Introduction
In 2011, 370 million people suffered from diabetes worldwide and 80% of them were from developing countries, according to the estimates released by the International Diabetes Federation. The prevalence has reached 11.6% in China, according to the 2010 report from Chinese Center for Disease Control and Prevention. Patients with type 2 diabetes mellitus (T2DM) roughly account for 90% of all diabetic cases. Insulin is an important means of diabetes treatment. Insulin antibodies (IA) can be developed upon initiation of insulin treatment [
1]. Their binding with insulins, forming insulin-antibody complexes which compete with insulins for the binding site of insulin receptors or serve as an insulin pool to reduce insulin activity and irregularly release insulin, ultimately results in hyperinsulinemia and hypoglycemia. Unfortunately, hyperinsulinemia has been proven to be a risk factor of wide glycemic fluctuation [
2], increased blood pressure [
3], and the occurrence of several tumors [
4], Alzheimer’s disease [
5] and microvascular dysfunction that leads to cardiovascular diseases [
6,
7]. Both insulin-antibody complexes and exogenous insulin antibodies (IAs) have been shown to involve in the development of lipoatrophy and microangiopathy, except for glycemic control, via altered insulin pharmacokinetics, insulin resistance, and other mechanisms [
8].
As for diabetic complications, patients detected exogenous IA positive due to intake of sulfhydryl-containing agents often suffer from insulin autoimmune syndrome (IAS), which is characterized by spontaneous hypoglycemia and endogenous hyperinsulinemia with a high titer of anti-insulin antibodies. For T2DM patients with insulin therapy, IAs may cause high glucose fluctuation and exogenous hyperinsulinemia, which is called exogenous insulin-related insulin autoimmune syndrome (EIAS). IAs preclude the possibility of glucose control and act as a barrier to avoid related complications.
Exogenous IAs have a non-negligible impact on T2DM in China. Dong et al. reported a relatively high ratio of 220/742 exogenous IA-positive patients in Shandong Province [
9]. However, the only two studies of exogenous IAs in China were both conducted in urban tertiary 3A hospitals. Any study that provides data for IA-associated measurements of rural patients is urgently needed, who have known risks of treatment incompliance. Therefore, the goal of this study is twofolded: to detect blood IA levels in rural T2DM patients, to explore relationships of exogenous IAs with clinical characteristics of patients and insulin usage in this patient group.
Methods
Subjects and designs
Between August 2019 and June 2020, patients who were residents from the Danxi community and six towns (Dongchen, Xizhou, Maoyang, Sizhoutou, Juexi, and Qiangtou) in Xiangshan County Zhejiang Province and diagnosed with T2DM and treated with insulins at Xiangshan Hospital of TCM Medical and Health Group were selected. Patients diagnosed with T2DM and receiving insulin therapy for at least 2 weeks were included. But they were excluded if they had (1) IAS at or before diagnosis; (2) a history of treatment with sulfhydryl-containing agents in the past 6 months; (3) moderate-to-severe liver or renal dysfunction with alanine aminotransferase (ALT) or glutamic oxalacetic transaminase (AST) > 100 U/L, or estimated glomerular filtration rate (eGFR)< 45mL/(min*1.73m2); (4) myocardial infarction, cerebral infarction, trauma, operation, and stress state occurred in the past 6 months; (5) acute complications; and (6) types of insulin changed in recent 2 years. The study protocol was approved by the ethical committee of Xiangshan Hospital of TCM Medical and Health Group (p2019-[k]-10).
All subjects were asked to undergo the 75-g oral glucose tolerance test (OGTT) after 10–12 h of fasting. No insulin injection or oral antidiabetic agents (OADs) were administered the night before and on the morning of the test and after the test, with a continuation of insulins. Blood samples were collected during the fasting glucose test and OGTT. We assessed fasting and 2-h post-OGTT glucose, insulin, and C-peptide levels. Fasting blood IA, glutamic acid decarboxylase antibody (GADA), islet cell antibody (ICA), and glycated hemoglobin (HbA1c) levels were quantitated.
Biochemical assays
Serum insulin and C-peptide were determined by immunoelectrochemiluminescence (Roche Diagnostics GmbH, Mannheim, Roche E601, Germany), and hexokinase method was performed to quantitate fasting and 2-h blood glucose levels (Meikang biology, Beckman AU5800, China). We employed high-performance liquid chromatography (Huizhong, Medicine, MQ-2000PT, China) to detect HbA1c. Serum IA was tested with radioimmunoassay (North Institute, Gamma counter sn-6105, China), while ICA and GADA were detected by indirect immunofluorescence assay (Euroimmun, fluorescence microscope EURostarIII, China).
Statistical analysis
Continuous variables were expressed as mean ± standard error of the mean (SEM). Differences between IA-positive and IA-negative groups were compared with the Student t-test if data were normally distributed. The chi-square test was applied for categorical variables. All statistical analyses were performed using SPSS24.0 (SPSS, Palo Alto, CA, USA) and Graphpad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). The significance level was set at p < 0.05.
Discussion
IAs are immunoglobulin, antibodies against insulin, of which IgG-class IAs are most common, followed by IgM-, IgA-, and IgD-class IAs. Insulin often binds to plasma proteins to form multimers. Insulins are primarily targeted by specific antibodies, with binding capacity reaching up to 10,000μU/ml. Once supersaturated insulin is formed, one molecule of IA will antibody can bind to two molecules of insulin, forming an Ab1Ag2 complex (molecular weight 162,000). Specifically, IA-insulin binding can form Ab1Ag2 complexes when a low amount of blood IAs are present or form Ab2Ag1 complexes (molecular weight 306,000) when there are excessive IAs, or constitutes (AbAg)n immune macromolecular complexes when the amounts of insulin and IAs are comparable. However, Ab1Ag2 complexes are unstable, cannot be cleared by the reticuloendothelial system, completely contrary to (AbAg)n complexes [
10]. Insulin-receiving individuals with high IA levels and recurrent hypoglycemia had a higher dissociation constant for insulin; furthermore, this study reported that IA characteristics were among the causative factors in hypoglycemic episodes [
11]. Therefore, monitoring IA expression monitoring and follow-up treatments of potential complications are particularly important.
In 1960, Harwood first reported the presence of IAs in patients receiving exogenous insulin therapy [
12]. As we mentioned above, IA+ patients are more likely to develop hyperinsulinemia more often than IA− patients without IA expression. Due to the mitotic effect of insulin, hyperinsulinemia can promote cell proliferation in vascular wall cells, resulting in thickening of the vascular wall and local deposition of cholesterol. Therefore, hyperinsulinemia has a strong correlation with cardio-cerebrovascular diseases. In addition, IA+ immune macromolecular complexes can cause vascular damage and contribute to diabetic complications.
In this study, blood IAs were detectable in 40.2% (314/781) of all patients, very close to 44% reported by Fineberg et al. [
13] in an Indiana population, but much higher than the percentage achieved in an urban Chinese population [
9]. This difference can be explained by several reasons. Physicians in county hospitals lack knowledge of the clinical significance of blood IA in T2DM. Glucose control among IA+ patients is unsatisfactory due to glucose fluctuation without regular glucose tests. Furthermore, patients in rural areas often have higher risks of poor compliance with tests or loss to follow-up.
We found that FBG and 2hBG levels significantly decreased and 0’ and 120’ serum insulin and insulin/C peptide ratio markedly increased in the IA+ versus IA− groups. It can be explained by the biological characteristics of IA [
1,
14]. IA accumulation or IA+ complexes in blood have been shown to have a robust ability to bind to a vast array of endogenous and exogenous insulin. Insulin can only be slowly dissociated from the complexes slowly in the absence of exogenous insulins and perform pharmacological actions later. This phenomenon is called a reservoir-like effect [
14], which contributes to lower concentrations of FBG and 2HBG in T2DM patients (Table
1). Although IA+ patients exhibited lower FPG and 2hPG levels and greater glucose fluctuation than IA− patients, both of which could not be discriminated by HbA1c.
Previous studies have ascertained that chronic hyperinsulinemia can lead to the decrease of insulin sensitivity [
15]. It is reasonable to speculate that hyperinsulinemia in T2DM patients with IA will lead to an inadequate response to insulins, stimulating endogenous insulin production, as manifested by elevated serum C-peptide.
Overweight or obesity may trigger specific and nonspecific autoimmunity [
16]. Therefore, BMI may be positively correlated with IA levels. Consistently, we found that BMI in the IA+ group was indeed than that in the IA− group.
Rajan et al. reported that chronic hyperinsulinemia could reduce insulin sensitivity in T2DM patients [
16], which required increased insulin dosage. Furthermore, the binding of IA binds to free insulin may reduce the amount of functional insulin in blood, resulting in increased insulin requirement. When insulin combines with IAs with different ratios, insulin-IA complexes of different sizes and molecular weights may exert different biological effects. Their macromolecular complexes can stimulate downstream signaling cascades upon extensive insulin-IA binding. Therefore, blood IAs are more associated with insulin dosage than insulin treatment duration.
In this study, the proportion of IA+ patients among all T2DM patients was comparable between those receiving premixed human insulins and rapid-acting analogs, consistent with the findings reported by Home et al. [
17] that there was similar immunogenicity between the two patient groups. As insulin stimulates IA production, patients treated with premixed human insulins group and rapid-acting analogs exhibited significantly higher proportions of IA+ patients than those receiving basal insulin support. The underlying mechanisms can be persistent low serum insulin levels induced by long-acting insulin analogs (insulin multimers) under the skin and weak antigenicity induced by the modified allergenic amino acid B30 in long-acting insulin analogs. Patients receiving a combination of basal and rapid-acting insulin analogs also revealed a higher proportion of IA+ cases than those treated with basal insulin therapy. This may be due to various immune responses triggered by types of insulin analogs [
18] and increased risk of IA development by repeated insulin injections [
19]. However, mechanisms responsible for a high percentage of IA+ patients among the three insulin regimens have not yet been fully explored.
As for physicians in county hospitals, personalized therapeutic decision-making in T2DM patients to reduce the rate of IA+ patients is feasible to improve blood glucose management and the use of insulin analogs. Thus, diabetic complications can be prevented or delayed to circumvent irreversible organ damage to ameliorate the prognosis and life quality in T2DM patients.
Declarations
We the undersigned declare that this manuscript entitled “Analysis of the status of insulin antibody positive rate in T2DM patients in county hospitals in China” is original, has not been published before, and is not currently being considered for publication elsewhere.
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