weaker was the relationship between HbA1c and CGM-related metrics. TIR, TAR, and average glucose levels accurately reflected HbA1c values in Japanese patients with type 1 diabetes with stable glycemic control. Glucose CV and TBR complemented the limitation of HbA1c to detect glucose variability and hypoglycemia. Stable glycemic control with minimal hypoglycemia depended on residual β-cell function.

Abstract Adrenocortical carcinoma (ACC) is a rare tumor, and some histological variants (oncocytic, myxoid, and sarcomatoid ACCs) have been reported in addition to the conventional ACC. Among these subtypes, oncocytic ACC is histologically characterized by the presence of abundant eosinophilic granular cytoplasm in the carcinoma cells owing to the accumulation of mitochondria, which generally yields high 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET). Herein, we report the case of a 21-year-old woman with oncocytic ACC with low FDG uptake on PET scan. Her circulating levels of androgens were high, and androgen-synthesis enzymes were detected in carcinoma cells. The patient also had hypocholesterolemia. However, glucose transporter 1 (GLUT1) was not detected in the tumor, which was considered to account for the low FDG uptake by the tumor. To the best of our knowledge, this is the first case of low FDG uptake by oncocytic ACC without GLUT1 expression. Additionally, since hypocholesterolemia was reported in 3 previous reports of androgen-producing tumors, a possible correlation between androgenicity in adrenal tumors and the development of hypocholesterolemia could be postulated; however, further investigations are needed for clarification. This case highlights important information regarding the diversity of ACC and its impact on hypocholesterolemia.

Mixed corticomedullary tumors (MCMTs) are rare and comprise medullary and cortical cells in a single adrenal tumor. The mechanisms underlying their development have not been fully elucidated. Here, we report a case of MCMT in a 42-year-old woman. Based on the preoperative clinical findings, the patient was diagnosed as having a pheochromocytoma with subclinical Cushing syndrome. Postoperative pathological diagnosis revealed that the tumor demonstrated morphologically distinct medullary and cortical components, which produced catecholamines and cortisol, respectively. Hybrid tumor cells producing both catecholamines and cortisol were not detected. Adrenocorticotropin (ACTH)-positive tumor cells were identified to be present in the pheochromocytoma. This ectopic production of ACTH can contribute to an autonomous cortisol production in a paracrine manner. In addition, micronodules producing aldosterone were detected in the adrenal tissue adjacent to the tumor. The simultaneous development of these 2 lesions may not be correlated with each other; however, this case confirms the importance of a detailed histopathological examination of the adrenal lesions harboring complicated hormonal abnormalities by providing pivotal and indispensable information on their pathogenesis and the possible interaction of the hormones produced in the adrenal gland.

The rate of glucose metabolism changes drastically after partial pancreatectomy. This work aims to analyze changes in patients’ glucose metabolism and endocrine and exocrine function before and after partial pancreatectomy relative to different resection types (Kindai Prospective Study on Metabolism and Endocrinology after Pancreatectomy: KIP-MEP study). A series of 278 consecutive patients with scheduled pancreatectomy were enrolled into our prospective study. Of them, 109 individuals without diabetes, who underwent partial pancreatectomy, were investigated. Data were compared between patients with pancreaticoduodenectomy (PD, n = 73) and those with distal pancreatectomy (DP, n = 36). Blood glucose levels during the 75-g oral glucose tolerance test (75gOGTT) significantly decreased after pancreatectomy in the PD group (area under the curve [AUC] –9.3%, P &lt; .01), and significantly increased in the DP population (AUC + 16.8%, P &lt; .01). Insulin secretion rate during the 75gOGTT and glucagon stimulation test significantly decreased after pancreatectomy both in the PD and DP groups (P &lt; .001). Both groups showed similar homeostasis model assessment of insulin resistance (HOMA-IR) values after pancreatectomy. Decrease in exocrine function quality after pancreatectomy was more marked in association with PD than DP (P &lt; .01). Multiple regression analysis indicated that resection type and preoperative HOMA-IR independently influenced glucose tolerance-related postoperative outcomes. Blood glucose levels after the OGTT differed markedly between PD and DP populations. The observed differences between PD and DP suggest the importance of individualization in the management of metabolism and nutrition after partial pancreatectomy.

Characterization of adrenocortical disorders is challenging because of varying origins, laterality, the presence or absence of hormone production, and unclarity about the benign or malignant nature of the lesion. Histopathological examination in conjunction with immunohistochemistry is generally considered mandatory in this characterization. We report a rare case of bilateral adrenocortical adenomas associated with unilateral adrenal endothelial cysts in a 65-year-old woman whose condition was not diagnosed before surgery. Detailed histological examination of the resected adrenal glands revealed hyperplasia in the zona glomerulosa. Despite hyperplasia, the patient had normal serum aldosterone levels and renin activity without clinical evidence of hypertension. The patient was treated with a sodium-glucose cotransporter protein 2 (SGLT2) inhibitor. This may have stimulated the renin-angiotensin-aldosterone system. To the best of our knowledge, this is the first case in which both relatively large bilateral adrenocortical adenomas and unilateral adrenal endothelial cysts were detected. This case also highlights the complexity and difficulty of preoperative diagnosis. Furthermore, this case reports the first detailed histopathological examination of adrenal lesions with SGLT2 treatment and the possibility of SGLT2 inhibitor treatment resulting in histological hyperplasia in the zona glomerulosa; however, it is difficult to prove a causative relationship between SGLT2 inhibitors and hyperplasia of the zona glomerulosa based on the data of this case. It can be confirmed only under limited conditions; therefore, further studies on adrenal gland histology employing SGLT2 inhibition are warranted.

OBJECTIVE: Glomerular filtration rate (GFR) decreases without or prior to the development of albuminuria in many patients with diabetes. Therefore, albuminuria and/or a low GFR in patients with diabetes is referred to as diabetic kidney disease (DKD). A certain proportion of patients with diabetes show a rapid progressive decline in renal function in a unidirectional manner and are termed early decliners. This study aimed to elucidate the prevalence of DKD and early decliners and clarify their risk factors. RESEARCH DESIGN AND METHODS: This combination cross-sectional and cohort study included 2385 patients with diabetes from 15 hospitals. We defined DKD as a urinary albumin to creatinine ratio (ACR) ≥30 mg/gCr and/or estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m². We classified patients into four groups based on the presence or absence of albuminuria and a decrease in eGFR to reveal the risk factors for DKD. We also performed a trajectory analysis and specified the prevalence and risk factors of early decliners with sequential eGFR data of 1955 patients in five facilities. RESULTS: Of our cohort, 52% had DKD. Above all, 12% with a low eGFR but no albuminuria had no traditional risk factors, such as elevated glycated hemoglobin, elevated blood pressure, or diabetic retinopathy in contrast to patients with albuminuria but normal eGFR. Additionally, 14% of our patients were early decliners. Older age, higher basal eGFR, higher ACR, and higher systolic blood pressure were significantly associated with early decliners. CONCLUSIONS: The prevalence of DKD in this cohort was larger than ever reported. By testing eGFR yearly and identifying risk factors in the early phase of diabetes, we can identify patients at high risk of developing end-stage renal disease.

AIMS/INTRODUCTION: Patients with a total pancreatectomy and type 1 diabetes are similar in regard to absolute insulin deficiency, but different in regard to glucagon, providing a unique opportunity to study the contribution of glucagon to glucose metabolism in an insulin-dependent state. The aim of the present study was to investigate the contribution of glucagon to glucose homeostasis in complete insulin deficiency in vivo. METHODS: A total of 38 individuals with a complete lack of endogenous insulin (fasting C-peptide <0.0066 nmol/L) and whose glycemic control was optimized with an insulin pump during hospitalization were retrospectively studied. The basal insulin requirement, time-to-time adjustment of the basal insulin infusion rate, prandial insulin requirement and fasting plasma glucagon were compared between patients with a total pancreatectomy (n = 10) and those with type 1 diabetes (n = 28) after achievement of optimal glycemic control. RESULTS: Total daily insulin (P = 0.03) and basal insulin (P = 0.000006), but not prandial insulin requirements, were significantly lower in total pancreatectomy patients than in type 1 diabetes patients. The basal percentage (basal insulin/total daily insulin) was also significantly lower in total pancreatectomy patients than in type 1 diabetes patients (15.8 ± 7.8 vs 32.9 ± 10.1%, P = 0.00003). An increase in the insulin infusion rate early in the morning was not necessary in most patients with a pancreatectomy. The fasting plasma glucagon concentration was significantly lower in total pancreatectomy patients than in type 1 diabetes patients (P = 0.00007), and was positively correlated with the basal insulin requirement (P = 0.038). CONCLUSIONS: The difference in insulin requirements between total pancreatectomy and type 1 diabetes patients suggests a contribution of glucagon to the basal insulin requirement and dawn phenomenon.

Aims/Introduction: Differences in the efficacy and safety of antidiabetic drugs among different ethnic groups are well documented. Metformin is widely used in the treatment of type 2 diabetes in Western countries, but high doses of metformin have been approved only recently for clinical use in Japan. The aim of the present study was to investigate the effects of dosage and dosing frequency on the efficacy and safety of high-dose metformin in Japanese patients. Materials and Methods: A total of 71 Japanese patients with type 2 diabetes were prospectively studied for the effects of dosage and dosing frequency on the efficacy and safety of metformin during hospitalization. Dose effects were studied in 27 patients treated with 0, 500, 1,000, 1,500 and 2,250 mg/day of metformin. The effect of dosing frequency was compared in 56 patients with 1,500 mg/day of metformin administered either two or three times per day. Results: Significant dose-dependent improvement in daily profiles of blood glucose was observed with metformin dosages up to 1,500 mg/day, with a trend towards further improvement observed at 2,250 mg/day. The efficacy of 1,500 mg of metformin was comparable when the drug was administered either two or three times per day. The most frequently reported side-effects were gastrointestinal symptoms, which were not affected by the dosage or dosing frequency of metformin. Conclusions: These results show that the efficacy of high-dose metformin is dose-dependent in Japanese patients. The efficacy and safety of metformin were similar when the drug was administered either two or three times per day.

Background: We report a rare case of a juxta-adrenal schwannoma that could not be discriminated from an adrenal tumor before surgical resection and was complicated by bilateral hyperaldosteronism. To the best of our knowledge, this is first case in which both a juxta-adrenal schwannoma and hyperaldosteronism co-existed. Case presentation: A 69-year-old male treated for hypertension was found to have a left supra-renal mass (5.8 x 5.2 cm) by abdominal computed tomography. His laboratory data showed that his plasma aldosterone concentration (PAC) was within the normal range, but his plasma renin activity (PRA) was reduced, resulting in an increased aldosterone/renin ratio (ARR). Load tests of captopril or furosemide in the standing position demonstrated autonomous aldosterone secretion and renin suppression. Adrenal venous sampling (AVS) with ACTH stimulation indicated bilateral hypersecretion of aldosterone. A left supra-renal tumor was resected because of the possibility of malignancy and was found to be a benign schwannoma arising from the juxta-adrenal region together with an adrenal gland. The dissected left adrenal gland was morphologically hyperplastic in the zona glomerulosa, but was immunohistochemically negative for CYP11B2 (aldosterone synthase). Multiple CYP11B2positive adrenocortical micronodules were detected in the adrenal gland, indicating micronodular hyperplasia. Although bilateral aldosteronism was indicated by AVS before the operation, the PRA, PAC and ARR values were within their respective reference ranges after resection of the unilateral tumor, suggesting that the slight increase in hormone secretion from the remaining right-sided lesion could not be detected after resection. Conclusion: A clinical and morphologic diagnosis of juxta-adrenal schwannoma is difficult, particularly in a case of hyperaldosteronism, as shown in this case. These data suggest the complexity and difficulty diagnosing adrenal incidentaloma.

Programmed cell death-1 (PD-1) is a co-stimulatory molecule that inhibits T cell proliferation. We aimed to clarify PD-1 expression in CD4(+) T cells and the association between PD-1 expression and the 7785C/T polymorphism of PDCD1, with a focus on the two subtypes of type 1 diabetes, type 1A diabetes (T1AD) and fulminant type 1 diabetes (FT1D), in the Japanese population. We examined 22 patients with T1AD, 15 with FT1D, 19 with type 2 diabetes (T2D) and 29 healthy control (HC) subjects. Fluorescence-activated cell sorting (FACS) and real-time PCR were utilized to analyse PD-1 expression quantitatively. Genotyping of 7785C/T in PDCD1 was performed using the TaqMan method in a total of 63 subjects (21 with T1AD, 15 with FT1D and 27 HC). FACS revealed a significant reduction in PD-1 expression in CD4(+) T cells in patients with T1AD (mean: 42 vs. 60% in FT1D, P=00450; vs. 58% in T2D, P = 00098; vs. 60% in HC, P=00018). PD-1 mRNA expression in CD4(+) T cells was also significantly lower in patients with T1AD than in the HC subjects. Of the 63 subjects, PD-1 expression was significantly lower in individuals with the 7785C/C genotype than in those with the C/T and T/T genotypes (mean: 41 vs. 59%, P=00016). Our results indicate that lower PD-1 expression in CD4(+) T-cells might contribute to the development of T1AD through T cell activation.

Background: A susceptibility locus, Nidd2n, for type 2 diabetes has been mapped to mouse chromosome 14 (Chr 14) and confirmed using the consomic strain (C3H-Chr 14(NSY)) of the Nagoya-Shibata-Yasuda (NSY) mouse, an animal model of spontaneous type 2 diabetes. The aim of this study was to localize and characterize Nidd2n. Results: We constructed two novel congenic strains homozygous for different segments of NSY-Chr 14 on the control C3H/HeNcrj (C3H) background: R1 (C3H.NSY-(D14Mit206-D14Mit5)) possesses the proximal and middle segment, and R2 (C3H.NSY-(D14Mit206-D14Mit186)) possesses the most proximal segment of NSY-Chr 14. Diabetes-related phenotypes were studied in comparison with those of consomic C3H-Chr 14(NSY) (R0) and parental NSY and C3H strains. Congenic R1 and R2 showed significantly higher post-challenge glucose than that in C3H mice. Fasting glucose, in contrast, was significantly lower in R1 and R2 than in C3H mice. Insulin sensitivity was significantly impaired in R1 and R2 compared to C3H mice. R2 showed significantly higher body weight and fat-pad weight than those in C3H and R1. Leptin level was significantly higher in R0, R1 and R2 than in C3H mice, with R2 showing the highest level, similar to that in NSY mice. Serum adiponectin level was significantly lower in R0, R1 and R2 than in C3H mice, while it was significantly higher in NSY than in C3H mice. Conclusions: These data indicate that Chr 14 harbors multiple genes for diabetes-related phenotypes. The original Nidd2n, which is located in the middle region of Chr 14, was divided into two segments; Nidd2.1n in proximal Chr 14 and Nidd2.2n in distal Chr 14. Nidd2.1n contributes to post-challenge hyperglycemia, insulin resistance and adiposity. Nidd2.2n contributes to fasting as well as post-challenge hyperglycemia and insulin resistance. Adp1n, which contributes to decreased adiposity and increased insulin sensitivity, rather than a diabetogenic gene, was mapped in the middle segment.

Miglitol, an α-glucosidase inhibitor (α-GI), is useful for controlling the early postprandial elevation of blood glucose, and can be administered in combination not only with other oral hypoglycemic agents but also with insulin preparations. We investigated the effectiveness of combination therapy with miglitol and insulin on Japanese type 2 diabetic patients. Subjects receiving any insulin therapy, including intensive insulin therapy (n=43), mixed insulin therapy (n=22), and basal supported oral therapy (BOT) (n=24), were additionally treated with miglitol for 12 weeks. The HbA1c (NGSP) was significantly lowered from 8.0±1.0% to 7.7±1.1% (p<0.01). We evaluated the daily glucose profile by self-monitoring blood glucose (SMBG) at seven points before and after miglitol treatment. Although there was little change in plasma glucose levels before each meal, those at 1 hr after each meal were significantly lowered. Mean AUC0-1hr of postprandial glucose and M-value were also significantly lowered, suggesting that additional treatment with miglitol improved the daily glucose excursion. When subjects were divided into three groups by the types of insulin therapy, similar improvements in the daily glucose excursion were shown in any group. Body weight and daily dosage of insulin were unchanged from the start of miglitol treatment. We conclude that the combination therapy with miglitol and broad types of insulin therapy is effective for reducing the daily glycemic fluctuation and achieving a much better control.

[抄録]目的: 円形脱毛症患者における甲状腺自己免疫および膵島自己免疫の臨床的・遺伝的実態を明らかにする. 方法: 円形脱毛症患者110例について臨床的特徴および自己免疫疾患の合併率を検討し, 血清学的に抗サイログロブリン（Tg）抗体, 抗甲状腺ペルオキシダーゼ（TPO）抗体, 甲状腺刺激ホルモン受容体抗体（TRAb)抗glutamic acid decarboxylase（GAD）抗体, 抗insulinoma-associated antigen2（IA-2抗体）, インスリン自己抗体（IAA）を測定した. またHLA-DRB1, -DQB1, -A, -B, -C 遺伝子型を決定した. 結果: 円形脱毛症患者は健常対照者に比し, 抗Tg抗体, 抗IA-2抗体, IAA, 抗GAD抗体陽性率は同等であったが, 抗TPO抗体（29.1％ vs. 11.6％, P＜0.001）, TRAb（42.7％ vs. 1.2％, P＜0.001）の陽性率は有意に高値を示した. 膵島関連自己抗体価の比較では抗GAD抗体, 抗IA-2抗体, IAAはいずれも健常対照者との間に差を認めず, 自己免疫性甲状腺疾患患者に比し有意に低値であった. 遺伝子解析において円形脱毛症患者は健常対照者に比し, A, 33: 03 が有意に低頻度であり（3.2％ vs. 9.7％, Pc＝0.036）, DRB1, 04: 05 -DQB1, 04:01 は低頻度の傾向, DRB1, 15: 01-DQB1, 06: 02 は高頻度の傾向を示した. 結語: 円形脱毛症には甲状腺自己免疫を高率に合併するが, 膵島自己免疫・１型糖尿病の合併は稀であること、遺伝子解析でも円形脱毛症では１型糖尿病の疾患感受性ハプロタイプが低頻度、疾患抵抗性ハプロタイプが高頻度であるという今回の結果から、円形脱毛症が自己免疫性甲状腺疾患とは共通性を有するのに対し、１型糖尿病とは臨床的にも遺伝的にも異質性を有することが示唆された.

[抄録]目的：自己免疫性甲状腺疾患（AITD）発症へのHLAクラスII領域とクラスI領域の関与を明らかにする． 方法：AITD患者281人と健常対照者198人を対象に，HLAクラスII領域のDRB1 とDQB1 アリルおよびクラスI領域のA，B とC アリルを決定し，アリル頻度およびハプロタイプ頻度を比較検討した．成績：DRB1 についてはDRB1*08:03 がAITD患者において有意に高頻度（14.4％ vs.7.6％，Pc＜0.01），DRB1*01:01 は有意に低頻度（2.3％ vs.8.8％，Pc＜0.0001）であった．DQB1 については，DQB1*05:01 が患者群において有意に低頻度（2.7％ vs.10.6％，Pc＜0.00001）であった．DRB1-DQB1 ハプロタイプについては，DRB1*08:03 -DQB1*06:01 が患者群において有意に高頻度（14.2％ vs.7.3％，Pc＜0.01），DRB1*01:01-DQB1*05:01が有意に低頻度（2.3％ vs.8.8％，Pc＜0.0001）であった．A については，いずれのアリルについても統計学的有意差を認めなかった．B についてはB *35:01 が患者群において有意に高頻度（13.2％ vs.6.8％，Pc＝0.04），B*07:02 が有意に低頻度（1.6％ vs.6.8％，Pc＜0.01）であった．C については，C *03:03 が患者群において有意に高頻度（17.4％ vs.8.1％，Pc＜0.01）であった．B -C ハプロタイプについては，B*35:01-C*03:03 が患者群において有意に高頻度（11.9％ vs.4.7％，Pc＜0.001），B *07:02 -C*07:02 が患者群で有意に低頻度（1.6％ vs.6.6％，Pc＝0.02）であった．DRB1

The quantitative trait locus (QTL) mapping in segregating crosses of NSY (Nagoya-Shibata-Yasuda) mice, an animal model of type 2 diabetes, with nondiabetic strain C3H/He mice has identified diabetogenic QTLs on multiple chromosomes. The QTL on chromosome 11 (Chr11) (Nidd1n) showing the largest effect on hyperglycemia was confirmed by our previous studies with homozygous consomic mice, C3H-11(NSY), in which the NSY-derived whole Chr11 was introgressed onto control C3H background genes. C3H-11(NSY) mice also showed a streptozotocin (STZ) sensitivity. In the present study, we constructed heterozygous C3H-11(NSY) mice and the phenotypes were analyzed in detail in comparison with those of homozygous C3H-11(NSY) and C3H mice. Heterozygous C3H-11(NSY) mice had significantly higher blood glucose levels and STZ sensitivity than those in C3H mice. Hyperglycemia and STZ sensitivity in heterozygous C3H-11(NSY) mice, however, were not as severe as in homozygous C3H-11(NSY) mice. The body weight and fat pad weight in heterozygous C3H-11(NSY) mice were similar to those in C3H and homozygous C3H-11(NSY) mice. These data indicated that the introgression of Chr11 of the diabetes-susceptible NSY strain onto diabetes-resistant C3H caused marked changes in the glucose tolerance and STZ susceptibility even in a heterozygous state, and suggested that the mode of inheritance of a gene or genes on Chr11 for hyperglycemia and STZ sensitivity is additive.

We studied the time course of serum insulin level in a patient who injected large amounts of regular insulin in an attempted suicide. A 58-year-old woman attempted suicide by subcutaneously injecting herself with 2400 U regular insulin. On arrival, the serum glucose level was 2.4 mmol/L (44 mg/dL) and the serum insulin level was 40,000 pmol/L (5700 mu IU/mL). The serum insulin level was high, with a maximum of 110,000 pmol/L (16,000 mu IU/mL) at 13 h after injection, followed by an initial rapid decrease and a subsequent slow decrease, with hyperinsulinemia lasting as long as 5 days after injection. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00211.x, 2012)

Type 1 diabetes is etiologically a multifactorial disease caused by a complex interaction of genetic and environmental factors, with the former consisting of multiple susceptibility genes. Identification of genes conferring susceptibility to type 1 diabetes would clarify etiological pathways in the development and progression of type 1 diabetes, leading to the establishment of effective methods for prevention and intervention of the disease. Among multiple susceptibility genes, HLA and INS are particularly important because of their contribution to tissue specificity in the autoimmune process. DRB1*04:05-DQB1*04:01 is associated with autoimmune type 1 diabetes, idiopathic fulminant type 1 diabetes and anti-islet autoimmunity in autoimmune thyroid diseases, suggesting that this haplotype is associated with beta-cell specificity in autoimmune diseases. Genes involved in the expression of insulin in the thymus contribute to beta-cell-specific autoimmune mechanisms in type 1 diabetes. These genes and pathways are important targets for tissue-specific prevention and intervention of type 1 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00176.x, 2011)

In contrast to the large number of studies on autoimmunity against the thyroid gland in patients with type 1 diabetes mellitus, little is known about the anti-islet autoimmune status in patients with autoimmune thyroid diseases (AITDs). We therefore studied the anti-islet autoimmune status in patients with AITD and the clinical and genetic characteristics of AITD patients with anti-islet autoimmunity. The positivity and titer of glutamic acid decarboxylase antibody (GAD Ab) were studied in 866 Japanese patients with AITD (546 with Graves disease and 320 with Hashimoto thyroiditis), 221 patients with thyroid disease of nonautoimmune origin, and 282 control subjects. The clinical characteristics and genotypes of HLA-DRB1, DQB1, and CTLA4 were compared between AITD patients with and without GAD Ab. The prevalence of GAD Ab was significantly higher in AITD patients than in control subjects (5.8% vs 2.1%, P = .01), particularly in Graves disease (7.1% vs 2.1%, P = .0019). The prevalence of diabetes mellitus was significantly higher in AITD patients with GAD Ab than in those without (40.0% vs 10.1%, P < .0001), particularly in those with a high titer of GAD Ab (high vs low titer: 64% vs 16%, P = .001) and also in those positive for insulinoma-associated antigen 2 (IA-2) Ab (IA-2 positive vs negative: 75.0% vs 31.3%, P = .016). The AITD patients with GAD Ab were characterized by younger age at onset of diabetes, lower body mass index, higher hemoglobin AI, level, and higher frequency of insulin therapy than those without GAD Ab. The frequency of the DRB1*0405-DQB1*0401 haplotype was significantly higher in AITD patients with GAD Ab than in those without GAD Ab and control subjects. A single nucleotide polymorphism (rs3087243) of CTLA4 was significantly associated with AITD, but not with positivity of GAD Ab. These results indicate that patients with AITD, and in particular Graves disease, are prone to develop beta-cell autoimmunity and insulin-requiring diabetes, particularly those with a high titer of GAD Ab and/or positive for both GAD and 1A-2 Ab. Glutamic acid decarboxylase Ab positivity in AITD patients was associated with HLA, conferring susceptibility to type 1 diabetes mellitus. (C) 2011 Elsevier Inc. All rights reserved.

OBJECTIVE-Tissue-specific self-antigens are ectopically expressed within the thymus and play an important role in the induction of central tolerance. Insulin is expressed in both pancreatic islets and the thymus and is considered to be the primary antigen for type 1 diabetes. Here, we report the role of the insulin transactivator MafA in the expression of insulin in the thymus and susceptibility to type 1 diabetes. RESEARCH DESIGN AND METHODS-The expression profiles of transcriptional factors (Rix I, NeuroD, Mafa, and Aire) in pancreatic islets and the thymus were examined in nonobese diabetic (NOD) and control mice. Thymic Ins2 expression and serum autoantibodies were examined in Mafa knockout mice. Luciferase reporter assay was performed for newly identified polymorphisms of mouse Mafa and human MAFA. A case-control study was applied for human MAFA polymorphisms. RESULTS-Mafa, Ins2, and Aire expression was detected in the thymus. Mafa expression was lower in NOD thymus than in the control and was correlated with Ins2 expression. Targeted disruption of MafA reduced thymic Ins2 expression and induced autoantibodies against pancreatic islets. Functional polymorphisms of MafA were newly identified in NOD mice and humans, and polymorphisms of human MAFA were associated with susceptibility to type 1 diabetes but not to autoimmune thyroid disease. CONCLUSIONS-These data indicate that functional polymorphisms of MafA are associated with reduced expression of insulin in the thymus and susceptibility to type 1 diabetes in the NOD mouse as well as human type 1 diabetes. Diabetes 59: 2579-2587, 2010

Diabetogenic loci for type 2 diabetes have been mapped to mouse chromosome (Chr) 11 and 14 in the Nagoya-Shibata-Yasuda (NSY) mouse, an animal model of type 2 diabetes. We aimed to obtain direct evidence of these genes on each chromosome and to clarify their function and interaction in conferring susceptibility to type 2 diabetes. We established three consomic strains homozygous for diabetogenic NSY-Chr11, NSY-Chr14 or both on the control C3H background (C3H-11(NSY), C3H-14(NSY) and C3H-11(NSY)14(NSY), respectively), and monitored diabetes-related phenotypes longitudinally. The glucokinase gene was sequenced as a positional candidate gene on Chr11. C3H-11(NSY) mice showed hyperglycaemia associated with impaired insulin secretion and age-dependent insulin resistance without obesity. C3H-14(NSY) mice exhibited hyperglycaemia mainly due to insulin resistance, with a slight increase in percentage body fat. C3H-11(NSY)14(NSY) double consomic mice showed marked hyperglycaemia and obesity, which was not observed in single consomic strains. Sequences of the glucokinase gene were allelically variant between NSY and C3H mice. These data provide direct evidence that Chr11 and Chr14 harbour major susceptibility genes for type 2 diabetes. These two chromosomes interact to cause more severe hyperglycaemia and obesity, which was not observed with the presence of either single chromosome, indicating different modes of gene-gene interaction depending on the phenotype. Marked changes in the phenotypes retained in the consomic strains will facilitate fine mapping and the identification of the responsible genes and their interaction with each other, other genes and environmental factors.

[抄録] 低Na血症は臨床上遭遇する電解質異常のなかでも頻度が高く，軽症では倦怠感や食欲不振の訴えにとどまるが，ひとたび重症化すると，けいれんや意識障害をきたし，生命維持に危機に関わる重要な病態である．今回我々は著明な低Na血症に加えて低血糖を伴った症例を経験し，速やかに加療を開始すると共に鑑別診断を行い，下垂体性副腎皮質機能低下症と診断し得たので報告する．

[抄録] 目的：自己免疫性甲状腺疾患（AITD）患者における膵島自己免疫の実態を明らかにする．方法：AITD 患者866人（バセドウ病患者546人，橋本病患者320人）と非自己免疫性甲状腺疾患患者221人を対象にGAD 抗体陽性率と抗体価を比較するとともに，抗体陽性AITD 患者の臨床的特徴及びHLA-DRB1，DQB1 とCTLA4 の遺伝子型を陰性者と比較．成績：GAD 抗体陽性率はAITD 患者で対照者に比し有意に高率（5.8％ vs. 0.6％，P＜0.001）．糖尿病有病率はGAD 抗体陽性者において陰性者に比し有意に高率（40.0％ vs. 10.1％，p＜0.0001），陽性者の中では高抗体価群で低抗体価群に比し有意に高率（64％ vs. 16％，p＝0.001）．GAD 抗体陽性AITD 患者は陰性患者に比し，糖尿病発症年齢が有意に若く，BMIが有意に低く，HbA1c値，インスリン使用率が有意に高値．DRB1＊0405-DQB1＊0401ハプロタイプはGAD 抗体陽性AITD 患者で抗体陰性者，対照者に比し有意に高頻度．CTLA4 の6230G＞A 多型（rs3087243）はAITD と有意の関連を示したが，GAD 抗体の有無とは関連を認めなかった．結論：AITD 患者ではGAD 抗体が高率に陽性を示し，抗体陽性者は１型糖尿病の臨床的，遺伝的特徴を有することが示された．

[抄録]高齢者糖尿病の治療においても血糖の正常化に努めるべきであるが，同時に治療がQOL を低下させることがないよう，患者の身体的，精神・心理的，社会的背景を十分に考慮した治療を実施すべきである．今回，我々は，血糖コントールのため一旦インスリンを導入したが，在宅自己注射が困難な状況を考慮し，経口薬でのコントロールに戻し得た高齢者２型糖尿病の１症例を経験したので報告する．

Type 1 diabetes is a multifactorial disease caused by a complex interaction of genetic and environmental factors. The genetic factors involved consist of multiple susceptibility genes, at least five of which, HLA, INS, CTLA4, PTPN22 and IL2RA/CD25, have been shown to be associated with type 1 diabetes in Caucasian (Western) populations, as has recently been confirmed by genome-wide association studies. It has been proposed, however, that the contribution of these genes to type 1 diabetes susceptibility may be different in Asian (Eastern) populations. HLA and INS genes are consistently associated with type 1 diabetes in both Caucasian and Asian populations, but apparent differences in disease-associated alleles and haplotypes are observed between Japanese and Caucasian subjects. The association of CTLA4 with type 1 diabetes is concentrated in a subset of patients with autoimmune thyroid disease (AITD) in both Japanese and Caucasian populations, while the association of PTPN22 with type 1 diabetes in Japanese and most Asian populations is not as clear as in Caucasians. IL2RA/CD25 genes seem to be similarly distributed in type 1 diabetes patients in the two populations, whereas genetic heterogeneity may exist regarding SUMO4, with an association of the M55V variant with type 1 diabetes observed in Asians, but not in Caucasians. Genome-wide association studies (GWA) are largely outstanding for Asian populations but they are now underway in Japan. This review reports on the discovered similarities and differences in susceptibility genes for type 1 diabetes between East and West and discusses the most recent observations made by the involved investigators. Copyright © by the SBDR.

Major histocompatibility complex (MHC)-linked susceptibility to type 1 diabetes consists of multiple components. Previous studies with NOD mice congenic for the MHC from a sister strain, the CTS mouse, mapped Idd16, a second component of MHC-linked susceptibility to type 1 diabetes, in the region adjacent to, but distinct from Idd1 in the class II region. In this study, three lines of subcongenic strains were established from the original congenic strain. Phenotypic analysis of the strains indicated that MHC-linked susceptibility to type 1 diabetes consists of at least three components: Idd1, Idd16.2 adjacent to Idd1, and Idd16.1 telomeric to Idd16.2.

Among candidate genes for type I diabetes, HLA, INS, CTLA4, PTPN22 and SUM04 have been shown to be associated with the disease in Caucasian populations. To clarify the similarities and differences in the contribution of these genes to type 1 diabetes between Asian and Caucasian populations, association of these genes with type 1 diabetes was studied in a large number of samples in Japanese and Korean populations. Class 11 HLA was strongly associated with type 1 diabetes in both Asian and Caucasian populations, but haplotypes associated with type 1 diabetes were markedly different due to difference in the presence and absence of haplotypes in each population. INS was consistently associated with type 1 diabetes in both Japanese and Caucasian populations, but frequency of disease-associated haplotype was markedly high in Japanese general population. CTLA4 was associated with type 1 diabetes only in a subset of patients with type 1 diabetes complicated with AITD in Japanese. A variant (R620W) of PTPN22 was associated with type 1 diabetes and other autoimmune diseases in Caucasians, but the variant was absent in Asians. SUM04 was associated with type 1 diabetes in Asians, but not in Caucasian, suggesting a genetic heterogeneity among diverse ethnic groups. Trans-racial study with a large number of samples in both Asian and Caucasian populations will contribute to genetic dissection of type 1 diabetes and identification of causative variants. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

A recent dramatic increase in elderly patients with diabetes mellitus has made the proper management of the disease in this population more important. Here, we discuss the present status of diabetes management in the elderly in Japan. As a characteristic feature of elderly persons, body weight reduction is difficult, because of the profound adaptive reduction in resting energy expenditure under calorie restriction in the elderly. However, hyperglycemia increases the risk for diabetic complications, except proliferative retinopathy, similarly in elderly and non-elderly. Of note, there is marked clinical heterogeneity in this generation in the following aspects: duration, complication status (past aspect), insulin secretion, insulin sensitivity, familial support and physical exercise/activity (present aspect), as well as the expected lifespan (future aspect). This heterogeneity among the elderly should render diabetes treatment diverse, and in fact, one of the largest surveys in Japan demonstrated significant diversity in diabetes management in the elderly. In Japan, thus, the present management of diabetes in the elderly is considerably diverse, reflecting the clinical heterogeneity among elderly patients with diabetes. Further clinical evidence is awaited for the establishment of proper and safe management of diabetes in the elderly. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Aims/hypothesis Obesity and fatty liver are commonly associated with type 2 diabetes, but the genetic and functional bases linking fatty liver with obesity and diabetes are largely unknown. Our aim was to investigate the association of fatty liver with obesity and other diabetes-related phenotypes and to define the genetic control of obesity and fatty liver. Materials and methods We established 306 F2 mice by crossing Nagoya-Shibata-Yasuda (NSY) mice, an animal model of type 2 diabetes, with control C3H mice, and analysed their phenotypes. Whole-genome screening of F2 mice was performed to identify the loci responsible for fatty liver and obesity. Results A strong association of fatty liver with obesity, hyperinsulinaemia and hyperglycaemia was observed in F2 mice. Using whole-genome screening in 306 F2 mice, we mapped a new locus for fatty liver (Fl1n) on chromosome 6 (maximum logarithm of odds score [MLS] 10.0) and one for body weight (Bw1n) on chromosome 7 (MLS 5.1). Fl1n was linked to epididymal fat weight as well as fatty liver, but its effects were opposite in the two tissues in that the NSY allele increased liver fat but decreased epididymal fat, suggesting a role of FlIn in partitioning of fat mass. The sequence of peroxisome proliferator-activated receptor Y (Pparg), a candidate for Fl1n, showed alletic variation between NSY and C3H mice. Conclusions/interpretation These data suggest that fatty liver and obesity are phenotypically related but genetically independent. Loci homologous to Fl1n and Bw1n are good candidate genes for susceptibility to fatty liver and obesity in humans.

Multiple genes are involved in conferring susceptibility to autoimmune type I diabetes mellitus. The immunoreceptor programmed cell death-1 (PDCD-1). an inhibitory costimulatory molecule regulating peripheral toterance, is reported to play an important role in the development of type I diabetes mellitus, making the human PDCD-1 gene, PDCD1, a candidate for disease susceptibility. The aim of this study was to clarify the contribution of PDCD1 to genetic susceptibility to type I diabetes mellitus in humans. To screen for sequence variants, we sequenced all 5 exons and exon-intron junctions of PDCD1 in Japanese subjects, 16 with type 1 diabetes mellitus and 16 without the disease. Some of the sequence variations identified were genotyped in larger samples (n = 275) with and without type 1 diabetes mellitus by polymerase chain reaction restriction fragment length polymorphism method or a fluorescence-based method. The distributions of polymorphisms were compared between patients with type 1 diabetes mellitus and healthy controls by contingency table analysis and Pearson %(2) test. In this study, we found 16 sequence variants, including a TGC repeating variant in the 3' untranslated region. We found this variant to be associated with the development of type 1 diabetes mellitus. These data suggest the contribution of PDCD1 and its gene product to the development of type 1 diabetes mellitus. (c) 2007 Elsevier Inc. All rights reserved.

Objective: This study aimed to investigate the renoprotective effect on diabetic nephropathy of a novel class of Ca2+ channel blocker, cilnidipine, that inhibits both L-type and N-type Ca2+ channels; a conventional L-type Ca2+ Channel blocker was substituted with cilnidipine in type 2 diabetic patients with albuminuria. Methods: Urinary albumin index (UAI), serum creatinine, and blood pressure were measured in 38 outpatients with type 2 diabetes receiving amlodipine, an L-type Ca2+ channel blocker, in addition to an angiotensin 1 converting enzyme inhibitor and/or an angiotensin type 1 receptor blocker. Amlodipine was then substituted with cilnidipine, and the same parameters were measured after 3 months. Results: Although blood pressure was not significantly changed after substitution with cilnidipine, log-transformed UAI was significantly decreased (P=.004) with a mean reduction of 28% [95% confidence interval (CI)=11-42]. Serum creatinine was significantly (P=.04) increased (from 0.82 +/- 0.22 to 0.86 +/- 0.23 mg/dl). When the subjects were divided into two groups according to the change in serum creatinine, UAI change was significant only in those with an increase in serum creatinine, who exhibited a mean reduction of UAI of 39% (95% CI=16-56, P=.005), but not in those without an increase in serum creatinine, whose mean reduction of UAI was 18% (95% Cl=- 12 to 40, P=.2). Conclusions: In patients with diabetic nephropathy, blocking N-type Ca 2+ channels with a new class of Ca 2+ channel blocker resulted in a significant reduction in albuminuria, suggesting a renoprotective effect of N-type Ca 2+ channel blockade, even when combined with renin-angiotensin inhibition. (C) 2007 Elsevier Inc. All rights reserved.

Abstract Context: Despite distinct differences in the pathogenesis, epidemiological data have indicated familial clustering of type 1 and type 2 diabetes, suggesting a common genetic basis between these two types of diabetes. Few shared susceptibility genes, however, have been reported to date. Objective: Small ubiquitin-like modifier 4 (SUMO4) has been identified as a candidate gene for the IDDM5 locus and suggested to have possible involvement in immune responses, such as autoimmunity and inflammation. Recent reports demonstrated that a polymorphism with an amino acid substitution (Met55Val) in SUMO4 was associated with type 1 diabetes in Asian populations, although no association was reproduced in subjects of Caucasian descent. The present study aimed to clarify the contribution of SUMO4 to type 2 diabetes susceptibility in the Japanese population. Subjects: The 753 subjects included 355 cases and 398 control subjects. Methods: The SUMO4 Met55Val (rs237025) and 001Msp (rs577001) polymorphisms were genotyped. Results: Strong linkage disequilibrium (D′: 1.0 in each pair of single-nucleotide polymorphisms) across the MAP3K7IP2/SUMO4 region was shown in the Japanese population. The frequency of genotypes with the G allele of the SUMO4 Met55Val polymorphism was significantly higher in patients with type 2 diabetes [odds ratio, 1.46; 95% confidence interval (CI), 1.08–1.93; P = 0.01, χ2 test]. The association was concentrated in patients without insulin therapy (odds ratio, 1.56; 95% CI, 1.13–2.15; P = 0.0072), but not in those with insulin (odds ratio, 1.24; 95% CI, 0.81–1.89; not significant). Conclusions: These data, together with previous reports, suggest the contribution of the SUMO4 Met55Val polymorphism to both type 1 and type 2 diabetes susceptibility in the Japanese population.

A recent study in the nonobese diabetic (NOD) mouse demonstrated the involvement of interleukin (IL)-21 in the pathogenesis of type 1 diabetes. A strong susceptibility locus, Idd3, has also been mapped to the interval containing the routine gene for IL-21 (I/21), making I/21 and the human orthologue IL21 a functional and positional candidate gene for type 1 diabetes. To investigate the contribution of the human genes for IL-21 and its receptor (IL-21R) to susceptibility to type 1 diabetes, we re-sequenced IL21 to identify novel sequence variants, searched for informative variants of IL21R, and studied the association of theme variants with the disease. Two polymorphisms, a single nucleotide polymorphism (SNP) and a mononucleotide repeat polymorphism, were identified for IL21, and an allele of the mononucleotide repeat polymorphism was positively associated with the IL21R were identified, one of which was associated with the disease. Scoring of individuals according to the status of these alleles showed a significant trend for high scores for susceptibility in diabetes patients, suggesting the contribution of IL21 and IL21R to disease susceptibility in an additive manner. These data suggest a contribution of IL21 and IL21R to genetic susceptibility to type 1 diabetes and possible involvement of IL-21 and its receptor system in the disease pathogenesis.

lestimide, have been clinically used as cholesterol-lowering agents. These agents bind bile acids in the intestine and reduce enterohepatic circulation of bile acids, leading to accelerated conversion of cholesterol to bile acids. A significant improvement in glycemic control was reported in patients with type 2 diabetes whose hyperlipidemia was treated with bile acid-binding resins. To confirm the effect of such drugs on glucose metabolism and to investigate the underlying mechanisms, an animal model of type 2 diabetes was given a high-fat diet with and without colestimide. Diet-induced obesity and fatty liver were markedly ameliorated by colestimide without decreasing the food intake. Hyperglycemia, insulin resistance, and insulin response to glucose, as well as dyslipidemia, were markedly and significantly ameliorated by the treatment. Gene expression of the liver indicated reduced expression of small heterodimer partner, a pleiotropic regulator of diverse metabolic pathways, as well as genes for both fatty acid synthesis and gluconeogenesis, by treatment with colestimide. This study provides a molecular basis for a link between bile acids and glucose metabolism and suggests the bile acid metabolism pathway as a novel therapeutic target for the treatment of obesity, insulin resistance, and type 2 diabetes.

Context: Recently, an association of a single nucleotide polymorphism, 163A > G encoding M55V, in the gene SUMO4, which has been shown to be a negative feedback regulator for nuclear factor kappa B, has been reported in type 1 diabetes. Objective: To establish whether SUMO4 locus contributes to the genetic susceptibility to other autoimmune disorders, a case-control analysis was carried out using genomic DNA from type 1 diabetes, autoimmune thyroid disease (AITD), rheumatoid arthritis (RA), and primary Sjogren's syndrome. Subjects: A total of 1480 samples, including 929 cases ( 411 patients with type 1 diabetes, 292 AITD, 172 RA, and 54 primary Sjogren's syndrome) and 551 healthy control subjects of Japanese origin participated in the study. Methods: The 163A > G (rs237025, M55V) polymorphism of SUMO4 was genotyped. Results: SUMO4 M55V variant was associated not only with type 1 diabetes [odds ratio (OR), 1.42; 95% confidence interval (CI), 1.09-1.84; P = 0.0072], but also with increased risk of other autoimmune diseases, AITD (OR, 1.52; 95% CI, 1.14-2.03; P = 0.0041) and RA without amyloidosis (OR, 1.53; 95% CI, 1.65-2.24; P = 0.027), but not primary Sjogren's syndrome. Furthermore, the association of SUMO4 M55V variant was stronger in type 1 diabetic patients complicated with AITD (OR, 1.62; 95% CI, 1.06-2.47; P = 0.023) and in patients who have neither type 1 diabetes-susceptible class II HLA, DRB1*0405 nor DRB1*0901 (OR, 2.28; 95% CI, 1.34-3.87; P = 0.0018). Conclusions: These results indicate that the SUMO4 is a more common autoimmune disease gene and a supplementary risk factor to type 1 diabetes in conjunction with class II HLA.

Type 1 diabetes is under polygenic control both in humans and the NOD mouse. Recently a possible role of IL-21 in the pathogenesis of type 1 diabetes was demonstrated in the NOD mouse. Furthermore, the murine IL-21 gene is mapped to the Idd3 interval, making the human IL-21 gene (IL21) a functional as well as positional candidate for susceptibility. We therefore screened sequence variants of IL21 and studied the association with type 1 diabetes. Preliminary data showed no association of IL21 polymorphisms with the disease, suggesting that IL21 plays little role in susceptibility to type 1 diabetes in Japanese.

A recombinant major histocompatibility complex (MHC) with the same class III region as the NOD mouse, but different class II region from the NOD mouse was identified in the NON mouse, and NOD mice congenic for this recombinant MHC, NOD.NON-H2, was established. None of the congenic mice homozygous for the NON MHC developed type 1 diabetes, indicating that the NOD MHC is necessary for the development of type 1 diabetes. A small portion of MHC heterozygotes developed late-onset type 1 diabetes, suggesting the contribution of class III MHC to type 1 diabetes susceptibility.

Although major histocompatibility complex (MHC)-linked susceptibility is the strongest component, recent studies demonstrated that MHC-linked susceptibility to type 1 diabetes consists of multiple components both in humans and non-obese diabetic (NOD) mouse. In the NOD mouse, Idd16 has been mapped to the region adjacent to, but distinct from Idd1 in the MHC class II region. Establishment of subcongenic NOD.CTS-H2 lines that possess the same MHC class II as the NOD mouse but non-NOD-derived chromosomal region in its adjacent regions, would facilitate further narrowing down of the localization of Idd16.

Type I diabetes is a polygenic disease with a major susceptibility locus, IDDM1, located in the human leukocyte antigen (HLA) region. Although class 11 loci, DR and DQ genes in particular, are major components of IDDM1, accumulating lines of evidence indicated that IDDM1 consists of multiple components and that non-class 11 genes in addition to class 11 genes contribute to susceptibility to and/or age-at-onset of type I diabetes. To identify a second component of IDDM1, we investigated the association of a panel of polymorphisms in 2.2 Mb region of the HLA encompassing from class 11 to class I regions with type 1 diabetes. Polymorphisms types were: DRB1 and DQB1 in class 11; two microsatellite markers, BAT2-GT and TNF alpha in class 111; and, five microsatellite markers, STR-MICA, MIB, C1-3-1, C2-4-4, and C3-2-10 in class I region. A total of > 200 Japanese patients and healthy control subjects were studied. Class 11 DRB1*0405 and DQB1*0401 were significantly associated with susceptibility to, but not with age-at-onset of, type 1 diabetes. C1-3-1, located near C locus, was significantly associated with not only susceptibility to, but also age-at-onset of type I diabetes. These data suggest that a second component of IDDM1 maps to the HLA class I region, contributing to susceptibility to as well as age-at-onset of type I diabetes.

Multiple genes are involved in the susceptibility to autoimmune type I diabetes. The immunoreceptor programmed cell death-1 (PDCD-1), an inhibitory costimulatory molecule regulating peripheral tolerance, was reported to play a role in the development of type 1 diabetes, making the human PDCD-1 gene, PDCD1, as a candidate for disease susceptibility. In this article, we sequenced all 5 exons and exon-intron junctions of PDCD1] in Japanese subjects, and found 10 sequence variants. Preliminary data suggested no association of these polymorphisms with type 1 diabetes. These sequence variants are valuable for further studies to clarify contribution of PDCD1] to susceptibility to type I diabetes.

Association studies are a potentially powerful approach to identifying susceptibility variants for common multifactorial diseases such as type 1 diabetes, but the results are not always consistently reproducible. The IDDM5 locus has recently been narrowed to an ∼200-kb interval on chromosome 6q25 by two independent groups. These studies demonstrated that alleles at markers in the mitogen-activating protein kinase 7 interacting protein 2 (MAP3K7IP2)/SUMO4 region were associated with susceptibility to type 1 diabetes. Subsequent studies, however, showed inconsistency in the association of the SUMO4 gene with type 1 diabetes. To clarify the contribution of the M55V polymorphism of the SUMO4 gene to type 1 diabetes susceptibility, 541 type 1 diabetic patients and 768 control subjects were studied in Asian populations. The M55V polymorphism was significantly associated with type 1 diabetes in Asian populations (summary odds ratio [OR] 1.46, P = 0.00083, Mantel-Haenszel test). Meta-analysis of published studies and the present data confirmed a highly significant association in Asian populations (summary OR 1.29, P = 7.0 × 10−6) but indicated heterogeneity in the genetic effect of the SUMO4/MAP3K7IP2 locus on type 1 diabetes among diverse ethnic groups. These data indicate that the MAP3K7IP2/SUMO4 locus in the IDDM5 interval is associated with type 1 diabetes in Asian populations.

Among polygenes conferring susceptibility to type I diabetes in the NOD mouse, Iddlo oil distal chromosome 3 has been shown to be important for disease susceptibility. In this Study, we investigated the candidacy of Fcgr1 and Cd101 for Idd10, by congenic mapping and candidate gene sequencing. Among seven NOD-related strains Studied. the IIS Mouse Was found to possess a recombinant Idd10 interval with the same sequence at Fegr1 as the NOD mouse, but a different sequence at Cdl10 from that in the NOD mouse with 10 amino acid Substitutions. The frequency of type 1 diabetes in NOD mice congenic for IIS hid10 (NOD.IISIdd10) was significantly reduced as compared to that in the NOD Mouse. despite the presence of the identical Fegr1 sequence. These data indicate that IIS mice possess a resistant allele at Idd10, and Suggest that Cd101. but not Fegr1. is responsi tile for the Idd10 effect. © 2005 Elsevier Inc. All rights reserved.

Background: To investigate the renoprotective effect of combination therapy with an angiotensin I converting enzyme inhibitor and an angiotensin type I receptor blocker (ARB) on diabetic kidney disease, half doses of each monotherapy were given to type 2 diabetic patients with albuminuria. Methods: Urinary albumin index (UAI) and blood pressure (BP) were measured in a total of 27 outpatients with type 2 diabetes mellitus receiving 10 mg imidapril or 8 mg candesartan per day. Either agent was then substituted with a combination of 5 mg imidapril and 4 mg candesartan. After 3 months of combination therapy, UAI and BP were measured. Changes in the parameters were assessed by paired t test. Results: Although BP was not significantly different prior to and at the end of combination therapy, log-transformed UAI was significantly reduced (P = 0.003) from an initial UAI (mean log-transformed UAI +/- SD) of 79.4 (27.4-231)mg/g Cre to 52.5 (17.1-161)mg/g Cre at the end of combination therapy. The reduction was not associated with the initial UAI, initial BP, decrease in BP, pretreatment medication or other concomitant antihypertensive agents. Conclusions: In patients with type 2 diabetes and nephropathy, dual blockade of the renin system with an angiotensin-converting enzyme inhibitor and angiotensin receptor blocker significantly reduces albuminuria and, thus, may be renoprotective even when the doses of the agents are reduced by one half. (C) 2005 American Journal of Hypertension, Ltd.

To investigate the intrafamilial clustering of type I and type 2 diabetes, an interview-based assessment of family history of diabetes was conducted. Outpatients with either type 1 (n = 23) or type 2 diabetes (n = 124), and non-diabetic subjects (n = 118) received an interview regarding the diabetic status of each of their family members. In patients with type 1 diabetes, 22% (5 out of 23) had a parental history of diabetes, and diabetes in these 5 parents was assessed as type 2 diabetes mellitus. The prevalence of parental diabetes in the type 1 diabetic probands (22%) was significantly higher (P < 0.05) than that in non-diabetic probands (7%, 8 out of 118). In probands with type 2 diabetes, the prevalence of parental diabetes was 39% (48 out of 124), which was significantly higher (P < 0.0005) than that in the non-diabetic probands (7%). In the type 2 diabetic probands, no significant difference was noted in the prevalence between paternal (19%, 23 out of 124) and maternal diabetes (23%, 28 out of 124), suggesting no preferential inheritance of maternal diabetes in this population. The present interview-based assessment of family history of diabetes suggested a common genetic basis between type 1 and type 2 diabetes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Aims/hypothesis. Recent 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. Methods. We 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. Results. While the K sequence of the NOD mouse was identical to that of K-d 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 K-K 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-K-d and anti-K-k 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 K-k- and K-b-like sequences. Conclusions/interpretation. These 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.

Aims/hypothesis. Type 1 diabetes mellitus, a T-cell-mediated autoimmune disease, results from the selective destruction of insulin-producing pancreatic beta cells. Autoantibodies against beta-cell components are used clinically as sensitive markers of this disease; however, their physiological role has not been clear. To investigate the role of glutamic acid decarboxylase 65 (GAD65) in the development of the Type 1 diabetes of non-obese diabetic (NOD) mice, we analysed and characterised NOD mice with targeted disruption of the GAD65 gene. Methods. GAD65-deficient mice were previously established. After backcrossing the knockout mutation onto the NOD genetic background for up to eight generations, female littermates of the three resulting genotypes were produced by intercrossing: GAD65 +/+ (n=23), GAD65 +/- (n=62), and GAD65 -/- (n=31). Results. The cumulative incidence of autoimmune diabetes showed no significant difference among the three groups in longitudinal studies using the Kaplan-Meier method. Islet morphology showed that the progression of islet infiltration did not differ significantly between the three groups. Conclusion/interpretation. The cumulative incidence of autoimmune diabetes was not influenced by the GAD65 deficiency. These data suggest that GAD65 is not a major regulatory target of beta-cell autoimmunity in NOD mice.

Nonobese diabetic (NOD) mice develop T cell-dependent autoimmune disease. Administration of interleukin-4 (I L-4), one of the T helper 2 (Th2) cytokines, is reported to prevent either insulitis or diabetes or both in NOD mice. We examined the effect of transferring an IL-4-expressing plasmid vector into muscle by in vivo electroporation on the progression of diabetes in NOD mice. Plasmid DNA expressing murine IL-4 (pCAGGS-IL-4) was introduced into the muscles of 4- and 6-week-old female NOD mice using an in vivo electroporation technique we developed previously. The serum IL-4 levels reached 2000-8000 pg/ml 3 days after the delivery of pCAGGS-IL-4 and remained detectable (>5 pg/ml) for over 4 weeks. In contrast to the previous reports, 88% of the mice treated with pCAGGS-IL-4 developed overt diabetes by 30 weeks of age, while only 25% of nontreated mice and 19% of the mice treated with control pCAGGS developed overt diabetes by then (p<0.01). Therefore, highly expressed IL-4 introduced by in vivo electroporation may have caused a Th1 shift, resulting in the promotion of diabetes in NOD mice. The high serum concentration of cytokines attained by our method is likely to unveil previously unknown cytokine functions. (C) 2003 Elsevier Science Ltd. All rights reserved.

Mutations in the hepatocyte nuclear factor-1 beta (HNF-1 beta) gene have been shown to be a cause of maturity-onset diabetes of the young (MODY). We studied the contribution of the HNF-1 beta gene to susceptibility to common forms of Type 2 diabetes in the genetically homogeneous Japanese population, by investigating the allelic association of Type 2 diabetes with two markers in the HNF-1 beta region. The frequency of a nonsense mutation, R177X, which was previously reported in a Japanese family, was also studied by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using a mismatch primer. A total of 200 subjects were studied. There was no significant difference in allele frequencies of either of the two polymorphisms studied between patients with Type 2 diabetes and control subjects, or between subgroups of patients subdivided by the presence of mild or severe diabetic nephropathy. None of the subjects studied had R177X mutation, giving a frequency of less than 1.1% in common forms of Type 2 diabetes in Japan. These results suggest that mutations in the HNF-1 beta gene derived from a limited number of founders are not a major cause of common forms of Type 2 diabetes, even in the genetically homogeneous Japanese population. (C) 2001, Editrice Kurtis.

To assess the contribution of the HLA class I region to susceptibility to and heterogeneity of type 1 diabetes, we investigated the association of polymorphism of MHC class I chain-related gene A (MICA) with age-at-onset as well as susceptibility to type 1 diabetes. One hundred one Japanese patients and 110 healthy control subjects were studied. The frequency of A4 allele was significantly higher and that of AG allele was significantly lower in patients than in control subjects. The frequency of A5.1 allele was highest in early-onset patients (23.0%), intermediate in intermediate-onset patients (9.2%) and lowest in late-onset patients (7.7%) (trend chi-squared test, p = 0.0098). A5.1 allele was strongly associated with HLA-B7 and Cw7, suggesting that MICA*A5.1-B7-Cw7 haplotype contains a gene responsible for age-at-onset. A4 allele was associated with a susceptible haplotype, DR4-DQB1*0401, and AG allele was associated with a protective haplotype, DR2-DQB1*0601, suggesting that the association of MICA with type 1 diabetes susceptibility may be due to linkage disequilibrium with class II haplotypes. These data suggest that MICA gene is associated with age-at-onset and that a gene (or genes) responsible fur age-at-onset of type 1 diabetes is located in the HLA class I region, probably near the region of MICA-B-C. (C) American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc.

Primer extension preamplification (PEP), which can amplify a limited quantity of DNA as a whole, regardless of their sequences, allows multiple DNA analyses by the polymerase chain reaction (PCR) even with very small amount of DNA. This method potentially make it possible to generate enough template DNA for multiple PCR-reactions in cases the amount of original genomic DNA is limited. To evaluate the accuracy of PEP, genotyping results of four microsatellite markers using PEP-amplified DNA as template for PCR were compared using DNA without PEP. Both genotyping results were identical to each other for each sample tested. Amplification of an initial genomic DNA by PEP was found to yield at least 50 times of the initial quantity. These results indicate that PEP can increase typing potential of PCR when only small amounts of genomic DNA are available and analyses of many loci are required.

To assess the contribution of three genes (IDDM7, IDDM12, IDDM13) predisposing to type 1 diabetes on chromosome 2q, we studied the association of markers in this region with diabetes in Japanese. A 137-mobility unit (mu) allele at D2S137, a microsatellite marker located in IDDM13 region on chromosome 2q34, was significantly associated with diabetes. In contrast, D2S152, reported to be in linkage disequilibrium with IDDM7, showed only a marginal association with diabetes. No significant association was observed for CTLA4 (IDDM12). These data suggest that IDDM13 contributes to genetic susceptibility to type 1 diabetes in Japanese.

Werner's syndrome is a rare disease with autosomal recessive inheritance characterized by an aged appearance, including glucose intolerance with extreme insulin resistance (1) Heterogeneity, however, in glucose tolerance is observed among patients with Werner's syndrome. To explore the reason for the heterogeneity, we investigated insulin secretary responses in a unique family in which two siblings shared the same mutation of the Werner helicase gene, but were discordant for diabetes. On OGTT, the proband (Case 1) was diagnosed as having diabetes, whereas the other (Case 2) was impaired glucose tolerance (IGT). Total insulin responses to OGTT and i.v. glucagon in the siblings were both increased, suggesting the presence of insulin resistance. The early-phase insulin response to intravenous glucose tolerance test (TVGTT) in Case 1 was defective, whereas it was increased in Case 2. Furthermore, by performing IVGTT in all members of this family, we found that a defect in the early-phase insulin response to IVGTT, similar to that in Case 1, was observed only in their father with type 2 diabetes, but not in other members. These data indicate the importance of impaired insulin secretion in addition to insulin resistance in the development of overt diabetes in Werner's syndrome.

Mutations in the hepatocyte nuclear factor genes have been shown to be a cause of maturity onset diabetes of the young (MODY). Genes responsible for MODY are candidate genes for common forms of type 2 diabetes. In this study, the contribution of the HNF-1 alpha and HNF-1 beta genes to susceptibility to common forms of type 2 diabetes was studied in the Japanese population. 253 diabetic patients and 103 control subjects were studied. There was no significant difference in allele frequencies of the polymorphic markers in or near HNF-1 alpha and HNF-1 beta genes studied between patients with type 2 diabetes and control subjects. No significant association was observed between allele frequencies and clinical characteristics in diabetic patients. The lack of association of type 2 diabetes with HNF-1 alpha and HNF-1 beta genes suggests that mutations in the HNF-1 alpha and HNF-1 beta genes derived from a limited number of founders are not major causes of common forms of type 2 diabetes in the Japanese population.

Aim To clarify the association of several clinical parameters, including plasma fibrinogen level, with diabetic retinopathy in patients with Type 2 diabetes mellitus (DM). Methods A total of 294 Japanese patients with Type 2 DM were studied; 53 patients with no diabetic retinopathy (NDR), 90 with background diabetic retinopathy (BDR), and 151 with proliferative diabetic retinopathy JPDR). Multiple logistic regression analysis was performed to assess variables independently associated with diabetic retinopathy in two settings: presence of retinopathy of any severity and presence of advanced retinopathy. Results The following parameters were identified as independent factors associated with the presence of diabetic retinopathy (NDR vs. BDR+PDR): type of therapy (P<0.0005), log-transformed plasma fibrinogen level (P<0.05), mean blood pressure (P<0.05), and duration of diabetes (P<0.05). The independent variables associated with advanced retinopathy were type of therapy (P<0.00005), age (P<0.0005) and nephropathy (P<0.05). Body mass index, smoking and hypertensive status, HbA(1c) and total cholesterol levels were not independently associated. Conclusions These data suggest that in patients with Type 2 DM, an increased blood viscosity due to high fibrinogen level as well as an elevated intravessel pressure play a role in the development of diabetic retinopathy, and that the progression to PDR is influenced or accompanied by the deterioration of renal status.

【目的】AITDにおける膵島自己免疫の実態やその臨床的・遺伝的背景を明らかにする。【方法】当科通院中のAITD患者 816例について病態、臨床背景、GAD抗体価、HLA遺伝子型（DRB1、DQB1）を比較検討した。【結果】AITD患者のGAD抗体陽性率は5.6%と高率であった。GAD抗体陽性AITD患者の糖尿病発症率は、GAD抗体陰性AITD患者に比し有意に高頻度であった。糖尿病合併AITD患者において、GAD抗体陽性者は陰性者に比し糖尿病発症年齢が有意に若く（、AITD発症年齢も有意に若齢であり、BMIが有意に低かった。非糖尿病AITD患者にける同様の比較では有意差を認めなかった。HLA解析ではDRB1*0405-DQB1*0401ハプロタイプが、GAD抗体陽性バセドウ病患者において、抗体陰性者及び健常対照者に比し有意に高頻度であった。GAD抗体陰性AITD患者では健常対象者に比し、DRB1*0803-DQB1*0601ハプロタイプ頻度が高頻度であった。【総括】AITD患者、特にバセドウ病ではGAD抗体陽性率が高く、糖尿病発症との関連が認められた。GAD抗

糖尿病を発症するマウス(Nagoya-Shibata-Yasuda mouse：NSY)の糖尿病感受性遺伝子を有する染色体を、非糖尿病マウス(C3H)へ導入した系統であるコンソミックマウス/コンジェニックマウスの作製により、糖尿病および糖尿病関連形質の疾患感受性領域を特定の染色体/領域に絞り込んできた(Kobayashi M, Babaya N, et al., BMC Genet 2020、Babaya N et al., Int J Endocrinol 2018、Babaya N et al., BMC Genet 2014、Babaya N et al., J Diabetes Res 2013、Babaya N et al., Diabetologia 2010 など)。今年度は、1番染色体コンジェニック系統の表現型解析を行い、加齢に伴う遺伝子座の影響を検討した。 ヒトにおいては２型糖尿病患者の表現型パネル作成を行っているが、その過程で副腎腫瘤をもつ個人を2例同定し、病理学的解析を行い報告した（Babaya N et al., J Endocr Soc 2021、Yoshida S, Babaya N et al., J Endocr Soc 2021、今村、馬場谷他, 第22回日本内分泌学会近畿支部学術集会）。また、ヒト糖尿病における新たな臨床ツールcontinuous glucose monitoring(CGM)の関連指標とHbA1c・残存膵β細胞機能との関連解析についての発表を行った（馬場谷他, 第94回日本内分泌学会学術集会）。さらには、膵部分切除術（膵頭十二指腸切除術と膵尾部切除術）後の糖尿病発症率の解析を行い、術式の違いによる糖尿病発症率に差があることを明らかにし報告した(Niwano F, Babaya N, et al., J Clin Endocrinol Metab)。

本研究では、１型糖尿病において治療困難の原因となる膵β細胞機能の完全廃絶を規定する遺伝因子を同定し、その分子メカニズムを明らかにするとともに、膵β細胞機能の完全廃絶を予知・予測するバイオマーカーを探索・同定することにより、内因性インスリンの完全廃絶阻止に資する基盤情報を得ることを目的として研究を進めている。 １）膵β細胞機能の完全廃絶を規定する遺伝因子の解析： 膵β細胞が発症時から完全廃絶する劇症１型糖尿病のゲノムワイド関連解析（GWAS）でゲノムワイド有意水準をクリアしたHLA領域とCSAD領域のターゲットリシークエンスを進め、劇症化・膵β細胞機能完全廃絶の直接原因となる変異・多型の同定・抽出を進めている。また、自己免疫性１型糖尿病（急性発症・緩徐進行）を対象としたGWASを新たに進めており、劇症１型糖尿病のGWAS結果と対比した解析を行うことで、１型糖尿病の劇症化・膵β細胞機能の完全廃絶に関与する遺伝子を抽出・同定し、膵β細胞機能完全廃絶を規定する体質・遺伝子の全貌解明へと展開する。 ２）膵β細胞機能の完全廃絶を予知・予測するバイオマーカーの探索： 劇症１型糖尿病のGWASで同定した染色体12q13.13のCSADがコードする遺伝子の機能に関連する血中バイオマーカーの探索を進めた結果、CSADが律速酵素として生合成する産物Taurineがバイオマーカーとなりうる可能性が示唆された。これと並行して血中メタボローム解析を施行し、アミノ酸以外の各種メタボライトも含めた解析で膵β細胞完全廃絶に関与するバイオマーカーの探索を進めている。

本研究は１型糖尿病の濃厚発症家系を対象とした責任遺伝子変異の解明を目的としている。我が国における１型糖尿病の有病率は欧米に比べて低いため、濃厚発症家系の集積は容易ではないが、本研究申請時以降も新規4家系13名の検体を上乗せすることに成功し、合計で13家系54名（罹患者35名、非罹患者19名）について、既に全エクソームシーケンス作業は完了している。本研究の解析対象者を選別する際に、糖尿病の病型診断として確実な１型糖尿病に限定して効率的に解析を進める必要がある。この目的のためには、高額な次世代シーケンス工程に入る前に、HLA遺伝子型タイピングをおこない、１型糖尿病の疾患感受性ハプロタイプを有することを確認するスクリーニング工程が非常に有効な手段であると考えられる。確実な１型糖尿病を選別するため、多数検体に対するHLAタイピングが必要であるが、不確実な病型診断を回避することにより、さらに高額な次世代シーケンス行程を必要かつ最小限に抑制するに足る十分な効果があった。また、独立基盤形成支援にともなう交付決定後増額を得たことにより各家系の発端者に対して全ゲノムシーケンス（WGS）を実施することができ、全エクソームシーケンス（WES）だけでは検出し得ない、染色体上の大きな構造変異の探索の探索やミスアライメントの検証など変異解析の精度を飛躍的に向上させることができた。 また次世代シーケンス解析によって同定された遺伝子変異が、技術的に生じるノイズではなく、実際に遺伝子配列の変異があることを検証するため、Taqman法を用いて遺伝子型を決定することにより、次世代シーケンスの結果を随時検証した。 上記の結果を、18th Immunology of Diabetes Society Congress(2021.11.2. Virtual conference)にて発表し、現在論文執筆中である。

本研究は１型糖尿病の濃厚発症家系を対象とした責任遺伝子変異の解明を目的としている。我が国における１型糖尿病の有病率は欧米に比べて低いため、濃厚発症家系の集積は容易ではないが、本研究申請時以降も新規4家系13名の検体を上乗せすることに成功し、合計で13家系54名（罹患者35名、非罹患者19名）について、既に全エクソームシーケンス作業は完了している。本研究の解析対象者を選別する際に、糖尿病の病型診断として確実な１型糖尿病に限定して効率的に解析を進める必要がある。この目的のためには、高額な次世代シーケンス工程に入る前に、HLA遺伝子型タイピングをおこない、１型糖尿病の疾患感受性ハプロタイプを有することを確認するスクリーニング工程が非常に有効な手段であると考えられる。確実な１型糖尿病を選別するため、多数検体に対するHLAタイピングが必要であるが、不確実な病型診断を回避することにより、さらに高額な次世代シーケンス行程を必要かつ最小限に抑制するに足る十分な効果があった。また、独立基盤形成支援にともなう交付決定後増額を得たことにより各家系の発端者に対して全ゲノムシーケンス（WGS）を実施することができ、全エクソームシーケンス（WES）だけでは検出し得ない、染色体上の大きな構造変異の探索の探索やミスアライメントの検証など変異解析の精度を飛躍的に向上させることができた。 また次世代シーケンス解析によって同定された遺伝子変異が、技術的に生じるノイズではなく、実際に遺伝子配列の変異があることを検証するため、Taqman法を用いて遺伝子型を決定することにより、次世代シーケンスの結果を随時検証した。 上記の結果を、18th Immunology of Diabetes Society Congress(2021.11.2. Virtual conference)にて発表し、現在論文執筆中である。

糖尿病を発症するマウス(NSY)の糖尿病感受性遺伝子を有する染色体を、非糖尿病マウス(C3H)へ導入した系統であるコンソミックマウス/コンジェニックマウスの作製により、糖尿病および糖尿病関連形質の疾患感受性領域を特定の染色体/領域に絞り込んできた(Babaya N et al., BMC Genet 2014、Babaya N et al., J Diabetes Res 2013、Babaya N et al., Diabetologia 2010、Babaya N et al., Biochem Biophys Res Commun 2005)。 今年度は、14番染色体コンジェニック・コンソミック系統の表現型解析が終了し、ストレプトゾトシン感受性（膵β細胞の脆弱性）領域が14番染色体のテロメア側に存在することを報告した（Babaya N et al., Int J Endocrinol 2018）。 また、NSYマウスとは異なる系統（A/J、SM系統）のコンジェニックマウスを用いて、ストレプトゾトシン感受性に関しての遺伝子同定をおこなった。この結果、NSYマウスと同様に別系統のマウスにおいても11番染色体上にストレプトゾトシン感受性領域が存在することが明らかとなった（Maegawa T, Babaya N, et al., Mamm Genome 2018）。 ヒトにおいては２型糖尿病患者の表現型パネル作成を行っているが、その過程で特異な表現型をもつ個人を1例同定し、遺伝子解析を行い報告した（Babaya N, et al., J Endocr Soc 2018）。これまで実験動物においてのみ証明されているIRS2遺伝子の糖尿病に対する影響が、ヒトにおいても関与している可能性を示した。

本研究では１型糖尿病の成因を分子レベルで解明し、それに基づいた予知・予防・根治療法の構築・確率を目的として、分子遺伝学的アプローチを駆使して１型糖尿病の疾患感受性遺伝子の同定・解析を進めた。 これまでに全国規模で収集を進めてきた濃厚発症家系においては、次世代シーケンス技術を用いて全エクソン塩基配列解析を行い、rare variantの同定を進めている。また、多数の症例・対照におけるゲノムワイド関連解析（GWAS）も並行して行い、多くの症例に共通するありふれた多型（common variant）の同定・解析を進めた。 rare variantの解析では家系内に３名以上の患者を有する濃厚家系の構成員（2n=66）と孤発の１型糖尿病孤発例（2n=676）、健常対照者（2n=792）を比較解析した結果、濃厚家系ではHLA-DR8ハプロタイプ（DRB1*08:02-DQB1 *03:02）が極めて高率であることが明らかとなった。HLA以外の新規遺伝子座を同定する目的で、濃厚家系における構成員のエクソームシークエンスを完了し、原因となる変異・多型の絞り込みを進めている。 common variant の解析では全国規模で集積した多数例の劇症１型糖尿病を対象としてGWAS解析を完了し、HLA以外にゲインムワイドの有意水準をクリアする新たな遺伝子を染色体12q13.13に同定し、報告した（Diabetes 68:665-675, 2019）.

1)ヒトにおける膵島破壊に関わる疾患感受性遺伝子の探索：１型糖尿病濃厚発症家系を7家系集積し、全エクソームシーケンス解析をおこなうことで候補遺伝子変異の探索をおこなった。そのうち1家系における変異が欧米白人には欠損し日本人特有の変異であること、孤発例日本人１型糖尿病の発症とも関連していることを見いだし（リスクアリル頻度：患者群6.1% vs 対照群0.7％、p<0.0003, オッズ比9.07、95％CI3.05-26.93）、また複数の家系に共通して1型糖尿病の発症に関与する遺伝子領域を連鎖解析にて同定し、その成果について第61回日本糖尿病学会年次学術集会シンポジウム5．1型糖尿病研究の最前線 －多様化する病態と診断上の課題－、「1型糖尿病の遺伝素因 Genetic factors of type 1 diabetes」 （東京、2018.5．24）にて口演発表をおこなった。その後、更に2家系の1型糖尿病濃厚発症家系を新規に見いだし、日本人に特有の膵島破壊に関わる遺伝素因の解明を進行中である。 2)マウスを用いた膵島破壊修飾モデルの作出：Mafa遺伝子ノックアウトNODマウスを作出し、8週齢における膵島炎スコアを検討した結果、ノックアウトマウスにて有意に膵島炎スコアが高いことを見いだした。さらに１型糖尿病の累積発症率を検討中である。 3)膵島破壊の新規バイオマーカーの探索：1型糖尿病19名、2型糖尿病6名、膵性糖尿病4名、バセドウ病6名の血漿サンプルの集積を完了し、このうち自己免疫性１型糖尿病（インスリン分泌能微小残存）、自己免疫性１型糖尿病（インスリン分泌完全枯渇）、２型糖尿病（インスリン非依存状態）において、miRNA解析をおこなった。

２型糖尿病は、複数の遺伝因子と環境因子との複雑な相互作用により発症する多因子疾患である。本研究では、疾患モデルマウスを用いて２型糖尿病関連形質の感受性遺伝子同定を行った。また、ヒトにおける表現型パネル作成を行い、その過程で得られた特異な症例について報告した(Babaya N, et al., BMC Endocrine Disorders、2017)。さらに、１型糖尿病、バセドウ病の疾患感受性遺伝子に関する報告を行った(Babaya N, et al., Hum Immunol、2017, and Babaya N, et al., J Clin Endocrinol Metab、2015)。

１型糖尿病の自己免疫と臓器特異性を一元的に説明する遺伝子の同定を目的に、標的臓器の異なる自己免疫の相互関係を解析した結果、膵β細胞と甲状腺、甲状腺と毛包自己免疫が相互に合併、膵β細胞と毛包自己免疫の合併は極めて稀であった。遺伝的背景を解析するとHLAが臓器特異性に大きく関与し、DR4 (DRB1*04:05-DQB1*04:01)が膵β細胞特異性に寄与することが示された。自己免疫と膵β細胞特異性の両者に関与するMAFAをノックアウトしたNODマウスでは発症が予想に反して発症が抑制されること、その機序として、膵β細胞特異的調節性T細胞がラ氏島炎部位に集積することが一因である可能性が示された。

現在50以上の1型糖尿病疾患感受性遺伝子座が報告されているが、これら全てをあわせても1型糖尿病の遺伝素因の70－80％しか説明できない。残されたmissing heritabilityの一部は、アリル頻度は低いが浸透率が高いrare variantにより説明できる。 日本人では１型糖尿病発症頻度が低いので3人以上の同胞発症家系は稀である。我々は4姉妹中3人に１型糖尿病を認め、残りの1人は膵島自己抗体が陽性である１家系を見いだした。これら4姉妹および両親を対象に全エクソームシークエンスを行い、全変異から11の候補変異に絞り込み、さらにゲノム連鎖解析の結果等から１つの変異を候補変異として絞り込んだ。

１型糖尿病における膵β細胞破壊のメカニズムを解明する目的で以下の解析をおこなった。(1) ヒトにおいて標的臓器を決定するHLA遺伝子とnon-HLA遺伝子について解明した（J Clin Endocrinol Metab 2015、Hum Immnol 2017）。また1型糖尿病発症家系を集めて日本人特有の新規Rare variantを同定した。(2) インスリンに対する免疫寛容を誘導する転写因子Mafa遺伝子の欠損マウスモデルを作出し、膵島炎は亢進するが免疫制御性リンパ球浸潤も増加するため糖尿病発症が抑制されることを解明した（第29回糖尿病・肥満動物学会、京都、平成27年2月14日にて発表）。

１型糖尿病は膵β細胞が免疫機序により破壊される臓器特異的自己免疫疾患であり、免疫調節機能を明らかにすることが治療につながる。１型糖尿病における免疫調節機能を解明するため、新規１型糖尿病感受性遺伝子の同定を行うとともに、複数の異なる臓器における自己免疫疾患の関連について解析を行い、遺伝子間の関連を明らかにした。新規１型糖尿病感受性遺伝子Mafa遺伝子では、１型のノックアウトマウスを作成し解析をしたところ、Mafa遺伝子が糖尿病発症と膵島炎に関わる遺伝子であることが明らかとなった。

２型糖尿病は、複数の遺伝子により構成される遺伝因子と、環境因子の複雑な相互作用により発症する多因子疾患であり、ヒトを用いた解析では感受性遺伝子同定は困難である。本研究では、糖尿病モデルマウスおよび交配実験において得られたコンソミック・コンジェニック系統を用いて、２型糖尿病の疾患感受性遺伝子領域を同定し、その作用機序を解明した。

「１型糖尿病でなぜ膵β細胞だけが破壊されるのか？」を分子レベルで解明することを目的として研究を進めた。胸腺で自己抗原インスリンの発現調節に寄与する転写因子MafAをノックアウトした１型糖尿病モデルNODマウスでは１型糖尿病発症が有意に抑制されること、ヒト対応遺伝子MAFAの機能亢進型多型は１型糖尿病と負の関連を示すことが明らかとなった。毛包に対する臓器特異的自己免疫疾患である円形脱毛症では甲状腺自己免疫を高率に合併するが、１型糖尿病との合併は極めて稀であること、その理由として両疾患に対するHLAクラスⅡハプロタイプの感受性、抵抗性が逆方向を向いていることが示された。

主要組織適合遺伝子複合体領域における１型糖尿病疾患感受性遺伝子を明らかにするために、HLAクラスII領域のDRB1とDQB1、クラスI 領域のA、B、Cと１型糖尿病との関連を詳細に検討した。その結果、DRB1*04:05－DQB1*04:01とDRB1*08:02－DQB1*03:02の組合せは、日本人一般人口での頻度は低いが、１型糖尿病発症に対して高リスクであることが示された。クラスIのB、Cで疾患感受性と関連を示したアリルはクラスIIとの連鎖不平衡によるものと考えられた。自己免疫性疾患における検討との比較により、クラスIとクラスIIの作用様式が、標的臓器により異なっている可能性が示された。

１型糖尿病は、膵臓でのインスリン分泌細胞（β細胞）に対して組織特異的自己免疫反応が生じ発症する。β細胞のみが破壊されるメカニズムを解明し治療に応用する目的で、１型糖尿病の候補遺伝子群と複数の臓器特異的自己免疫疾患（β細胞：１型糖尿病、甲状腺：バセドウ病・橋本病、毛包：円形脱毛症）との関連解析を行ない、膵島に対する組織特異的自己免疫反応に関連を示す遺伝因子を解明した。ヒトにおいて関連が示されたMafA遺伝子をノックアウトした１型糖尿病モデルマウスを作出したところ、１型糖尿病発症が著明に抑制され、Mafa遺伝子は１型糖尿病の発症機序に関わる重要な遺伝子であることがin vivoにて確認された。

１型糖尿病遺伝子の解析をヒトとマウスの染色体相同性を駆使して進めた結果、主効果遺伝子であるHLAとの関連にサブタイプ間で差を認めること(劇症と他のサブタイプ間で質的差異、緩徐進行と急性発症間で量的差異)、臓器特異性を決定する遺伝子としてインスリン特異的転写因子MafAが胸腺でのインスリン発現量の変化を介して１型糖尿病の疾患感受性に関与することが示された。

１型糖尿病と２型糖尿病は異なる機序により発症すると考えられてきたが、１型糖尿病患者の家族に２型糖尿病患者が高率に認められることが報告されており、両者に共通する遺伝基盤の存在が想定される。我々は日本人１型糖尿病、および２型糖尿病の疾患感受性遺伝子、および2型糖尿病モデルマウスにおける疾患感受性遺伝子座を同定することにより、両者共通の遺伝基盤を解析するためのスクリーニングパネルを作製した。さらにヒトおよびマウスにおける１型糖尿病の新規疾患感受性遺伝子を発見し、２型糖尿病との疾患感受性について検討し報告した。