Irisin and leptin concentrations in relation to obesity, and developing type 2 diabetes: A cross sectional and a prospective case-control study nested in the Normative Aging Study

    Irisin and Leptin Concentrations in relation to Obesity, and Developing Type 2 Diabetes: a cross sectional and a prospective case-control study nested in the Normative Aging Study Ayse Sahin-Efe, Jagriti Upadhyay, Byung-Joon Ko, Fadime Dincer, Kyung Hee Park, Alexandra Migdal, Pantel Vokonas, Christos Mantzoros PII: DOI: Reference:

S0026-0495(17)30299-8 doi: 10.1016/j.metabol.2017.10.011 YMETA 53667

To appear in:

Metabolism

Received date: Accepted date:

12 June 2017 21 October 2017

Please cite this article as: Sahin-Efe Ayse, Upadhyay Jagriti, Ko Byung-Joon, Dincer Fadime, Park Kyung Hee, Migdal Alexandra, Vokonas Pantel, Mantzoros Christos, Irisin and Leptin Concentrations in relation to Obesity, and Developing Type 2 Diabetes: a cross sectional and a prospective case-control study nested in the Normative Aging Study, Metabolism (2017), doi: 10.1016/j.metabol.2017.10.011

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ACCEPTED MANUSCRIPT Irisin and Leptin Concentrations in relation to Obesity, and Developing Type 2 Diabetes: a cross sectional and a prospective case-control study nested in the Normative Aging Study

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Ayse Sahin-Efe 1 2 3*, Jagriti Upadhyay1 2 3*, Byung-Joon Ko1, Fadime Dincer 1, Kyung Hee Park5, Alexandra Migdal1, Pantel Vokonas 4, Christos Mantzoros 1 2 3 1

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Division of Endocrinology, Beth-Israel Deaconess Medical Center/Harvard Medical School, Boston, MA, USA 2

Division of Endocrinology, Boston University Medical Center, Boston, MA, USA

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Section of Endocrinology, Department of Medicine, VA Boston Healthcare System, Boston, MA, USA 4

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Normative Aging Study, VA Boston Healthcare System and Boston University Schools of Public Health and Medicine, Boston, MA, USA 5

Department of Family Medicine, Hallym University Sacred Heart Hospital, Hallym University, Gyeonggi-do, Korea * These authors equally contributed to this article and the order of names is in alphabetical order

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Running Title: Role of irisin in prediction of type 2 diabetes

Word count: 3483

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Key words: Irisin, insulin resistance, glucose tolerance, diabetes

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Number of figures and tables: 5

Corresponding author and person to whom reprint requests should be addressed:

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Jagriti Upadhyay, MD Beth Israel Deaconess Medical Center 330 Brookline Ave, Stoneman 820 Boston, MA 02215 Phone: 617-667-8630 Fax: 617-667-8634 Email: [email protected] Disclosure Statement: The authors have nothing to disclose Funding: The Normative Aging Study is supported by Cooperative Studies Program/ERIC, US Department of Veterans Affairs and is a research component of the Massachusetts Veterans Epidemiology Research and Information Center. Mantzoros Lab is supported in part by NIH DK081913.

ACCEPTED MANUSCRIPT Abstract

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Objective: To investigate the associations between irisin and leptin levels in obesity and insulin resistance in a cross sectional study. To assess the potential role of irisin and leptin as a predictive marker of T2DM using a nested case-control study.

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Methods: Both studies were designed within the longitudinal VA NAS cohort. The cross sectional study involved 111 non obese and 105 obese subjects who were subdivided into two groups based on their fasting glucose tolerance. In the nested 1:3 case-control study, 47 subjects with T2DM and 140 non-diabetic controls were selected. Serum samples collected 3-5 years before the diagnosis of T2DM were analyzed. Irisin and leptin concentrations were measured using a validated ELISA and radioimmunoassay respectively.

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Results: In the cross-sectional study, irisin did not differ between groups based on their fasting glucose tolerance. When subjects were grouped based on obesity status, both irisin and leptin concentrations were significantly higher in obese compared to the non-obese group (p = 0.03 and <0.001, respectively). Irisin concentrations positively correlated with leptin concentrations (r= 0.392, P < 0.001). In the nested case control study, leptin concentrations were a significant predictor of developing diabetes (p = 0.005) in unadjusted models, but not after correcting for BMI, whereas irisin concentrations did not play a role of comparable significance.

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Conclusions: Leptin concentrations are higher in the obese group irrespective of their glucose tolerance. Obese individuals with impaired fasting glucose have higher concentrations of circulating irisin compared to non-obese subjects with normal glucose tolerance. Irisin concentrations do not predict risk of developing diabetes prospectively.

ACCEPTED MANUSCRIPT Introduction Myokines are proteins expressed and secreted by skeletal muscle, which is now recognized

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as an endocrine organ producing hormones, some of which regulate metabolism. Beneficial effects of

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regular physical exercise are considered to be, at least in part, mediated by myokines. Peroxisome proliferator-activated receptor (PPAR) γ coactivator (PGC)-1α is a transcriptional coactivator

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mediating many of the downstream molecular events induced by exercise such as oxidative metabolism (1). Irisin is a recently discovered exercise-regulated myokine, which is a cleavage

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product of the fibronectin type III domain containing 5 (FNDC5), and regulated by PGC1- α as initially described by in vitro and in vivo studies in mice (2). Similar to mice, in humans FNDC5 is predominantly expressed in muscle tissue. Age and muscle mass are the main predictors of circulating irisin concentrations (3), and both PGC1- α and FNDC5 expression levels are increased in the skeletal

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muscles of the patients that have better aerobic performance .

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It has been proposed that irisin induces white adipose tissue (WAT) “browning” or “beigeing” defined as generation of cells with brown adipocyte characteristics (e.g. high abundance of

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uncoupling protein-1, multilocular fat droplets, and mitochondria) from the precursor cells within the white adipose tissue. Beige or brown adipose tissue is metabolically much more active as it dissipates

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energy. An increase in the brown adipose tissue via transplant resulted in improved insulin sensitivity and decreased fat mass. Similarly, after a short irisin treatment interval, there is an increase in the energy consumption in obese mice resulting in improvement of diet induced insulin resistance and a slight weight loss (2). Therefore, the discovery of irisin raised expectations that this myokine may act as a potential therapeutic agent for diabetes and obesity. Correlations between irisin concentration and adverse metabolic phenotypes have been analyzed in humans by several studies with controversial results (3-6). Higher irisin concentrations were found in obese subjects compared to normal weight or anorexic individuals (6) and, irisin concentrations declined after weight loss either by bariatric surgery or caloric restriction (3). The human FNDC5 gene, encoding irisin, has been shown to have insulin-desensitizing potential (7) and a

ACCEPTED MANUSCRIPT positive association between irisin, fasting insulin levels and HOMA-IR has been reported (5). In addition, there is a lack of consensus in the literature concerning the role of irisin in insulin resistance

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and (type 2 diabetes) T2DM. Several studies showed higher irisin concentrations in individuals with

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impaired fasting glucose/T2DM (8), others showed lower concentrations (4,9,10) and others showed no association between irisin and T2DM (11). Elevated concentrations of irisin have been associated

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with increased risk of metabolic syndrome (MetS) and cardiovascular disease (5). A recent review by Perakakis et al has well summarized role of irisin in glucose homeostasis and association of irisin with

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obesity, metabolic syndrome, insulin resistance and type 2 diabetes mellitus (12). Since, no prospective studies that incorporate the time sequence criterion for causality have been performed to date, the precise role and physiology of this hormone in obesity, insulin resistance and T2DM requires further clarification.

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We studied the relationship between circulating irisin concentrations and obesity status as

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well as with impaired fasting glucose (IFG)/T2DM, utilizing a cross sectional study design. Importantly, we also investigated the potential role of irisin concentrations as a predictive marker of

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risk for developing T2DM prospectively utilizing a nested case control study design.

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Materials and Methods

Both studies were embedded in the ongoing longitudinal Veterans Affairs Normative Aging Study (VANAS) cohort of ageing established in 1961. 2280 healthy male veterans were recruited from the Greater Boston area and were re-evaluated every 3-5 years with medical history, physical examination, laboratory tests and questionnaires as previously described. Serum samples collected at every visit and were stored in -30oC freezers. Fasting blood glucose and total cholesterol were measured at the time of collection. The institutional review board of VA Boston Healthcare System has approved this study and all participants provided written informed consent. 1. Cross-sectional study: This study focused on the analysis of 111 obese and 105 nonobese male participant’s serum samples (collected between 1977 and 2011) assayed for irisin. All of

ACCEPTED MANUSCRIPT the obese subjects who had a control subject matched by age and year of sample collection was selected for analysis. A total of 216 serum samples were analyzed. The participants were divided into

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two groups based on their obesity status, then the two groups were subdivided into participants

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according to fasting glucose tolerance; with normal fasting blood glucose tolerance (FBG<100 mg/dL) and participants with abnormal FBG (≥100 mg/dL) and/or diabetes.

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2. Nested case-control study: All forty-seven subjects, who were diagnosed with T2DM

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between 1992 and 2008 and had data / samples stored and 140 controls (1:3 to maximize power) matched for age and year of sample collection were included. Serum samples had been collected 3-5 years before the diagnosis of T2DM.

Biochemical measurements: Irisin was measured using ELISA (#EK-067-52, initially

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developed by Aviscera, now commercialized by Phoenix Pharmaceuticals, Burlingame, CA, range 0.066–1024ng/mL) with intra-assay coefficient of variation (CV) of 4–6% and inter- assay CV of 8–

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10%), as previously described and validated (3). Serum leptin and adiponectin was measured using commercially available radio-immuno-assays (Millipore Co. Billerica, MA) with sensitivities 0.437

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ng/mL and 0.9375 ng/mL, intra-assay CV 3.4 - 8.3% and 1.78 - 3.59% and Inter-Assay CV: 3.0 - 6.2%

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and 6.90 - 9.25% accordingly) as previously described (13,14). All samples were measured in duplicates and were repeated if coefficient of variability for any sample was >15%. Statistical analysis

All statistical analyses were performed using SPSS (Version 19.0; SPSS Inc., Chicago, IL). Normality of the distributions was evaluated with frequency histograms and the Shapiro-Wilk test and the variables, which did not fulfill the normality assumptions, were log-transformed before parametric analysis. In the cross-sectional study, anthropometric data were compared using ANOVA and laboratory variables were compared using analysis of covariance (ANCOVA) after adjusting for the year of sample collection of the four groups which were divided based on the presence of obesity (BMI ≥ 30 kg/m2 or ≥ 27 with comorbidities) and impaired fasting glucose (IFG) defined as having

ACCEPTED MANUSCRIPT fasting plasma glucose concentration ≥ 100 mg/dL. Post-hoc analysis was performed by Bonferroni method, which was performed on the basis of 6 tests. Irisin and leptin concentrations were compared

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according to the obesity and IFG/T2DM status after adjusting for the year of sample collection by

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using generalized linear models. Pearson’s partial correlation analysis between continuous variables after controlling for the year of sample collection was performed. In the nested case-control study,

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anthropometric, laboratory, and clinical data were presented by the development of diabetes mellitus using generalized linear model and generalized estimating equation. Odds ratios (ORs) and 95%

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confidence intervals (CIs) were calculated using conditional logistic regression analysis for the development of diabetes mellitus in relation to the tertile of biomarkers. A similar logistic regression analysis was also performed using biomarkers as continuous variables after adjusting for the same covariates. Family history of diabetes mellitus, smoking status, dyslipidemia medication, high-density

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lipoprotein cholesterol, biomarkers, and body mass index were adjusted as covariates in the models. A two-sided P value of < 0.05 was considered as statistically significant for all the analyses after. Unlike

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leptin, there are no prior data on which to base power calculations on irisin. Thus we estimated that

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the required sample size, using one tailed analysis (nested case control); given conventional ratio of variance of 0.5; alpha error of 0.05 and a case control ratio of 3:1, would be at least 37 cases and 112

Results

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control subjects to obtain an actual power of 80%.

General characteristics of participants according to the presence of obesity and IFG in the cross-sectional study are presented in Table 1. Irisin concentrations were significantly higher in obese subjects with IFG/Diabetes as compared to the non-diabetic/non–obese group (p=0.03), whereas leptin concentrations in obese subjects with IFG and/or normal fasting glucose were higher than nonobese subjects with normal fasting glucose and/or IFG/DM. As shown in Figure 1, when we divided the subjects into non-obese and obese groups, obese subjects had higher irisin and leptin concentrations compared to non-obese group (P <0.001). The differences of irisin concentrations were not significant between normal fasting glucose and IFG/DM groups when we divided the participants

ACCEPTED MANUSCRIPT by the presence of IFG/DM (P = 0.21), although leptin concentrations were different among the two groups (p = 0.01). Figure 2 further illustrates the relationship of BMI and glucose (as continuous

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variables) with irisin and leptin respectively. There was a correlation between BMI and glucose with

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irisin which was significant (p<0.05). A significant relationship is also demonstrated between BMI and leptin and glucose and leptin (<0.01). Supplementary Table shows Pearson’s partial correlation

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coefficients between variables adjusted for the year of sample collection in the cross-sectional study. Irisin concentrations were positively correlated with leptin (r = 0.370, P <0.001), BMI (r = 0.18, P

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<0.05), Waist circumference (WC) (r = 0.19, P <0.001), Fasting Plasma Glucose (r = 0.18, P = 0.001) and Triglycerides (TG) (r = 0.18, P = 0.05). Leptin concentrations were directly correlated with BMI, WC, TG, FPG (P = 0.001), and negatively correlated with HDL (r = -0.30, P = 0.001). Table 2 demonstrates anthropometric, laboratory, and clinical characteristics of participants

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by the development of DM in the nested case-control study. Elevated leptin concentrations were

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observed in diabetes-developing cases compared to control (P = 0.005); however, the difference of irisin concentrations between diabetes-developing cases and controls was not significant (P = 0.41).

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In the nested case-control study, participants with the highest leptin concentrations had

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higher risk for developing DM compared to those with the lowest tertile after controlling for family history of DM, smoking, dyslipidemia medication, HDL-C and irisin as shown in model 2 of Table 3 (OR 3.50, 95% CI (1.19-10.37). However the significance disappeared after controlling for BMI as seen in model 3 (p=0.06). No association was observed between irisin concentrations and the risk of developing DM. A logistic regression analysis done on a similar model for continuous variables of biomarkers also did not show any significance in unadjusted (leptin; p= 0.09, irisin; p = 0.11) and adjusted models for the same covariates as above (leptin; p= 0.13, irisin; p = 0.13). Discussion: In this study, we report the association of leptin and irisin concentrations in regard to obesity; glucose tolerance; and their role in predicting future type 2 DM, in two studies. The first is a cross sectional study stratified by obesity status and glucose tolerance and the second is a prospective

ACCEPTED MANUSCRIPT cohort designed as a nested case control study focusing on future diabetes development. There is a significant upregulation in circulating irisin in obese individuals, which is even higher in individuals

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with impaired fasting blood glucose or diabetes. In addition, we demonstrate that although leptin is a

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predictor of future risk for developing T2DM, mainly through an underlying association with obesity, irisin concentrations do not predict the development of T2DM within 3-5 years. Using prospective

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data from this study to run power calculations one would estimate that to demonstrate a significant association with 80% power at the conventional one-tailed alpha 0.05, one would need 1158 subjects

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(290 cases and 868 controls). To our knowledge this is the first study to date that has evaluated whether circulating irisin concentrations have a possible role as a predictive marker of T2DM in a healthy male western population.

Irisin is a cleavage product of membrane protein FNDC5, secreted by muscle tissue and has

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been suggested to mimic the favorable metabolic effects of exercise by mediating the cross talk

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between contracting muscle and other tissues organs particularly the adipose tissue (2). Irisin has been proposed to induce beige/brown adipose tissue transformation by increasing the mitochondrial content

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and therefore the uncoupling protein-1 (UCP1) concentrations, which, in turn increases thermogenic function of the transforming white adipose tissue. These beige/brown cells have protective role

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against hypothermia, diabetes and obesity and improve glucose homeostasis (2). Increased expression of irisin is seen in skeletal muscle and blood concentrations of mice after acute exercise (15). Similar results were also seen in a cohort of obese children where irisin concentrations increased by 12 % after an exercise program and diet modification of 1 year (16) . Therefore, irisin’s potential role in metabolic health has gained significant attention since its discovery. Obesity Here, a significant upregulation in circulating irisin was found in obese individuals which was even higher in individuals with impaired fasting glucose or diabetes. There have been conflicting results on association of obesity, adiposity and irisin (4,9), however, recent interventional studies have shown a positive correlation between irisin, BMI and fat mass (17-19). In a randomized controlled

ACCEPTED MANUSCRIPT trial, significant decrease in irisin concentrations were seen after weight loss in overweight/obese population (20). However, this needs further exploration to establish the association between irisin

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secretion, adiposity and other metabolic parameters. We report a significantly positive correlation between irisin and leptin, supporting prior data (21,22). Leptin is an adipokine secreted by white adipocytes, and strongly correlates with fat mass and

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BMI (23). It is known to play a role in energy homeostasis by acting as a sensor via delivering a

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signal from adipose tissue to hypothalamus in regards to the status of energy storage. Hyperinsulinemia, insulin resistance and metabolic syndrome are known to have a strong association with leptin (24). Leptin administation to leptin deficient mice, enhanced irisin induced myogenesis, but interestingly downregulated the FNDC5 expression and prevented irisin induced upregulation of both brown and beige adipocyte specific genes, suggesting that leptin plays a regulatory role on

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FNDC5/irisin expression (25). In vitro studies have shown alteration of FNDC5 mRNA expression in

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subcutaneous adipose tissue after a 24hour incubation with leptin, demonstrating direct effect of leptin on FNDC5 regulation(26). In humans, obesity and metabolic syndrome are known as states of leptin

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resistance; similarly, irisin was shown to be elevated in metabolic syndrome. Leptin and irisin were observed to have positive correlation with BMI in patients with metabolic syndrome (27). These

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studies suggest a possible compensatory physiologic elevation of irisin and/or possible irisin resistance or a joint regulation by a common root cause (5). However, the exact link between irisin and leptin remains unknown.

Diabetes We report a significantly positive association between leptin concentration and risk of developing diabetes similar to the previous studies (28,29). Our study suggest that this is mainly through underlying mechanisms of obesity given that the association became non-significant after controlling for BMI in contrast to the study by Soderberg et al who showed that the association is independent of other factors in men (28). Previous studies have shown positive correlation of leptin

ACCEPTED MANUSCRIPT concentrations with fasting glucose, adiposity markers like BMI, weight, waist and hip circumference, percentage body fat and metabolic risk factor score (30). However, high leptin concentrations have

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not been shown to have any significant independent association with CVD risk or mortality in diabetic

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women in a 12 year follow up study (31). A cross-sectional study of 103 non-diabetic subjects had shown positive correlation of leptin concentrations with hyperinsulinemia and insulin resistance (24).

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Predictive role of leptin in identification of metabolic syndrome (MetS) has been demonstrated in studies and has been comparable to that of insulin resistance in identification of MetS (32). High

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circulating leptin concentrations, one day after myocardial infarction have shown to be associated with abnormal glucose tolerance at discharge in patients with no known history of diabetes (33). A 2 year prospective study on young men had shown predictive role of leptin and leptin binding protein (sOB-R) in prediction of fasting glucose, metabolic syndrome score, systolic blood pressure and

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adiposity (30). Associations with metabolic complications of obesity and / or diabetes and CVD are largely attributable to uncontrolled confounding by obesity and more specifically fat mass which is

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strongly associated with leptin.

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Data on irisin concentrations and diabetic state remains controversial. In this study, irisin concentrations were significantly different between non obese /normal glucose tolerance groups and

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obese with impaired fasting glucose or diabetes. Given that prospectively irisin was not an independent predictor of developing diabetes we conclude that any associations demonstrated herein or in the past may reflect an underlying association with obesity. The irisin-BMI correlation is apparently a weaker correlation compared to leptin-BMI correlation. In human gene expression studies, it was shown that FNDC5 expressed in muscle tissues and minimally expressed in the adipose tissue (3). In vitro studies revealed that irisin plays a role in recovery of insulin action and signaling in palmitic acid treated muscle (34). When irisin was introduced to the culture media, it inhibited glucotoxicity-induced apoptosis in human vascular epithelial cells. Furthermore, protection against endothelial injury and atherosclerosis and improvement in vascular function has also been demonstrated with administration of irisin in mice (35). Several, mouse studies have focused on irisin

ACCEPTED MANUSCRIPT administration showing favorable metabolic outcomes. An observational study has demonstrated that high fat diet-induced diabetic mice have decreased irisin secretion, which might contribute to the

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development of muscle insulin resistance. In insulin deficient mice, blood glucose decreased after

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irisin administration (36). Irisin treated diabetic mice also had a decrease in fasting glucose, fat mass, total cholesterol, triglycerides and improvement of glucose tolerance, uptake and insulin sensitivity

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(37). Contrary to these data in mice, multiple cross-sectional and case-control human studies have shown a negative correlation of irisin concentrations and diabetes (4,5,9,10). Irisin concentrations

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were also noted to be lower in impaired fasting, impaired glucose tolerance and diabetic state as compared to normal glucose tolerance subjects (19). Lower irisin concentrations were also seen in gestational diabetes patients as compared to healthy controls. Interestingly, in obese people, the negative association between T2DM and irisin was not shown and in nondiabetic individuals, irisin

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was found to be correlated with signs of insulin resistance (5,10)

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A few other studies have also demonstrated no relationship between circulating irisin concentrations and T2DM cross sectionally (11) A study comparing irisin concentrations between

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healthy controls and subjects with polycystic ovarian syndrome (PCOS), which is an insulin resistant state, has not demonstrated any significant difference (37). There are also two prospective studies

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showing conflicting data in regard to irisin and future diabetes risk. Erol et al. showed that lower irisin levels in early pregnancy may predict the development of gestational diabetes in a small cohort (38) whereas higher irisin levels were shown to be associated with future DM in a healthy rural Korean cohort (39). Since authors did not use dual energy absorptiometry, they could not provide information, and thus rule out uncontrolled confounding from, any association between circulating irisin levels and fat mass. In defining incident DM, authors used HgbA1c and a rather short period of observation, i.e. 2.6 years. Since DM develops progressively over time it remains uncertain whether patients might have had early stages of diabetes at the time of bllod draw and whether this might have affected the reported associations with irisin. To avoid such pitfall in our study, we excluded subjects diagnosed within 3 years prior to the study conclusion. Finally, since prior study participants were

ACCEPTED MANUSCRIPT rural Korean adults, results may not be applicable to other ethnic groups including western populations.

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Indeed, in several other cross sectional observational human studies, irisin was found to be positively correlated with BMI, and related with increased risk of metabolic syndrome and cardiovascular disease (4,5,9,10). Positive correlation is observed in several other insulin resistant

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states. Women with PCOS in a study by Li et al had higher irisin concentration, which decreased after

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treatment with metformin, hence, improvement in insulin sensitivity (37). Irisin concentrations were significantly high in individuals with acanthosis nigricans, which is a sign of insulin resistance, compared to healthy controls and positively correlated with fasting insulin and BMI (18). Significantly higher irisin concentrations were seen in diabetic population with hypertriglyceridemia

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and 8 weeks of fenofibrate treatment resulted in a significant decrease (37). Our results are consistent with this study. In our study serum irisin is positively correlated with serum triglyceride

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concentrations. A few previous studies showed no immediate feedback on circulating irisin following acute lipid infusion, that causes both lipotoxicity and insulin resistance (40).

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One limitation of our study is relatively small size for an epidemiological study. The study

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maximized power, however, by utilizing a 1:3 ratio and was able to reveal statistically significant changes where these were expected on the basis of prior literature. The samples that were used for the assays have been stored for a long time in -30oC. Although we have demonstrated previously that irisin degrades slowly overtime (41), it is stable with freeze and thaw cycles (3). In order to overcome this limitation, we matched the cases and controls for the year of sample collection. Year of sample collection was also controlled in the analysis, eliminating any potential bias that would be caused by the long-term storage. The subjects analyzed herein were only male veterans and hence comparisons across genders could not be performed. These results cannot necessarily be generalized to the general population. We measured irisin and leptin at least 3 years prior to the development of diabetes. The question still remains whether irisin and leptin closer to development of diabetes mellitus are better predictors. We avoided doing so given that diabetes may remain undiagnosed for a long period of

ACCEPTED MANUSCRIPT time and thus the closer the study time point to the timing of diagnosis the higher the chances of eliminating the time sequence criterion for causality inherent in cohort studies.

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In conclusion this is the first study that investigated the possible predictive role of irisin in T2DM with a prospective study in a western male population, which unlike prior observational

significant predictive value of irisin in diabetes.

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studies offers the time sequence criterion for causality. The study failed to show a statistically

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Recent studies have found correlations between irisin concentrations, the metabolic syndrome, cardiovascular disease, and insulin resistance (4,5,9,10,41,42). Our prospective study does not support an independent causal role for irisin in these associations. Future studies are required to evaluate further any causal or prognostic role of irisin in metabolic dysregulation and type 2 diabetes.

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Disclosure Statement: The authors have nothing to disclose.

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Author Contributions: All authors have read and agreed with the content of the manuscript. ASE wrote the manuscript, collected data and performed the assays. JU wrote the manuscript and contributed to statistical analysis. BK performed the statistical analysis and wrote the manuscript. FD performed the biochemical analysis. KHP contributed to the statistical analysis. AM contributed to assays. PV coordinated the Normative Ageing Study. CM supervised the study.

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Figure legends

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Figure 1. Circulating irisin and leptin concentrations according to the obesity and IFG/DM status after adjusting for the year of sample collection. (a) Irisin concentrations according to the obesity status; (b) Leptin concentrations according to the obesity status; (c) Irisin concentrations according to the IFG/DM status; (d) Leptin concentrations according to the IFG/DM status. Abbreviation: IFG, impaired fasting glucose, DM- Type 2 Diabetes Mellitus

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Figure 2. Circulating irisin and leptin concentrations as continuous variables and their association with BMI and fasting blood glucose.

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* - p < 0.05, ** - p < 0.01

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Figure 1

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Figure 2

ACCEPTED MANUSCRIPT Table 1. Characteristics of participants in the cross-sectional study. Obese subjects Obese subjects with normal with IFG/DM P* fasting glucose (n = 64) (n = 41)

69.5 ± 9.2

66.9 ± 7.9

69.4 ± 8.6

T

Non-obese subjects with IFG/DM (n = 38)

RI P

Age (y)

Non-obese subjects with normal fasting glucose (n = 73)

67.7 ± 7.4

0.33

32.7 (32.0-33.5) <0.001 110.4 (108.5<0.001 112.4) 132.4 ± 17.3 0.03

DBP (mmHg)

73.9 ± 8.6

77.9 ± 11.6

77.8 ± 10.4

0.06

TC (mg/dL)

214.8 ± 5.0

216.9 ± 6.9

216.9 ± 6.7

204.4 ± 5.4

0.34

121.6 ± 11.3

133.2 ± 11.0

177.2 ± 8.7

<0.001

54.3 ± 2.2

46.8 ± 2.1

44.2 ± 1.7

<0.001

113.8 ± 3.1

91.8 ± 3.0

129.3 ± 2.4

<0.001

124.8 ± 16.8

147.0 ± 16.2

172.5 ± 13.0

0.03

13.6 ± 1.2

13.2 ± 1.1

12.4 ± 0.9

0.86

6.1 ± 1.7

13.9 ± 1.7

16.5 ± 1.4

<0.001

FPG

(mg/dL)

acdef

†,

91.8 ± 2.3

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Irisin (ng/mL)†c 123.6 ± 12.1 Adiponectin 13.8 ± 0.8 (μg/mL)† †, Leptin (ng/mL) 5.3 ± 1.3 bcde

78.4 ± 11.8

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TG (mg/dL)†, bce 101.4 ± 8.2 †, HDL-C (mg/dL) 54.8 ± 1.6 bce

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BMI (kg/m2)†, bcdef 24.0 (23.3-24.7) 24.4 (23.3-25.4) 30.5 (29.6-31.5) 107.0 (104.6WC (cm)†, bcde 91.1 (89.3-92.9) 92.2 (89.7-94.7) 109.4) SBP (mmHg) 125.5 ± 15.9 134.4 ± 19.1 128.3 ± 17.1

Anthropometric data are presented as mean ± SD for continuous parametric variables or median

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(interquartile range) for continuous nonparametric variables. Laboratory variables were adjusted for the year of sample collection and are presented as mean ± SE or geometric mean ± SE. Impaired

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fasting glucose was defined as having fasting plasma glucose concentration ≥ 100 mg/dL. Abbreviation: BMI, body mass index; DBP, diastolic blood pressure; DM, diabetes mellitus; FPG, fasting plasma glucose; HDL-C, high-density lipoprotein cholesterol; IFG, impaired fasting glucose; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride; WC, waist circumference. *

Calculated by ANOVA or analysis of covariance (ANCOVA) adjusted for the year of sample

collection. †

Log-transformed before analysis.

ACCEPTED MANUSCRIPT Post-hoc analysis was performed by using Bonferroni method. “a” indicates there is a significant difference (P < 0.05) between non-obese subjects with normal fasting glucose and non-obese subjects with IFG; “b” indicates there is a significant difference between non-obese subjects with normal

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fasting glucose and obese subjects with normal fasting glucose; “c” indicates there is a significant

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difference between non-obese subjects with normal fasting glucose and obese subjects with IFG; “d” indicates there is a significant difference between non-obese subjects with IFG and obese subjects with normal fasting glucose; “e” indicates there is a significant difference between non-obese subjects

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with IFG and obese subjects with IFG; “f” indicates there is a significant difference between obese

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subjects with normal fasting glucose and obese subjects with IFG.

ACCEPTED MANUSCRIPT Table 2. Characteristics of participants according to the development of DM in the nested case-control study. P*

Controls (n = 140)

Age (y)

70.1 ± 0.9

70.0 ± 0.06

BMI (kg/m2)†

28.9 ± 0.5

27.4 ± 0.3

WC (cm)†

103.5 ± 1.3

99.1 ± 0.78

0.01

TC (mg/dL)

196.1 ± 6.0

207.5 ± 3.5

0.10

TG (mg/dL)†

166.2 ± 11.7

137.5 ± 6.8

0.06

HDL-C (mg/dL)†

43.4 ± 1.8

48.8 ± 1.0

0.01

FPG (mg/dL)†

129.7 ± 2.9

HbA1c (%)

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DM (n = 47)

1.0 0.01

<0.001

6.04 ± 0.4 (n = 14)

5.4 ± 0.4 (n = 42)

<0.001

Creatinine (mg/dL)†

1.1 ± 0.03

1.0 ± 0.01

0.24

Irisin (ng/mL)†

79.64± 4.03

84.31±2.34

0.41

Leptin (ng/mL)†

18.70±1.6

13.08±0.90

0.01

Family history of DM

14 (30.4)

33 (23.9)

0.34

ED

98.8 ± 1.7

Smoking status

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Hypertension medication (yes)

Dyslipidemia medication (yes)

30 (28.6)

25 (69.4)

67 (63.8)

3 (8.3)

8 (7.6)

28 (59.6)

67 (47.9)

0.16

23 (48.9)

42 (30.0)

0.02

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Ex-smoker Current smoker

8 (22.2)

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Non-smoker

0.56

Data are presented as mean ± SE or geometric mean ± SE for continuous variables and n (%) for categorical variables. Abbreviation: BMI, body mass index; DM, diabetes mellitus; FPG, fasting plasma glucose; HDL-C, high-density lipoprotein cholesterol; HbA1c, glycosylated hemoglobin, type A1c; TC, total cholesterol; TG, triglyceride; WC, waist circumference. Calculated by generalized linear model or generalized estimating equation. †Log-transformed before

*

analysis

ACCEPTED MANUSCRIPT Table 3. Odds ratios and 95% confidence intervals for the development of DM in relation to biomarker tertile (T) in the nested case-control study.

73.9-86.5

1.28 (0.58- 0.91 (0.40-

el 1

2.85)

Mod

1.06 (0.37- 0.87 (0.33-

2.10)

*

3.02)

Mod

1.11 (0.34- 0.72 (0.25-

el 3

3.27)

2.04)

*

P

86.7-

trend

for 62) 0.9-7.5

1

1

1

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el 2

*

2.3)

62)

235.6

Mod

*

T1 (n =

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T3 (n =

T2 (n = 62)

7.7-15.7

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T1 (n = 62) T2 (n = 62)

T

Leptin (ng/mL)

RI P

Irisin (ng/mL)

0.69

1

0.93*

1

0.70*

1

T3 (n = 62) P

trend 16.3-49.1

3.74 (1.45- 3.74 (1.459.67)

9.67)

3.43 (1.18- 3.50 (1.19†

†

9.98)

10.37)

3.64 (1.18- 2.98 †

11.24)

for

(0.8†

10.92)

0.005

0.028†

0.058†

cholesterol.

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Model 1 is unadjusted.

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Abbreviation: BMI, body mass index; DM, diabetes mellitus; HDL-C, high-density lipoprotein

Model 2 is adjusted for family history of DM, smoking, dyslipidemia medication, HDL-C, and mutual

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biomarkers (*leptin and †irisin). Model 3 is adjusted for family history of DM, smoking, dyslipidemia medication, HDL-C, mutual biomarkers (*leptin and †irisin), and BMI.