Abstract
Islet-cell associated antibodies are predictive and diagnostic markers for type 1 diabetes. We studied the differences in the early clinical course of children with type 1 diabetes with a single antibody and those with multiple antibodies against pancreatic β-cells. Sixty-seven children with type 1 diabetes aged less than 15 years diagnosed between 2010 and 2021 were included in the study and subdivided into two subgroups: children who were single positive for either glutamic acid decarboxylase (GAD) antibodies (n = 16) or insulinoma-associated antigen-2 (IA-2) antibodies (n = 13) and those positive for both antibodies (n = 38) at diagnosis. We compared the patients’ clinical characteristics, pancreatic β-cell function, and glycemic control during the 5 years after diagnosis. All clinical characteristics at diagnosis were similar between the two groups. One and two years after diagnosis, children who tested positive for both antibodies showed significantly lower postprandial serum C-peptide (CPR) levels than those who tested positive for either GAD or IA-2 antibodies (p < 0.05). In other periods, there was no significant difference in CPR levels between the two groups. There was a significant improvement in glycosylated hemoglobin (HbA1c) levels after starting insulin treatment in both groups (p < 0.05), but no significant difference in HbA1c levels between the groups. Residual endogenous insulin secretion may be predicted based on the number of positive islet-cell associated antibodies at diagnosis. Although there are differences in serum CPR levels, optimal glycemic control can be achieved by individualized appropriate insulin treatment, even in children with type 1 diabetes.
TYPE 1 DIABETES (T1D) is caused by autoimmune destruction of pancreatic β-cells, wherein endogenous insulin secretion gradually decreases and becomes depleted [1]. Serologic markers for autoimmunity toward pancreatic β-cells include insulin autoantibodies (IAA), glutamic acid decarboxylase (GAD) antibodies, insulinoma-associated antigen-2 (IA-2) antibodies, and zinc transporter 8 (ZnT8) antibodies [2]. Conversely, there is a difference in the appearance of islet-cell associated antibodies in the preclinical and clinical courses of type 1 diabetes. The first appearance of islet-cell associated antibodies may include IAA or GAD [3, 4]. It is reported that children with multiple islet antibodies develop diabetes, but it may take up to 20 years before the clinical onset [5]. Kawasaki et al. [6] demonstrated that the prevalence of IA-2 and ZnT8 antibodies was higher for children than for adults, while that of GAD was higher for adults than for children. Moreover, a high titer of GAD was associated with the progression of T1D requiring insulin therapy in individuals at high risk of T1D [7]. High titers of IA-2 antibodies were related to dysglycemia and were found to predict the clinical onset of T1D [8]. On the other hand, the presence of ZnT8 antibodies seems to be associated with a more advanced deterioration of β-cell function [9]. There may be differences in the clinical course of diabetes according to the type of islet-cell associated antibodies. Detection of multiple islet-cell associated antibodies is a genetic risk marker for developing T1D at the preclinical stage of the disease [5]. It was also reported that patients with multiple antibodies had a lower first-phase insulin response than those with single-positive antibodies. This study supports the view that β-cell function has deteriorated, and the disturbance in insulin production is more severe [9, 10]. However, there are no studies reporting the different clinical course by the kind and number of positive islet-cell associated antibodies in patients with T1D at the time of diagnosis.
T1D is a chronic disease associated with serious long-term health complications, including heart disease, vascular disease, kidney disease, blindness, and neuropathy [11]. If we elucidate the relationship between the number of positive islet-cell associated antibodies and clinical characteristics at the time of diagnosis and the subsequent clinical course, we can offer appropriate insulin therapy according to the disease condition and residual β-cell function during the course of diabetes in patients with T1D. Therefore, we examined the association between the number of positive islet-cell associated antibodies and the clinical course during the first 5 years after diagnosis in children with T1D.
Materials and Methods
Materials
Sixty-seven children with new onset T1D aged less than 15 years, who were consecutively recruited at the Department of Pediatrics, Nihon University Hospital, Tokyo, Japan between 2010 and 2021, were included in the study. T1D was diagnosed according to the criteria of the Japan Diabetes Society and American Diabetes Association [2], and was defined as the presence of one or more islet-cell associated antibodies, including GAD and IA-2 antibodies. All patients had acute-onset T1D exhibiting typical symptoms of diabetes and/or ketosis at the time of diagnosis and requiring insulin treatment to improve their metabolic intolerance [12-14]. In the present study, the male/female ratio was 28/39 (male, 42%), the mean age was 7.6 ± 4.2 years, and the mean of standardized body mass index (BMI SDS) was –0.36 ± 1.30 SD at the time of diagnosis. Forty-four children were treated with multiple daily injections of insulin (MDI) and 23 were treated with continuous subcutaneous insulin infusion (CSII) after the diagnosis of T1D. Diabetic ketoacidosis (DKA) was defined by the presence of all of the following in a patient with diabetes, as outlined in a consensus statement from the International Society for Pediatric and Adolescent Diabetes in 2018: hyperglycemia – blood glucose of >200 mg/dL; metabolic acidosis – venous pH <7.3 or a plasma bicarbonate <15 mEq/L; and ketosis – determined by the presence of ketones in the blood or urine [15]. Twenty-five children had diabetic ketoacidosis at the time of diagnosis.
Methods
We compared patients’ characteristics at the time of diagnosis, including age, sex, BMI SDS, the method of insulin therapy (MDI or CSII), and presence of DKA at diagnosis between children with a single positive (GAD or IA-2) antibody, and those positive for both antibodies. In addition, changes in pancreatic β-cell function evaluated by serum levels of postprandial C-peptide immunoreactivity (CPR) and glycemic control evaluated by glycosylated hemoglobin (HbA1c) levels over 5 years after diagnosis were compared between the two patient groups.
The time of blood-sample collection to measure serum CPR was 1–2 h after meals. Serum CPR levels were determined by radioimmunoassay (RIA) until 2013 and were determined by enzyme-linked immunosorbent assay (ELISA) after 2013. The RIA had a detection limit of 0.16 ng/mL, and met the linearity acceptable criteria from 0.78 to 2.33 ng/mL. The ELISA had a detection limit of 0.03 ng/mL, and met the linearity acceptable criteria from 0.08 to 0.85 ng/mL [16]. HbA1c levels were measured using a high-performance liquid chromatography method (normal range: 4.6–6.2%).
GAD detection
GAD antibodies were determined by RIA before December 2015 and determined by ELISA after December 2015. The cut-off value was 1.5 U/mL for RIA, and 5.0 U/mL for ELISA. Higher levels of GAD antibodies were defined as ≥10 U/mL for RIA, and ≥180 U/mL for ELISA [17].
IA-2 detection
IA-2 antibodies were determined by RIA before October 2018 and determined by ELISA after October 2018. The cut-off value was 0.4 U/mL for RIA, and 0.6 U/mL for ELISA. Higher levels of IA-2 antibodies were defined as ≥1.5 U/mL for RIA and ≥4.5 U/mL for ELISA [18, 19].
Statistical analysis
The results are expressed as the mean ± SD. The frequencies of patients’ characteristics (sex, the method of insulin therapy, and presence of DKA at diagnosis) were compared using a Pearson-Chi Square test. Differences in nonparametric data of age and BMI SDS between the two groups were tested using the Mann-Whitney U test. The statistical difference in HbA1c or CPR at each timepoint between the two groups were tested using the Mann-Whitney U test. Comparisons of changes in serum CPR and HbA1c levels were performed using analysis of variance and a Tukey’s honest significant difference test. Statistical significance was set at 0.05. All statistical analyses were performed using the JMP Pro 14 software (SAS Institute Japan, Japan).
Ethical approval
This study was approved by the review board of the Nihon University Hospital (211006; October 2021).
Results
Comparison of clinical characteristics at diagnosis between children with single positive GAD or IA-2 antibody and those positive for both antibodies
The patients were subdivided into groups: children who were single positive for GAD antibodies (n = 16) or those who were single positive for IA-2 antibodies (n = 13), and those who tested positive for both antibodies (n = 38) at the time of diagnosis. We compared the clinical characteristics of children who tested positive for either antibody and those who were positive for multiple antibodies and found no significant differences in the clinical characteristics at diagnosis between the groups (Table 1).
Table 1
Clinical characteristics
|
Single (n = 29) |
Multiple (n = 38) |
p value |
| Age (years) |
8.1 ± 3.7 |
7.3 ± 4.5 |
0.44 |
| Males/Females |
10/19 |
18/20 |
0.29 |
| BMI-SDS |
–0.57 ± 1.56 |
–0.24 ± 1.13 |
0.36 |
| Method of insulin therapy: MDI/CSII |
21/8 |
23/15 |
0.31 |
| Presence of DKA |
10 (39%) |
15 (34%) |
0.68 |
| HbA1c (%) |
11.0 ± 2.9 |
11.2 ± 2.5 |
0.87 |
| C-peptide (ng/mL) |
0.68 ± 0.52 |
0.68 ± 0.79 |
0.52 |
Data are shown as mean ± standard deviation.
BMI-SDS, the mean of standardized body mass index; MDI, multiple daily injections of insulin; CSII, continuous subcutaneous insulin injection; DKA, diabetic ketoacidosis; HbA1c, glycosylated hemoglobin
We compared the clinical characteristics of children with high and low titers of GAD and IA-2 antibodies. We found no significant differences in the clinical characteristics at diagnosis between the groups with high and low titers of GAD antibodies. Children who had a high titer of IA-2 antibodies had significantly higher HbA1c levels than those with a low titer of IA-2 antibodies. Otherwise, we found no significant differences in the clinical characteristics at diagnosis between the groups with high and low titers of GAD antibodies (Supplementary Tables 1, 2).
Relationship between the number of positive islet-cell associated antibodies and the clinical course during the first 5 years after diagnosis
We analyzed the relationship between the number of positive islet-cell associated antibodies and the clinical course over 5 years after diagnosis. We classified the patients into two groups according to the number of islet-cell associated antibodies: children who were positive for either antibody (GAD or IA-2 antibody) and those who were positive for both antibodies at diagnosis. First, we compared the postprandial serum CPR levels at diagnosis and at 3 months, 6 months, 1 year, 2 years, 3 years, and 5 years after diagnosis between children who were single positive for either antibody and those who were multiple positive for both antibodies. One and two years after diagnosis, children who were positive for both antibodies had significantly lower postprandial serum CPR levels than those who were single positive for either antibody (p < 0.05; Fig. 1A). There was no significant difference in CPR levels in other periods (Fig. 1A). Secondly, we compared the HbA1c levels during the same period. There was significant improvement in HbA1c levels from the time of diagnosis to that after starting insulin treatment: at 3 months, 6 months, 1 year, 2 years, 3 years, and 5 years after diagnosis (p < 0.05; Fig. 1B), and no significant difference of HbA1c levels between the two groups was observed during the entire period (Fig. 1B).
We evaluated the relationship between the GAD antibody titer and the clinical course over 5 years after diagnosis. We classified the patients according to high and low titers of GAD antibodies at diagnosis. We compared the postprandial serum CPR levels at diagnosis and at 3 months, 6 months, 1 year, 2 years, 3 years, and 5 years after diagnosis. There was no significant difference in CPR levels over 5 years after diagnosis between the two groups (Supplementary Fig. 1A). Secondly, we compared HbA1c levels during the same period between children with high and low titers of GAD antibodies. There was no significant difference in HbA1c levels over 5 years after diagnosis between the two groups (Supplementary Fig. 1B). However, there was a significant improvement in HbA1c levels after starting insulin treatment in both groups (p < 0.05; Supplementary Fig. 1B).
We also evaluated the relationship between the IA-2 antibody titer and the clinical course over 5 years after diagnosis. We classified the patients according to high and low titers of IA-2 antibodies at diagnosis. First, we compared postprandial serum CPR levels during the same period and analyzed them as GAD antibodies. There was no significant difference in CPR levels over 5 years after diagnosis between the two groups (Supplementary Fig. 2A). Secondly, we compared HbA1c levels during the same period in children with high and low titers of IA-2 antibodies between the groups. Children who had a high titer of IA-2 antibodies had significantly higher HbA1c levels than those with a low titer of IA-2 antibodies at diagnosis. Otherwise, there were no significant differences between the groups (Supplementary Fig. 2B). However, there was a significant improvement in HbA1c levels after starting insulin treatment in children with high titers of IA-2 antibodies (p < 0.05; Supplementary Fig. 2B). There was also a significant improvement in HbA1c at diagnosis and at 3 months, 6 months, and 1 year period after diagnosis in children with low titers of IA-2 antibodies (p < 0.05; Supplementary Fig. 2B).
Discussion
We evaluated the relationship between the number of positive islet-cell associated antibodies and the clinical course over 5 years after diagnosis in children with T1D. One and two years after diagnosis, children who were positive for multiple islet-cell associated antibodies showed significantly lower serum CPR levels than those who were positive for single islet-cell associated antibodies. Zinger et al. [5] reported that children at risk of T1D with the human leukocyte antigen DR/DQ genotype who had multiple islet-cell associated antibodies were likely to progress to diabetes over the next 15 years. Therefore, children who had multiple islet-cell associated antibodies showed faster progression to T1D and were younger than those with single positive islet-cell associated antibodies. Islet-cell associated antibodies are produced as a consequence of islet-cell destruction by the autoimmune process. The islet-cell associated antibodies bind and form immune complexes, which are likely to promote islet inflammation [5]. IA-2 antibodies appear later than GAD antibodies in the prediabetic period; therefore, the appearance of IA-2 antibodies predicts progression to diabetes within the early period [20]. It has been reported that individuals positive for only single islet-cell associated antibodies present no histopathological evidence of immune-mediated β-cell destruction. Insulitis appears to be more among those who were positive for multiple antibodies and was a high risk factor for developing symptomatic T1D [21]. Children with multiple islet-cell associated antibodies have a lower first-phase insulin response than those with single islet-cell associated antibodies. This study supports the view that the more severe the disturbance is in insulin production, the more likely it is for the development of symptomatic T1D [10]. Children with T1D with multiple islet-cell associated antibodies were associated with a more rapid loss in β-cell secretory capacity [22]. However, the underlying mechanism remain unknown. Our results are similar to those of previous reports [22, 23]. Those who are positive for multiple islet-cell associated antibodies have higher immune activity and a greater decline in β-cell function than those who are positive for single islet-cell associated antibodies.
There was a significant improvement in HbA1c levels after starting insulin treatment in all patients. We demonstrated an improvement in glycemic control and maintenance of optimal glycemic control by performing appropriate intensive insulin therapy in all patients. We adjusted basal insulin regimen to achieve fasting blood glucose levels of 100–120 mg/dL. Moreover, we adjusted bolus insulin regimen to achieve postprandial blood glucose levels of ≤150 mg/dL using carbo-counting method. Sugihara et al. [24] reported that patients with childhood-onset T1D who reached 18–20 years of age may have difficulty in controlling their blood glucose levels. The control of blood glucose levels in female adolescents is considered more difficult than that in male adolescents because insulin resistance is greater in females adolescents than in male adolescents [25]. Younger children are likely to engage more in physical activity and consume less food. They lack counterregulatory hormone responses to subsequent hypoglycemia via an automatic mechanism. Moreover, younger children tend to have more frequent and serious episodes of hypoglycemia than later-onset patients with T1D [26-29]. Due to the increased use of basal-bolus insulin regimens using appropriate insulin analogs, a previous study indicated an improvement in glycemic control and a decrease in the incidence rate of severe hypoglycemic events [30]. Regular self-monitoring of glucose is essential for diabetes management in all children with T1D to reduce the risk of acute and chronic disease complications [31]. Yokoyama et al. [32] reported that the cumulative incidence of nephropathy after 30 years of post-pubertal diabetes was significantly higher in early-onset diabetes. Therefore, it is important to maintain appropriate blood glucose levels during childhood and adolescence to reduce the risk of developing chronic diabetic complications.
Our study has several limitations. First, the number of children with T1D was small. Due to the different times of diagnosis, some data, including serum CPR and HbA1c levels, were deficient in some children during the 5-year study period. Second, we did not examine ZnT8 antibodies. Previous studies have reported that ZnT8 antibodies are associated with acute-onset and childhood-onset in patients with T1D and are considered a more specific marker of autoimmune-mediated β-cell destruction [33, 34]. IAA was also not examined in the present study. It is an important marker for developing T1D [35]. Third, the measurements of CPR levels, GAD, and IA-2 antibodies were analyzed in different clinical laboratories with different reference levels.
In conclusion, residual insulin secretion ability may be predicted based on the number of positive islet-cell associated antibodies. Patients with multiple islet-cell associated antibodies tend to have deteriorated β-cell function earlier than those with a single islet-cell associated antibody. Intensive insulin treatment is essential and can help achieve optimal glycemic control in children with T1D. However, it is difficult for young children and adolescents to maintain appropriate glycemic control because of their irregular lifestyle and eating habits. Complete deficiency of endogenous insulin is also one of the factors causing difficulty in achieving appropriate glycemic control. Although there are differences in residual β-cell function, optimal glycemic control can be achieved by individualized appropriate insulin regimens, even in children with T1D.
Acknowledgment
Not applicable.
Disclosure
T.U. declares having received funding from Novo Nordisk Pharma Ltd., Eli Lilly Japan K.K., Terumo Corp., and JCR Pharmaceuticals Co., Ltd. Outside the submitted work, I.M. has received lecture fees from AstraZeneca K.K., MSD Co., Ltd., Pfizer Japan, Inc., Novo Nordisk Pharma Ltd., Shionogi Co., Ltd., AbbVie LLC, Sandoz AG., Shino-Test Corp., JCR Pharmaceuticals Co., Ltd., Alexion Pharmaceutical Inc., and Atom Medical Corp. Other authors have no relevant conflicts of interest to disclose.
References
- 1 Cooper JJ, Haller MJ, Greenbaum CJ, Ziegler AG, Wherrett DK, et al. (2018) ISPAD Clinical Practice Consensus Guidelines 2018: stages of type 1 diabetes in children and adolescents. Pediatr Diabetes 19 Suppl 27: 20–27.
- 2 Mayer-Davis EJ, Kahkoska AR, Jefferies C, Dabelea D, Balde N, et al. (2018) ISPAD Clinical Practice Consensus Guidelines 2018: definition, epidemiology, and classification of diabetes in children and adolescents. Pediatr Diabetes 19 Suppl 27: 7–19.
- 3 Regnell SE, Lernmark A (2017) Early prediction of autoimmune (type 1) diabetes. Diabetologia 60: 1370–1381.
- 4 Vehik K, Bonifacio E, Lernmark A, Yu L, Williams A, et al. (2020) Hierarchical order of distinct autoantibody spreading and progression type 1 diabetes in the TEDDY study. Diabetes Care 43: 2066–2073.
- 5 Ziegler AG, Rewers M, Simell O, Simell T, Lempainen J, et al. (2013) Seroconversion to multiple islet autoantibodies and risk of progression to disease in children. JAMA 309: 2473–2479.
- 6 Kawasaki E, Nakamura K, Kuriya G, Satoh T, Kobayashi M, et al. (2011) Differences in the humoral autoreactivity to zinc transporter 8 between childhood- and adult-onset type 1 diabetes in Japanese patients. Clin Immunol 138: 146–153.
- 7 Kawasaki E, Nakamura K, Kuriya G, Satoh T, Kuwahara H, et al. (2010) Autoantibodies to insulin, insulinoma-associated antigen-2, and zinc transporter 8 improve the prediction of early insulin requirement in adult-onset autoimmune diabetes. J Clin Endocrinol Metab 95: 707–713.
- 8 Larsson HE, Larsson C, Lernmark A (2015) Baseline heterogeneity in glucose metabolism marks the risk for type 1 diabetes and complicates secondary prevention. Acta Diabetol 52: 473–481.
- 9 Martinez MM, Spiliopoulos L, Salami F, Agardh D, Toppari J, et al. (2022) Heterogeneity of beta-cell function in subjects with multiple islet autoantibodies in the TEDDY family prevention study-TEFA. Clin Diabetes Endocrinol 7: 23.
- 10 Martinez MM, Salami F, Larsson HE, Toppari J, Lernmark A, et al. (2020) Beta-cell function in participants with single or multiple islet autoantibodies at baseline in the TEDDY Family Prevention Study: TEFA. Endocrinol Diab Metab 4: e00198.
- 11 Deshpande A, Harris-Hayes M, Schootman M (2008) Epidemiology of diabetes and diabetes-related complications. Phys Ther 88: 1254–1264.
- 12 Urakami T, Suzuki J, Yoshida A, Saito H, Mugishima H (2008) Incidence of children with slowly progressive form of type 1 diabetes detected by the urine glucose screening at schools in the Tokyo Metropolitan Area. Diabetes Res Clin Pract 80: 473–476.
- 13 Urakami T, Miyamoto Y, Fujita H, Kitagawa T (1989) Type 1 (insulin-dependent) diabetes in Japanese children is not a uniform disease. Diabetologia 32: 312–315.
- 14 Urakami T, Miyamoto Y, Matsunaga H, Owada M, Kitagawa T (1995) Serial changes in the prevalence of islet cell antibodies and islet cell antibody titer in children with IDDM of abrupt or slow onset. Diabetes Care 18: 1095–1099.
- 15 Wolfsdorf JI, Glaser N, Agus M, Fritsch M, Hanas R, et al. (2018) ISPAD Clinical Practice Consensus Guidelines 2018: diabetic ketoacidosis and the hyperglycemic hyperosmolar state. Pediatr Diabetes 19 Suppl 27: 155–177.
- 16 Gresch SC, Mutch LA, Janecek LJ, Hegstad-Davies RL, Graham ML (2017) Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum. Xenotransplantation 24: e12320.
- 17 Yasui J, Kawasaki E, Tanaka S, Awata T, Ikegami H, et al. (2016) Clinical and genetic characteristics of non-insulin-requiring glutamic acid decarboxylase (GAD) autoantibody-positive diabetes: a nationwide survey in Japan. PLoS One 11: e0155643.
- 18 Ota T, Takamura T, Bando Y, Usuda R, Nagai Y (2003) Predictive value of autoantibodies to IA-2 for insulin requirements in Japanese subjects with type 1 diabetes. Diabetes Care 26: 3188–3189.
- 19 Kawasaki E, Oikawa Y, Okada A, Kanatsuna N, Kawamura T, et al. (2018) Evaluation of IA-2Ab ELISA “Cosmic” kit in type 1 diabetes—Comparison with IA-2Ab “Cosmic” (RIA method)—. Jpn J Med Pharm Sci 75: 669–680 (In Japanese).
- 20 De Grijse J, Asanghanwa M, Nouthe B, Albrecher N, Goubert P, et al. (2010) Predictive power of screening for antibodies against insulinoma-associated protein 2 beta (IA-2β) and zinc transporter-8 to select first-degree relatives of type 1 diabetic patients with risk of rapid progression to clinical onset of the disease: implications for prevention trials. Diabetologia 53: 517–524.
- 21 Smeets S, Paep DLD, Stange G, Verhaeghen K, Van der Auwera B, et al. (2021) Insulitis in the pancreas of non-diabetic organ donars under age 25 years with multiple circulating autoantibodies against islet cell antigens. Virchows Arch 479: 295–304.
- 22 Koskinen MK, Mikk ML, Laine AP, Lempainen J, Loyttyniemi E, et al. (2020) Longitudinal pattern of first-phase insulin response is associated with genetic variants outside the class Ⅱ HLA region in children with multiple autoantibodies. Diabetes 69: 12–19.
- 23 Kasuga A, Maruyama T, Nakamoto S, Ozawa Y, Suzuki Y, et al. (1999) High-titer autoantibodies against glutamic acid decarboxylase plus autoantibodies against insulin and IA-2 predicts insulin requirement in adult diabetic patients. J Autoimmune 12: 131–135.
- 24 Sugihara S, Kikuchi T, Urakami T, Yokota I, Kikuchi N, et al. (2021) Residual endogenous insulin secretion in Japanese children with type 1A diabetes. Clin Pediatr Endocrinol 30: 27–33.
- 25 Hoffman RP, Vicini P, Sivitz WI, Cobelli C (2000) Pubertal adolescent male-female differences in insulin sensitivity and glucose effectiveness determined by the one compartment minimal model. Pediatr Res 48: 384–388.
- 26 Cody D (2007) Infant and toddler diabetes. Arch Dis Child 92: 716–719.
- 27 Abraham MB, Jones TW, Naranjo D, Karges B, Oduwole A, et al. (2018) ISPAD Clinical Practice Consensus Guidelines 2018: assessment and management of hypoglycemia in children and adolescents with Diabetes. Pediatr Diabetes 19 Suppl 27: 178–192.
- 28 Matyka KA, Crowne EC, Havel PJ, Macdonald IA, Matthews D, et al. (1999) Counterregulation during spontaneous nocturnal hypoglycemia in prepubertal children with type 1 diabetes. Diabetes Care 22: 1144–1150.
- 29 Urakami T (2020) Severe hypoglycemia: is it still a threat for children and adolescents with type 1 diabetes? Front Endocrinol (Lausanne) 11: 609.
- 30 Mochizuki M, Kikuchi T, Urakami T, Kikuchi N, Kawamura T, et al. (2017) Improvement in glycemic control through changes in insulin regimens: findings from a Japanese cohort of children and adolescents with type 1 diabetes. Pediatr Diabetes 18: 435–442.
- 31 DiMeglio LA, Acerini CL, Codner E, Craig ME, Hofer SE, et al. (2018) ISPAD Clinical Practice Consensus Guidelines 2018: glycemic control targets and glucose monitoring for children, adolescents, and young adults with diabetes. Pediatr Diabetes 19 Suppl 27: 105–114.
- 32 Yokoyama H, Okudaira M, Otani T, Sato A, Miura J, et al. (2000) Higher incidence of diabetic nephropathy in type 2 than in type 1 diabetes in early-onset diabetes in Japan. Kidney Int 58: 302–311.
- 33 Kawasaki E, Oikawa Y, Okada A, Kanatsuna N, Kawamura T, et al. (2020) Zinc transporter 8 autoantibodies complement glutamic acid decarboxylase and insulinoma-associated antigen-2 autoantibodies in the identification and characterization of Japanese type 1 diabetes. J Diabetes Investig 11: 1181–1187.
- 34 Kawasaki E (2012) ZnT8 and type 1 diabetes. Endocr J 59: 531–537.
- 35 Brooks-Worrell B, Gersuk VH, Greenbaum C, Palmer JP (2001) Intermolecular antigen spreading occurs during the preclinical period of human type 1 diabetes. J Immunol 166: 5265–5270.
