Latent Autoimmune Diabetes in Adults in North Indian Region: Assessment of β-Cell Function,Metabolic and Immunological Features

Abstract

Background:

We undertook a study to assess β-cell function, metabolic and immunological features of patients with latent autoimmune diabetes in adults (LADA) and investigate heterogeneity within LADA based on low and high glutamic acid decarboxylase autoantibodies (GADA) titers.

Methods:

A total of 139 patients with adult-onset diabetes were examined cross-sectionally in the National capital region of Northern India. Medical history of all subjects was reviewed with the aim of collecting clinical data. Glucose, glycosylated hemoglobin, lipid profile, creatinine, C-peptide, and GADA were measured in 10–12 hrs fasting blood sample.

Results:

Assessment of metabolic features revealed lower mean systolic blood pressure in subjects with LADA than in those with type 2 diabetes (DM2). Mean triglyceride levels were lower in LADA subjects compared to DM2 subjects. Compared to DM2 subjects, prevalence of metabolic syndrome (MS) was also lower in LADA subjects. Compared to GADA-low, all GADA-high patients were male, had lower waist circumference, fasting C-peptide (FCP), and prevalence of MS. Compared to DM2 patients, GADA-high patients were younger, had lower age at onset, body mass index, waist circumference, systolic blood pressure, triglycerides, FCP, and prevalence of MS. The rate of patients on insulin was higher in GADA-high compared to DM2. There were no significant differences between characteristics of DM2 and GADA-low patients.

Conclusions:

Our results indicate that LADA patients have distinct metabolic features with lower residual β-cell function than DM2 patients. GADA titer is important parameter in defining the severity of the disease as patients with high GADA titer tend to have significant β-cell impairment.

Introduction

In the past, diabetes was considered to be a disease specific to the developed world but in the last few decades, the prevalence of diabetes in developing countries has increased exponentially. Almost three quarter of diabetic population live in low- or middle-income economies. In India, the prevalence of diabetes in adults is 9.3%.¹

Type 2 diabetes (DM2) is the prevalent form of diabetes among adults; however, adulthood does not exclude autoimmune diabetes.² A subset of adults, who are initially presumed to have DM2, show islet autoantibodies in their sera and are termed as latent autoimmune diabetes in adults (LADA).³ These patients do not require insulin at the diagnosis of diabetes but generally progress toward insulin requirement earlier than DM2 patients.^4,5 Compared to the high prevalence of 9.7% reported by a large European Action LADA study, lower prevalence of LADA was observed in Asians ranging from 2.6% to 5.7%.^6

–10 Glutamic acid decarboxylase autoantibodies (GADA) are considered to be the ideal marker for screening of LADA.¹¹ In the Western and Chinese populations, heterogeneity has been observed in the clinical and metabolic features of low GADA titer LADA patients and high GADA titer LADA patients.¹²

Although, LADA is autoimmune in nature it also shares some metabolic features with DM2.^2,13 Data about association of metabolic syndrome (MS) with LADA are inconclusive. A Chinese study demonstrated a close association of LADA with MS, which was comparable to DM2 patients.¹⁴ In contrast, a large multicenter European study showed significantly higher prevalence of MS in DM2 patients than in LADA patients and concluded that MS is not a characteristic of LADA.¹⁵ Features of LADA differ between different populations.¹⁶ Data related to β-cell function, MS, and features based on GADA titer in LADA patients are not available in North Indians. Information on various characteristics of LADA may help in better understanding of the disease process and finding appropriate treatment strategies in this population.

Therefore, we aimed to define β-cell function metabolic and immunological features and assess heterogeneity within LADA based on low and high GADA titers. All features of LADA were compared with DM2 patients.

Materials and Methods

This study was conducted in the National capital region of North India. The study design was cross-sectional and recruited consecutive patients with adult-onset diabetes who attended Diabetes Clinic and fulfilled the inclusion criteria. All subjects were aged 30–70 years and diagnosed within last 5 years. Total of 139 subjects that included 9 LADA and 130 DM2 subjects were studied. LADA and DM2 in the studied subjects were defined on the basis of GADA status and time to insulin initiation. GADA negative subjects were diagnosed as DM2, and subjects with positive GADA who did not require insulin at least in first 6 months after diagnosis were defined as LADA.

Medical history of all subjects was reviewed with the aim of collecting clinical data. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured twice in sitting position. An average of both reading was used for analysis. Glucose, glycosylated hemoglobin (HbA1c), lipid profile, creatinine, C-peptide, and GAD-65 antibody (GADA) were measured from a 10–12 hrs fasting blood sample.

Diagnosis of MS

MS was assessed according to the revised National Cholesterol Education Program (NCEP): Adult Treatment Panel III criteria¹⁷ All subjects fulfilled the criteria of hyperglycemia. In addition, two of the following criteria were required for the diagnosis of MS:

1. Waist circumference ≥90 cm (Asian male) or ≥80 cm (Asian female).

2. Serum triglyceride ≥150 mg/dL or use of drug treatment for dyslipidemia.

3. Serum high-density lipoprotein cholesterol (HDL-C) ≤40 mg/dL (male) or ≤50 mg/dL (female).

4. Systolic blood pressure (SBP) ≥130 mmHg and/or DBP ≥85 mmHg or use of antihypertensive medication.

Biochemical analysis and antibody assay

A fasting blood sample was collected and analyzed locally, using standardized assays to measure glucose, HbA1c, the lipid profile, including the measurement of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and HDL-C, creatinine, C-peptide, and GAD-65 antibody. HbA1c was performed using Bio-Rad Laboratories, Inc. VARIANT™ II Dual (A2/F/A1c) instrument by ion-exchange high performance liquid chromatography, a National glycohemoglobin Standardization program (NGSP) certified method. C-peptide was measured to assess insulin secretion. It was analyzed using Siemens ADVIA Centaur XP^® immunoassay system by direct chemiluminescent technology with coefficient of variation <10% and normal reference range of 0.48 to 5.05 ng/mL.¹⁸

GAD-65 antibody

GAD-65 antibody was analyzed using RSR-ELISA Kits (RSR Limited, Cardiff, UK) with 98% specificity and 92% sensitivity in the Diabetes Antibody Standardization Program (DASP) 2005.^19,20 Assay cutoff of <5 U/mL was considered negative and ≥5 U/mL was considered positive.²¹ Our results did not show a bimodal distribution of GADA titer. Hence, to analyze the characteristics of LADA subjects according to GADA titer, based on the median value of GADA titer, LADA subjects were stratified into two subgroups: GADA-high titer (>13.6 U/mL) and GADA-low titer (≤13.6 U/mL). Due to small sample size, LADA subjects were not divided into tertiles, that is, low, medium, and high titer levels. Specimens for C-Peptide and GADA were transported, refrigerated, and processed same day or were frozen at or below −20°C if the sample was not assayed within 24 hrs.

Statistical analysis

Quantitative variables are described as means ± standard deviations or medians and interquartile ranges. Categorical variables were described as n (%). Mean values between groups were compared using Independent t-test/Mann–Whitney U test. The comparison of categorical variables was analyzed using Chi-square/Fisher's exact test.

IBM SPSS statistics for windows software (version 21.0; Armonk, NY) and an α value of 0.05 for statistical significance were used for all analyses.

For the purpose of C-peptide secretion analysis, disease duration (in months) was calculated as the period between the date of diagnosis and the date of the study assessment. The disease duration was stratified into two periods (<36 months and >36 months) for the two categories analyzed, that is, LADA and DM2.

Results

Metabolic characteristics

The metabolic characteristics of LADA and DM2 patients are summarized in Table 1. LADA subjects presented a different metabolic profile than DM2 subjects. Assessment of metabolic features revealed lower mean SBP in subjects with LADA than in those with DM2 (115.3 ± 16.4 vs. 126.5 ± 14.0 mmHg; P = 0.033). Mean triglyceride levels (107 (92–141) vs. 151 (112.3–210) mg/dL; P = 0.033) were lower in LADA subjects compared to DM2 subjects. Compared to DM2 subjects, prevalence of MS was also lower in LADA subjects (44.4% vs. 88.5%; P = 0.003). There was no significant difference in fasting plasma glucose (P = 0.898) and HbA1c (P = 0.096) between LADA and DM2 subjects, but both parameters were higher in LADA patients than in DM2 patients. Other parameters like cholesterol-total, LDL-C, HDL-C, and DBP did not differ between two groups. Not significantly different but the proportion of those on insulin treatment was higher in LADA subjects than in DM2 subjects (22.2% vs. 4.6%; P = 0.085). Similarly, mean time to start insulin was lower in LADA subjects than in DM2 subjects (0.449). The characteristics of LADA and DM2 patients are summarized in Table 1.

Table 1.

Characteristics of LADA Versus DM2 Subjects

Cases	LADA (n = 9)	DM2 (n = 130)	P
SBP (mmHg)	115.3 ± 16.4	126.5 ± 14.0	0.023^*
DBP (mmHg)	80.0 [65.5–82.0]	80.0 [75.0–84.3]	0.330
Antihypertensive treatment, n (%)	2 (22.2)	44 (33.8)	0.718
Triglycerides (mg/dL)	107.0 [92.0–141.0]	151.0 [112.3–210]	0.033^*
Total cholesterol (mg/dL)	160.4 ± 35.7	167.8 ± 38.1	0.577
HDL-C (mg/dL)	46.1 ± 7.5	43.2 ± 10.8	0.425
LDL-C (mg/dL)	104 ± 33.9	103.3 ± 31.2	0.948
MS, n (%)	4 (44.4)	115 (88.5)	0.003^*
Fasting plasma glucose (mg/dL)	139 [117.5–162.5]	137 [120–169.3]	0.898
HbA1c (%)	8.1 [7.0–10.6]	7.1 [6.6–8.5]	0.096
FCP (ng/mL)	1.5 ± 0.9	2.3 ± 0.8	0.009^*
Insulin treatment, n (%)	2 (22.2)	6 (4.6)	0.085
Time to insulin (months)	25.0 ± 26.8	47.2 ± 5.3	0.449

Data are means ± SD, median [IQR] or n (%), ^*Significant P values.

DBP, diastolic blood pressure; DM2, type 2 diabetes; FCP, fasting C-peptide; HbA1c, glycosylated hemoglobin; HDL-C, high-density lipoprotein cholesterol; IQR, interquartile range; LADA, latent autoimmune diabetes in adults; LDL-C, low-density lipoprotein cholesterol; MS, metabolic syndrome; SBP, systolic blood pressure; SD, standard deviations.

Immunologic characteristics

LADA GADA-high subjects versus GADA-low subjects

Patients with GADA at high titer (GADA-high, n = 4) were compared with GADA at low titer (GADA-low, n = 5) group. Compared to GADA-low patients, all GADA-high patients were male (0% vs. 80%; P = 0.048), had lower body mass index (BMI) (22.6 ± 4.1 vs. 28.1 ± 2.4 kg/m²; P = 0.040), waist circumference (82.6 ± 7.4 vs. 94.0 ± 4.8 cm; P = 0.026), fasting C-peptide (FCP) (0.8 ± 0.7 vs. 2.1 ± 0.6 ng/mL; P = 0.025), and prevalence of MS (0% vs. 80%; P = 0.048). No statistical significance was observed between two groups although LADA GADA-high patients required insulin more frequently (50% vs. 0%; P = 0.167). The characteristics of GADA-high versus GADA-low titer are presented in Table 2.

Table 2.

Characteristics of GADA-High Versus GADA-Low LADA in Adult Subjects

Cases	GADA-high (n = 4)	GADA-low (n = 5)	P
Female, n (%)	0 (0)	4 (80)
Male, n (%)	4 (100)	1 (20)	0.048^*
Age (years)	37.3 ± 3.6	43.6 ± 9.2	0.236
Age at diagnosis (years)	33.3 ± 3.0	40.2 ± 8.8	0.178
Disease duration (months)	50 ± 11.2	42.8 ± 14.1	0.433
BMI (kg/m²)	22.6 ± 4.1	28.1 ± 2.4	0.040^*
Waist (cm)	82.6 ± 7.4	94.0 ± 4.8	0.026
SBP (mmHg)	105.5 ± 9.0	123.2 ± 17.3	0.109
DBP (mmHg)^*	70.5 ± 9.5	79.2 ± 12.5	0.288
Antihypertensive treatment, n (%)	0 (0)	2 (40)	0.444
Triglycerides (mg/dL)^*	96.8 ± 12.7	131.2 ± 34.7	0.092
Total cholesterol (mg/dL)	157.5 ± 37.2	163 ± 38.6	0.841
HDL-C (mg/dL)	47.8 ± 6	44.8 ± 9.0	0.590
LDL-C (mg/dL)	100 ± 39.4	107.2 ± 33.5	0.775
MS, n (%)	0 (0)	4 (80)	0.048^*
Fasting plasma glucose (mg/dL)^*	140 ± 29.9	154.8 ± 57.8	0.659
HbA1c (%)^*	8.9 ± 3.2	8.8 ± 1.7	0.953
Family history of diabetes, n (%)	1 (25)	3 (60)	0.524
FCP (ng/mL)	0.8 ± 0.7	2.1 ± 0.6	0.025^*
Insulin treatment	2 (50)	0 (0)	0.167

Data are means ± SD or n (%), ^*Significant P values.

BMI, body mass index; GADA, glutamic acid decarboxylase autoantibodies.

LADA subjects with GADA at high titer (GADA-high) versus DM2 subjects

Patients with GADA at high titer (GADA-high, n = 4) were compared with DM2 (n = 130) group. Compared to DM2 patients, GADA-high patients were younger (37.3 ± 3.6 vs. 47.2 ± 9.3 years; P = 0.035), had lower age at onset (33.3 ± 3.0 vs. 44.4 ± 9.4 years; P = 0.020), BMI (22.6 ± 4.1 vs. 28.3 ± 4.8 kg/m²; P = 0.040), waist circumference (82.6 ± 7.4 vs. 96.8 ± 9.8 cm; P = 0.005), SBP (105.5 ± 9.0 vs. 126.5 ± 14.0 mmHg; P = 0.003), triglycerides (99.5 [83.8–107.0] vs. 151 [210–112.3] mg/dL; P = 0.026), FCP (0.8 ± 0.7 vs. 2.3 ± 0.8 ng/mL; P = 0.001), and prevalence of MS (0% vs. 88.5%; P < 0.001). The rate of patients on insulin was higher in GADA high compared to DM2 (50% vs. 4.6%; P = 0.018). The characteristics of GADA high versus DM2 subjects are shown in Table 3.

Table 3.

Characteristics of GADA-High LADA Subjects Versus DM2 Subjects

Cases	GADA-high (n = 4)	DM2 (n = 130)	P
Females, n (%)	0 (0)	55 (42.3)
Males, n (%)	4 (100)	75 (57.7)	0.144
Age (years)	37.3 ± 3.6	47.2 ± 9.3	0.035^*
Age at diagnosis (years)	33.3 ± 3.0	44.4 ± 9.4	0.020^*
Disease duration (months)	50 ± 11.2	33.7 ± 17.9	0.074
BMI (kg/m²)	22.6 ± 4.1	28.3 ± 4.8	0.020^*
Waist (cm)	82.6 ± 7.4	96.8 ± 9.8	0.005^*
SBP (mmHg)	105.5 ± 9.0	126.5 ± 14.0	0.003^*
DBP (mmHg)^*	69.5 [62.3–79.8]	80 [75.0–84.3]	0.075
Antihypertensive treatment, n (%)	0 (0)	44 (33.8)	0.302
Triglycerides (mg/dL)^*	99.5 [83.8–107.0]	151 [112.3–210]	0.026
Total cholesterol (mg/dL)	157.5 ± 37.2	167.8 ± 38.1	0.596
HDL-C (mg/dL)	47.8 ± 6	43.2 ± 10.8	0.403
LDL-C (mg/dL)	100 ± 39.4	103.3 ± 31.2	0.837
MS, n (%)	0 (0)	115 (88.5)	<0.001^*
Fasting plasma glucose (mg/dL)^*	143 [109.8–167.3]	137 [120–169.3]	0.927
HbA1c (%)^*	8.4 [6.2–12.2]	7.1 [6.6–8.5]	0.596
Family history of diabetes, n (%)	1 (25)	86 (66.2)	0.124
FCP (ng/mL)	0.8 ± 0.7	2.3 ± 0.8	0.001^*
Insulin treatment	2 (50)	6 (4.6)	0.018^*

Data expressed as means ± SD, medians [IQR] or n (%), ^*Significant P values.

GADA-low LADA subjects versus DM2 subjects

Patients with GADA at low titer (GADA-low, n = 4) were compared with DM2 patients (n = 130) group. There were no significant differences between characteristics of DM2 and GADA-low patients. The characteristics of GADA low versus DM2 patients are presented in Table 4.

Table 4.

Characteristics of GADA-Low LADA Subjects Versus DM2 Subjects

Cases	GADA-low (n = 5)	DM2 (n = 130)	P
Females, n (%)	4 (80)	55 (42.3)	0.167
Males, n (%)	1 (20)	75 (57.7)	0.400
Age (years)	43.6 ± 9.2	47.2 ± 9.3	0.329
Age at diagnosis (years)	40.2 ± 8.8	44.4 ± 9.4	0.265
Disease duration (months)	42.8 ± 14.1	33.7 ± 17.9	0.922
BMI (kg/m²)	28.1 ± 2.4	28.3 ± 4.8	0.531
Waist (cm)	94.0 ± 4.8	96.8 ± 9.8	0.605
SBP (mmHg)	123.2 ± 17.3	126.5 ± 14.0	0.792
DBP (mmHg)	80 [70–88]	80 [75.0–84.3]	1.000
Antihypertensive treatment, n (%)	2 (40)	44 (33.8)	0.354
Triglycerides (mg/dL)	122 [100–167]	151 [112.3–210]	0.775
Total cholesterol (mg/dL)	163 ± 38.6	167.8 ± 38.1	0.741
HDL-C (mg/dL)	44.8 ± 9.0	43.2 ± 10.8	0.784
LDL-C (mg/dL)	107.2 ± 33.5	103.3 ± 31.2	0.473
MS, n (%)	4 (80)	115 (88.5)	0.798
Fasting plasma glucose (mg/dL)	139 [117–201]	137 [120–169.3]	0.073
HbA1c (%)	8.1 [7.5–11.5]	7.1 [6.6–8.5]	1.000
Family history of diabetes, n (%)	3 (60%)	86 (66.2%)	0.568
FCP (ng/mL)	2.1 ± 0.6	2.3 ± 0.8	1.000
Insulin treatment	0 (0)	6 (4.6)	0.167

Data expressed as means ± SD, medians [IQR] or n (%).

β-cell function

Above observations in Table 1 show that subjects with LADA had lower FCP levels compared with DM2 subjects (1.5 ± 0.9 vs. 2.3 ± 0.8 ng/mL; P = 0.009). For the purpose of assessment of C-peptide secretions according to disease duration, the LADA and DM2 groups were stratified according to the duration of diabetes into two periods (<36 months and >36 months) and compared. In comparison to DM2 subjects, LADA subjects displayed significantly lower FCP concentrations in those with <36 months duration (1.73 ± 0.10 vs. 2.29 ± 0.88 ng/mL; P ≤ 0.001; 95% confidence interval [CI]: −0.81 to −0.31) and the difference was also evident in patients with >36 months of disease duration (1.39 ± 1.10 vs. 2.25 ± 0.77 ng/mL; P = 0.016; 95% CI: −1.54 to −0.17) (Table 5).

Table 5.

C-Peptide Secretion According to Duration of Diabetes

<36 months					>36 months
Cases	LADA (n = 3)	DM2 (n = 71)	P	95% CI	LADA (n = 6)	DM2 (n = 59)	P	95% CI
C-peptide (ng/mL)	1.73 ± 0.10	2.29 ± 0.88	<0. 001	−0.81 to −0.31	1.39 ± 1.10	2.25 ± 0.77	0.016	−1.54 to −0.17

Data are means ± SD.

Significant P values are in bold.

CI, confidence interval.

Discussion

It has been observed that characteristics of LADA in European, Chinese, and Arab populations remarkably differ from GADA-negative DM2 subjects.^6,7,10 In accordance, patients with LADA in our cohort showed different features than DM2 patients. LADA patients had lower triglycerides, FCP, and frequency of MS than DM2 subjects. As in Caucasians and Chinese,^7,10 characteristics of LADA patients with high-GADA titer in our study were significantly different than low-GADA titer LADA patients. Patients with high-GADA titer, compared with low-GADA titer, were more likely to be male, leaner, and on insulin treatment with lower frequency of MS. FCP levels were lower in high-GADA titer patients. Age difference between high-GADA and low-GADA patients was not significant. Contrary to our findings, compared to low-GADA patients, European high-GADA patients tended to be female and younger. Other observations were nearly similar to those reported in large European Action LADA-7 and LADA China study.

Furthermore, our data show that compared to LADA high-GADA titer subjects, DM2 subjects had significantly higher SBP and BMI. Interestingly, this difference was not evident on comparison of total LADA patients with DM2 patients.

Like in a Chinese study,^22,23 LADA patients with low-GADA titer in the present study did not significantly differ from DM2 patients, although this observation was contradictory to the European data that showed a difference between the two groups.¹⁰ Our finding possibly indicates that LADA patients with low-GADA titer are similar to DM2 patients, at least in terms of metabolic features and β-cell function. However, future studies are required to ascertain if LADA patients with low-GADA titer can be actually equated with DM2 patients. Ethnic variation in this regard also needs to be further explored.

In agreement with previous reports, our data also indicate that based on GADA titer, different subgroups can be identified within LADA.^7,10,12,24 Besides distinct clinical and phenotypic features, GADA titer is important in defining the severity of disease process and may help in selection of appropriate therapeutic choices.

Similar to European and Chinese cohorts, current study showed that MS is more common in DM2 subjects than in LADA subjects. Frequency of MS among LADA subjects in our study was 44.4%, slightly higher than 41.9% in European LADA subjects,¹⁵ but much lower than 62% reported in Chinese LADA subjects.⁷ Our data further indicate that MS is not prevalent in LADA patients with high-GADA titer. Compared with LADA patients without MS, all LADA patients with MS tended to be female. The current study is the first to describe MS in Indian LADA patients.

Compared to DM2 patients, C-peptide concentration in the current study was lower in LADA patients. This difference was evident up to 36 months from diagnosis and contrary to Spanish data²⁵ the difference remained even after 36 months of duration.

We acknowledge that due to small number of subjects in LADA group, our results cannot be generalized and future research is needed with larger sample size. Nevertheless, to the best of our knowledge, this is the first study in the region of north India providing insights into clinical, metabolic, and immunological features of LADA.

In conclusion, our results indicate that LADA patients have distinct metabolic features with lower residual β-cell function than DM2 patients. As antibody titers define the severity of disease, LADA patients with high-GADA titers need to be closely monitored to assess deterioration in glycemic control and β-cell function. Due to similarities with DM2, LADA patients with low-GADA titers may additionally require therapies targeting insulin resistance.

With the ultimate aim of improving the management of LADA patients, in future, large-scale studies using validated methods are required to have an improved understanding on various aspects of LADA and establish optimal preventive and treatment strategies for LADA. Diagnosing LADA solely on the basis of autoantibody detection poses a challenge as titer of autoantibodies may fluctuate and disappear with time.²³

Due to overlapping of certain features, in addition to antibody assays, other novel markers should be identified to differentiate LADA and DM2 at an early stage. Global collaboration using standardized assays and diagnostic criteria is required to obtain insights into ethnic differences in LADA.

Footnotes

Acknowledgments

This work had been realized in the Medicine Doctorate framework of the Universitat Autònoma de Barcelona. Results of this study were presented in abstract form at the 26th American Association of Clinical Endocrinologists (AACE) annual scientific and clinical congress, Austin, Texas, May 3–7, 2017.

Ethical Approval

All procedures performed in this study involving human participants were in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Author Disclosure Statement

No conflicting financial interests exist.

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