Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians

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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres

Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians Kuppan Gokulakrishnan *, Kalaivani Manokaran, Gautam Kumar Pandey, Anandakumar Amutha, Harish Ranjani, Ranjit Mohan Anjana, Viswanathan Mohan Madras Diabetes Research Foundation & Dr Mohan’s Diabetes Specialities Centre, WHO Collaborating Centre for NonCommunicable Diseases Prevention and Control, IDF Centre for Education, Gopalapuram, Chennai 600086, India

article info

abstract

Article history:

Background and Aims: Betatrophin is emerging as a marker for compensatory beta cell

Received 28 October 2014

proliferation. While betatrophin has been mainly investigated in adults, there is a lack of

Received in revised form

data on betatrophin levels in youth-onset type 2 diabetes mellitus (T2DM-Y). The aim of this

23 March 2015

study was to determine levels of betatrophin and its association with T2DM-Y in Asian

Accepted 27 April 2015

Indian participants.

Available online xxx

Methods: We recruited 100 individuals with normal glucose tolerance (NGT; n = 50) and newly-diagnosed cases (within 18 months of first diagnosis) of T2DM-Y (n = 50) with onset

Keywords:

between 12 and 24 years of age from a large tertiary diabetes center in Chennai in southern

Betatrophin

India. Insulin resistance was measured by homeostatic model (HOMA-IR) and insulin

Insulin resistance

secretion by oral disposition index (DIO). Betatrophin levels were measured by enzyme-

Type 2 diabetes

linked immunosorbent assay.

Asians Indians

Results: Betatrophin levels were significantly lower in the T2DM-Y group compared with the NGT group (803 vs 1104 pg/ml, p < 0.001). Betatrophin showed a significant inverse correlation with waist circumference ( p = 0.035), HOMA-IR ( p < 0.001), fasting and 2 h postprandial glucose ( p < 0.01), glycated hemoglobin ( p = 0.019) and a positive correlation with fasting Cpeptide ( p < 0.001) and DIO ( p = 0.012). In regression analysis, betatrophin was independently associated with T2DM-Y even after adjustment for age, gender, and waist circumference (OR per standard deviation: 0.562, 95% CI: 0.342–0.899, p = 0.019). However, the association was lost when HOMA-IR was included in the model (OR: 1.141, 95% CI: 0.574– 2.249; p = 0.646). Conclusion: Betatrophin levels are lower in T2DM-Y and this association is likely mediated through insulin resistance. # 2015 Published by Elsevier Ireland Ltd.

* Corresponding author at: Department of Research Biochemistry, Madras Diabetes Research Foundation 4, Conran Smith Road, Gopalapuram, Chennai 600 086, India. Tel.: +91 44 4396 8888; fax: +91 44 2835 0935. E-mail addresses: [email protected], [email protected] (K. Gokulakrishnan). Abbreviations: T2DM-Y, youth-onset type 2 diabetes mellitus; NGT, normal glucose tolerance; DIO, oral disposition index; GAD, glutamic acid decarboxylase; WHO, World Health Organization. http://dx.doi.org/10.1016/j.diabres.2015.04.028 0168-8227/# 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Gokulakrishnan K, et al. Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.04.028

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1.

Introduction

Type 2 diabetes (T2DM) is a complex metabolic disorder characterized by insulin resistance and progressive pancreatic beta cell failure [1]. T2DM often remains undiagnosed for many years, and exposure to hyperglycemia over time could lead to the development of chronic micro- and macro-vascular complications [2]. There is evidence to show that insulin resistance causes compensatory expansion of pancreatic beta cell mass due to circulating growth factors [3–5]. Studies have also demonstrated that systemic hepatocyte-derived growth factors promote beta cell proliferation in mouse and human islets, supporting a liver-to-pancreas axis in the adaptive beta cell growth response to insulin resistance [6,7]. Yi et al. [5] recently identified a novel hormone, betatrophin, primarily expressed in the liver and adipose tissue that induces a 17-fold increase in the rate of pancreatic beta cell proliferation. Hepatic over-expression of betatrophin, in pharmacological and genetic mouse models of insulin resistance, causes an increase in the rate of beta cell proliferation, islet size, and insulin content with benefits on glucose homeostasis [5]. Betatrophin has emerged as a signaling molecule favoring a compensatory beta cell growth in response to insulin resistance [5,8]. Studies have reported that betatrophin levels are correlated significantly with an atherogenic lipid profile in high-risk cohorts with morbid obesity or T2DM [9,10]. It is also believed to play a role in dysfunctional lipid metabolism involving the regulation of hepatic very low-density lipoprotein secretion as well as in altered lipoprotein lipase activity [11,12]. Betatrophin has been mainly investigated in older adults [5], and there are no studies on youth-onset T2DM (T2DM-Y). Studies on patients with T2DM-Y may be especially informative, as they may be less affected by various comorbid factors. Moreover, decline in beta cell function may be more rapid compared with individuals with later-onset T2DM [13,14]. Asian Indians, in particular, are known to be more insulin resistant [15] and have high rates of type 2 diabetes at younger ages [16] but there is a lack of data on betatrophin levels in Asian Indians. The aim of this study was to determine betatrophin levels and its association with T2DM-Y among Asian Indians.

1.2.

Anthropometric measurements including weight, height, and waist circumference were obtained by trained data collectors using standardized methods [17]. Body mass index (BMI) was calculated as weight (kg)/height (m2). Blood pressure was recorded from the right arm in a sitting position to the nearest 2 mmHg using a mercury sphygmomanometer (Diamond Deluxe BP apparatus, Pune, India). Two readings were taken 5 min apart and the mean of the two was taken as the blood pressure.

1.3.

Biochemical tests

Fasting plasma glucose (hexokinase method), serum cholesterol (cholesterol oxidase–peroxidase-aminopyrine method), serum triglycerides (glycerol phosphate oxidase–peroxidaseaminopyrine method) and high density lipoprotein (HDL) cholesterol (direct method-polyethylene glycol-pretreated enzymes), were measured using Hitachi-912 Autoanalyser (Hitachi, Mannheim, Germany). Low density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Glycated hemoglobin (HbA1c) was measured by high pressure liquid chromatography using a Variant machine (BioRad, Hercules, California, USA). Serum insulin concentration was estimated using the electrochemiluminescence method (COBAS E 411; Roche Diagnostics). The intra- and inter-assay coefficients of variation for the biochemical assays ranged between 3.1 and 7.6%. All measurements were performed in a laboratory certified by the College of American Pathologists (Northfield, IL) and the National Accreditation Board for Testing and Calibration of Laboratories (New Delhi, India).

1.4.

Betatrophin measurements

Betatrophin was measured by competitive inhibition enzyme linked immunosorbent assay (Wuhan Eiaab Science, Wuhan, China) according to the manufacturer’s protocol. The values were expressed in pg/ml units. The intra- and inter-assay coefficients of variation were <5% and <10%, respectively.

1.5. 1.1.

Anthropometric measurements

Definitions

Research design and methods

Recruitment of study participants: Fifty individuals with newly-diagnosed T2DM with onset between 12 and 24 years of age (within 18 months of first diagnosis) and individuals with normal glucose tolerance (NGT; n = 50) were recruited from a large tertiary diabetes center at Chennai in southern India. Institutional Ethics Committee approval was obtained prior to the start of the study. Written informed consent was obtained from the individuals aged 18 years and above, and ‘‘assent’’ from the study participants along with written informed parental consent was obtained for those under 18 years of age. Participants completed interviewer-administered questionnaires, were examined for anthropometric and clinical measurements, and provided bio-specimens.

Diabetes was defined as fasting plasma glucose 126 mg/dl (7.0 mmol/l) and/or 2 h postprandial glucose level 200 mg/dl (11.1 mmol/l), or a past medical history (self-reported diabetes under treatment by a physician), or drug treatment for diabetes (insulin or oral hypoglycemic agents) [18]. T2DM-Y was classified based on the following criteria: onset before 25 years of age, recruitment within 18 months of diagnosis, adequate response to oral hypoglycemic agents and negative for glutamic acid decarboxylase (GAD), IA-2 and zinc transporter antibodies, absence of ketosis, good beta cell reserve as shown by C-peptide assay (fasting: 0.6 pmol/ml and stimulated: 1.6 pmol/ml), and absence of pancreatic calculi on abdominal X-ray [18,19]. NGT was defined as fasting plasma glucose <5.6 mmol/L (<100 mg/dl) and 2 h postprandial glucose value <7.8 mmol/L (<140 mg/dL) [18].

Please cite this article in press as: Gokulakrishnan K, et al. Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.04.028

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1.5.1.

Generalized obesity

For adults, the World Health Organization (WHO) Asia Pacific BMI cut-off points of <25.0 kg/m2 and 25.0 kg/m2 was used to define non-obese and obese groups, respectively [20]. For adolescents, the WHO recommended BMI z-score (cut-off: BMI +2SD) was used to define obesity [21,22].

1.5.2.

Insulin resistance

Insulin resistance was estimated using the homeostasis model assessment-insulin resistance (HOMA-IR) formula: HOMA-IR = fasting insulin [mIU/ml]* fasting glucose [mmol/ L]/22.5 [23].

1.5.3.

Oral disposition index (DIO)

Beta cell function was measured by the oral disposition index (DIO) which takes insulinogenic index and insulin sensitivity into account [DIO = (DI 0–30/DG0–30)  (1/fasting insulin)] [24,25].

1.6.

Statistical analysis

Table 1 – Clinical and biochemical characteristics of study participants. Parameters Age (years) Male n (%) Body mass index (kg/m2) Waist circumference (cm) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Fasting plasma glucose (mg/dl) 2 h postprandial glucose (mg/dl) HOMA-IR Oral disposition index (DIO) HbA1c (%) Duration of diabetes (years) Serum cholesterol (mg/dl) Serum triglycerides (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)

NGT (n = 50)

T2DM-Y (n = 50)

21  4.0 28 (56) 22.1  6.2 79.7  9.0 117  14 75  8 82  5.0 98  16 2.0  0.7 4.09  2.1 5.4  0.4 – 154  31 97  40 98  25 38  7

21  3.5 27 (54) 25.2  3.3 * 83.3  8.2 * 119  11 ** 76  8 ** 134  44 ** 257  90 ** 5.7  3.5 ** 0.53  0.36 ** 7.7  2.0 ** 0.72  0.5 159  35 115  50 95  30 36  7.0

Data presented as mean  SD. p < 0.05, **p < 0.001 compared with NGT.

*

We compared anthropometric, clinical, and biochemical characteristics of groups using one-way analysis of variance (with Tukey’s HSD) for continuous variables and Chi square test or Fisher’s exact test to compare proportions. We performed Pearson correlation analysis to examine the correlation of various risk factors with betatrophin. We checked the collinearity between HOMA-IR and betatrophin levels. The tolerance was >0.1, and variance inflation factor did not exceed >5.0, denoting that there was no collinearity among them [26]. Standardized multiple logistic regression analysis was done to assess how incremental changes in betatrophin were associated with T2DM-Y. We adjusted models for any demographic, anthropometric, clinical, or biochemical characteristics that were significantly different across groups. We calculated the propensity score to assess the association between betatrophin and T2DM-Y using a two-step procedure [27,28]. In the first step, a logistic regression analysis was performed with betatrophin, considered as dependent variable and the confounders as independent variables. The propensity score reflects the differences between the two groups (NGT vs T2DM-Y) on the confounders. In the second step, the effect of betatrophin on T2DM-Y was adjusted for the propensity score using an analysis of covariance. All analyses were performed using Windows based SPSS statistical package (version 12.0, Chicago, IL).

2.

Results

The clinical and biochemical characteristics of the study participants are shown in Table 1. A total of 100 individuals were included in the study, of whom 58% (n = 58) were males. BMI, waist circumference, systolic and diastolic blood pressure, and HOMA-IR were significantly higher, and oral disposition index was lower, in the T2DM-Y group ( p < 0.05) compared with the NGT group. All patients with T2DM-Y were only on oral anti-diabetic drugs, and 1 participant was on anti-hypertensive medication. Among patients taking oral anti-diabetic drugs, 16 patients

were on metformin, 9 were on sulphonylureas, and 25 were on both metformin and sulphonylureas. Compared with the NGT group, betatrophin levels were significantly lower in the T2DM-Y group (803 vs 1104 pg/ml, p < 0.001). Betatrophin showed a significant inverse correlation with waist circumference ( p = 0.035), insulin resistance ( p < 0.001), fasting and 2 h postprandial glucose ( p < 0.01), glycated hemoglobin ( p = 0.019), and a positive correlation with fasting C-peptide ( p < 0.001) and DIO ( p = 0.012) (Table 2). Betatrophin levels were next stratified according to obesity using BMI (cut-off value of 25.0 kg/m2 as this applies to the Asian Indian population) for adults and WHO recommended BMI z-score for adolescence. It was found that obese individuals had lower levels of betatrophin compared with Table 2 – Pearson correlation analysis of betatrophin with metabolic risk variables in total study participants. Variables

Betatrophin r-Value

Age Body mass index (BMI) Waist circumference Systolic blood pressure Diastolic blood pressure HOMA-IR Oral disposition Index Fasting plasma glucose 2 h postprandial glucose Glycated hemoglobin Duration of diabetes (years) a Serum cholesterol Serum triglycerides HDL cholesterol Fasting C-peptide Stimulated C-peptide a

0.146 0.158 0.212 0.016 0.087 0.403 0.252 0.258 0.296 0.234 0.174 0.050 0.016 0.064 0.413 0.172

p-Value 0.147 0.116 0.035 0.872 0.390 <0.001 0.012 0.010 0.003 0.019 0.224 0.619 0.706 0.528 <0.001 0.088

Analysis done only in patients with T2DM-Y.

Please cite this article in press as: Gokulakrishnan K, et al. Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.04.028

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Table 3 – Multiple logistic regression analysis using type 2 diabetes as dependent variable and betatrophin as independent variable. Variable

Fig. 1 – Mean betatrophin levels in relation to obesity.

Betatrophin – independent variable a Betatrophin – unadjusted Model 1: Adjusted for age and gender Model 2: Model 1 + waist circumference Model 3: Model 2 + HOMA-IR a

non-obese although this did not reach statistical significance. This trend was similar in both control participants as well as in patients with T2DM (Fig. 1). We further stratified the study participants as separate adolescent and adult groups and found that betatrophin levels followed a similar trend – obese individuals had lower levels of betatrophin compared with non-obese, although this did not reach statistical significance. However, as the number of participants was limited this would have to be further investigated in future studies (Supplementary Figs. 1a and b). To examine the association between HOMA-IR and betatrophin levels, study participants were stratified by the tertiles of HOMA-IR. Betatrophin levels significantly decreased with increasing tertiles of HOMA-IR in the overall group, in patients with T2DM and in controls ( p for trend <0.05) (Fig. 2). Logistic regression analysis was used to determine the association of betatrophin with T2DM-Y (Table 3). Betatrophin was independently associated with T2DM-Y (OR per standard deviation: 0.524, 95% CI: 0.328–0.837, p = 0.007). Adjustment for age, gender, and waist circumference did not substantially change the association between betatrophin and T2DM-Y [OR per standard deviation: 0.562, 95% CI: 0.342–0.899, p = 0.019]. However, the significance was lost when HOMA-IR included in

Fig. 2 – Mean betatrophin levels in tertiles of HOMA-IR. *p for trend <0.05. Data presented as mean WSD.

Odds ratio (OR)

95% confidence interval (CI)

p-Value

0.524

0.328–0.837

0.007

0.519

0.321–0.862

0.006

0.562

0.342–0.899

0.019

1.141

0.574–2.249

0.646

Per standard deviation changes in betatrophin.

the model (OR per standard deviation: 1.141, 95% CI: 0.574– 2.249; p = 0.646). We calculated the propensity score to assess the association between betatrophin and T2DM-Y [27,28]. Even after adjusting for the propensity score, betatrophin was significantly associated with T2DM-Y (OR per standard deviation: 0.524, 95% CI: 0.326–0.885, p = 0.014) (Supplementary Table 1).

3.

Discussion

While the link between beta cell dysfunction and T2DM is very well established [29], betatrophin, a marker of beta cell proliferation, is emerging as a potential biomarker for T2DM [5]. In this study, we report the following significant findings: (1) in Asian Indians with T2DM-Y, decreased levels of betatrophin are seen; (2) betatrophin shows a significant inverse correlation with insulin resistance, fasting and 2 h postprandial glucose, and glycated hemoglobin but is positively correlated with the DIO and fasting C-peptide; and (3) the association of betatrophin with T2DM-Y was statistically significant even after adjusting for age, gender, and waist circumference but lost its significance when adjusted for insulin resistance. T2DM-Y is a relatively recent clinical phenomenon, and hence the role of insulin resistance and beta cell function in its pathogenesis merits study. Studies have suggested that there could be an accelerated beta cell dysfunction in younger age groups, thus shortening the transition time between prediabetes and T2DM-Y [13,30]. Two recent cross-sectional studies indicate that Asian Indians may be susceptible to early decline in beta cell function even during stages of mild dysglycemia [31,32]. In this context, this study assumes significance as we report decreased betatrophin levels in Asian Indians with T2DM-Y. The decreased levels of betatrophin found in T2DM-Y could be related to hyperglycemia through insulin resistance [33]. Betatrophin levels were also negatively correlated with fasting glucose or 2 h postprandial glucose and positively with C-peptide. There is a growing body of evidence demonstrating that betatrophin is a promising marker to evaluate beta cell proliferation [5,8,34]. However, there are conflicting reports

Please cite this article in press as: Gokulakrishnan K, et al. Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.04.028

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with regard to its clinical utility in predicting the development of T2DM [35]. Betatrophin levels have been shown to be increased in patients with T2DM [36–38], and have also been shown to be associated with obesity [10]. While some studies have demonstrated that betatrophin is positively correlated with serum insulin levels in patients with T2DM [36], others have reported an inverse correlation [33]. However, another study reported decreased levels of betatrophin in T2DM [33]. These differences may be attributed to methodological differences between the immunoassays or due to ethnic differences [39]. Preclinical studies have demonstrated that betatrophin may act as a circulating factor that promotes beta cell proliferation in response to insulin resistance and raising hopes of potential therapeutic implications for the treatment of diabetes [8,40]. Moreover, a recent study by Gusarova et al. [41] reported that betatrophin may not be required for beta cell function or the compensatory beta cell growth response to insulin resistance. The authors also showed that overexpression of betatrophin does not increase beta cell area or improve glycemic control. In humans, it is not clear whether betatrophin is induced by insulin resistance or how this hormone is linked to glucose homeostasis as the exact molecular mechanisms of betatrophin are yet to be elucidated. Studies have demonstrated that insulin resistance induces an increase in beta cell response to compensate for the increased insulin demand in normal individuals [42]. Studies using humans and mouse models have reported that defects in insulin-signaling pathways in beta cells lead to decrease in mass and reduced secretory function [43]. Indeed, impaired beta cell responsiveness to insulin has been shown in insulin resistant patients [44]. In the present study, we report that, compared with individuals with NGT, patients with T2DM-Y had lower levels of betatrophin which may be related to insulin resistance as the association with T2DM-Y lost its significance when adjusted for insulin resistance. It is of particular interest that betatrophin levels were significantly decreased with increasing tertiles of HOMA-IR in the overall study group, in patients with type 2 diabetes and also in the control participants. We also observe a decreased beta cell function as measured by the oral disposition index in T2DM-Y compared with normal individuals and the positive correlation with C-peptide. These findings suggest that betatrophin may have a direct influence on beta cell function but further studies are needed to understand the mechanism underlying this association. Moreover, our analyses are based on single measurements of betatrophin, which may not reflect betatrophin levels over time. Serial measurements of betatrophin need to be done at different stages of insulin resistance/T2DM-Y to further clarify the role of betatrophin. Studies have shown that betatrophin levels could be affected by duration of T2DM [36]. However, in the present study the mean duration of T2DM was only 0.72  0.5 years. When we analyzed the data from patients with T2DM-Y with respect to treatment, there was no statistically significant difference in betatrophin levels between different treatment groups. However, as the number of patients is limited this warrants further investigation. One of the limitations of our study is that it is crosssectional and no causal relationships between betatrophin

5

and T2DM-Y can be established. One strength of the study is that we investigated youth onset T2DM with a similar age group of NGT controls thus minimizing the influence of age upon circulating levels of betatrophin. In summary, low betatrophin levels are associated with T2DM-Y perhaps mediated through insulin resistance. Further studies are needed to understand the mechanisms that contribute to decrease circulatory betatrophin levels in T2DM and its role as a biomarker in understanding the natural history of T2DM through longitudinal studies.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgment We gratefully acknowledge the research grants received from the Madras Diabetes Research Foundation – Intramural Research Funding (MIRF).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. diabres.2015.04.028.

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Please cite this article in press as: Gokulakrishnan K, et al. Relationship of betatrophin with youth onset type 2 diabetes among Asian Indians. Diabetes Res Clin Pract (2015), http://dx.doi.org/10.1016/j.diabres.2015.04.028