<p>ONLINE-ONLY APPENDIX</p><p>Iron metabolism is prospectively associated with insulin resistance and glucose intolerance over a 7-year follow-up period: the CODAM study</p><p>Authors:</p><p>Nick Wlazlo, MD1,2,3, Marleen MJ van Greevenbroek, PhD2,3, Isabel Ferreira, PhD2,3,4,5,</p><p>Eugene HJM Jansen, PhD6, Edith JM Feskens, PhD7, Carla JH van der Kallen, PhD2,3, Casper</p><p>G Schalkwijk, PhD2,3, Bert Bravenboer, MD, PhD1, Coen DA Stehouwer, MD, PhD2,3</p><p>1Department of Internal Medicine, Catharina Hospital, Eindhoven, 2CARIM School for</p><p>Cardiovascular Diseases, Maastricht University, Maastricht, 3Department of Internal</p><p>Medicine/Laboratory for Metabolism and Vascular Medicine, Maastricht University Medical</p><p>Centre (MUMC+), Maastricht, 4Department of Clinical Epidemiology and Medical</p><p>Technology Assessment (KEMTA), MUMC+, Maastricht, 5CAPHRI School for Public</p><p>Health and Primary Care, MUMC+, Maastricht, 6Laboratory of Health Protection Research,</p><p>National Institute for Public Health and the Environment (RIVM), Bilthoven, 7Division of</p><p>Human Nutrition – Section Nutrition and Epidemiology, Wageningen University,</p><p>Wageningen, The Netherlands</p><p>1 Supplementary Table 1: Associations of baseline transferrin saturation with insulin resistance over 7 years</p><p>Transferrin</p><p> saturation (per 1%) Outcome Model β 95%CI - HOMA2-IR 1 -1.17; -0.39‡ 0.78 - (% increase) 2 -0.96; -0.25† 0.60 - 3 -0.50; 0.10 0.20 - 4 -0.45; 0.15 0.15 Hepatic IR - 1 -0.96; -0.09* 0.53 (% increase) - 2 -0.86; 0.01 0.42 - 3 -0.49; 0.32 0.08 - 4 -0.49; 0.34 0.08 - Adipocyte IR 1 -1.44; -0.49‡ 0.97 - (% increase) 2 -1.02; -0.15† 0.59 - 3 -0.55; 0.23 0.16 - 4 -0.46; 0.28 0.09 βs are unstandardized regression coefficients and represent the longitudinal associations between baseline transferrin saturation on the one hand, and HOMA2-IR, hepatic insulin resistance (IR), and adipocyte insulin resistance (IR) at both baseline and follow up on the other hand. 95%CI: 95% confidence interval.</p><p>Model 1: crude associations</p><p>2 Model 2: model 1 + adjusted for age, sex, follow-up time, glucose metabolism status, baseline cardiovascular disease, baseline estimated glomerular filtration rate, smoking status, alcohol consumption, dietary energy intake, physical activity, family history of T2DM, use of medication, and ferritin and transferrin adjusted for each other</p><p>Model 3: model 2 + adjusted for waist circumference</p><p>Model 4: model 3 + adjusted for low-grade inflammation score</p><p>*p<0.05; †p<0.01; ‡p<0.001. </p><p>3 Supplementary Table 2: Associations of baseline transferrin saturation with glucose tolerance over 7 years</p><p>Transferrin</p><p> saturation (per 1%) Outcome Model β 95%CI Fasting - 1 -0.24; 0.01 glucose 0.12 - 2 -0.18; -0.01* 0.10 - 3 -0.12; 0.04 0.04 - 4 -0.11; 0.04 0.03 2h glucose - 1 -0.56; 0.08 0.24 - 2 -0.31; 0.17 0.07 3 0.02 -0.21; 0.25 4 0.04 -0.19; 0.27 - AUC glucose 1 -0.37; 0.06 0.16 - 2 -0.27; 0.06 0.10 - 3 -0.18; 0.14 0.02 - 4 -0.17; 0.15 0.01 βs are unstandardized regression coefficients and represent the longitudinal associations between baseline transferrin saturation on the one hand, and fasting glucose, 2h glucose, and area under the curve (AUC) for glucose (during oral glucose tolerance test) at both baseline and follow up on the other hand. 95%CI: 95% confidence interval.</p><p>Model 1: crude associations</p><p>Model 2: model 1 + adjusted for age, sex, follow-up time, baseline cardiovascular disease, baseline estimated glomerular filtration rate, smoking status, alcohol consumption, dietary energy intake,</p><p>4 physical activity, family history of T2DM, use of medication, and ferritin and transferrin adjusted for each other</p><p>Model 3: model 2 + adjusted for waist circumference</p><p>Model 4: model 3 + adjusted for low-grade inflammation score</p><p>*p<0.05; †p<0.01; ‡p<0.001. </p><p>5 Supplementary Table 3: Associations of baseline markers of iron metabolism with absolute changes in insulin resistance and glucose tolerance during 7-year follow-up</p><p>Ferritin Transferrin Serum iron NTBI (per 10 μg/L) (per 0.1 g/L) (per μmol/L) (per μmol/L) Outcome Group β 95%CI β 95%CI β 95%CI β 95%CI - - ΔHOMA2-IR Observational -0.001 -0.010; 0.007 -0.049; 0.003 -0.016; 0.013 0.002 -0.063; 0.067 0.023 0.002 - Treated -0.005 -0.019; 0.010 -0.093; 0.032 0.021 -0.014; 0.057 0.066 -0.068; 0.199 0.031 ΔHepatic IR - Observational 0.033 -0.084; 0.149 -0.510; 0.229 0.050 -0.156; 0.257 0.437 -0.476; 1.349 0.140 - - Treated 0.028 -0.110; 0.166 0.079 -0.516; 0.673 -0.403; 0.302 -1.537; 0.925 0.051 0.306 - - - ΔAdipocyte IR Observational -0.080 -0.342; 0.181 -1.570; 0.084 -0.680; 0.239 -2.639; 1.470 0.743 0.221 0.584 - - Treated -0.953 -1.441; -0.465‡ -3.289; 0.894 0.159 -1.098; 1.416 -5.636; 3.718 1.198 0.959 ΔFasting glucose Observational -0.001 -0.007; 0.004 0.011 -0.007; 0.028 0.002 -0.008; 0.012 0.047 0.005; 0.089* - Treated -0.006 -0.018; 0.005 -0.100; -0.001* 0.028 -0.001; 0.057 0.034 -0.074; 0.142 0.051 Δ2h glucose Observational 0.002 -0.023; 0.028 0.060 -0.020; 0.140 0.033 -0.012; 0.077 0.140 -0.058; 0.338 - Treated -0.014 -0.054; 0.026 -0.228; 0.119 0.091 -0.010; 0.193 0.046 -0.313; 0.406 0.055 ΔAUC glucose Observational -0.511 -2.441; 1.421 - -6.429; 5.814 2.963 -0.410; 6.337 20.09 5.358; 34.82†</p><p>6 0.307 - - Treated -1.228 -4.715; 2.258 -25.25; 4.598 7.144 -1.645; 15.93 -39.23; 23.67 10.32 7.776 βs are unstandardized regression coefficients and represent the associations between baseline ferritin, transferrin, serum iron, and non-transferrin-bound iron</p><p>(NTBI) on the one hand, and the absolute change in HOMA2-IR, hepatic insulin resistance (IR), adipocyte insulin resistance (IR), fasting glucose, 2h glucose, and area under the curve (AUC) for glucose (during oral glucose tolerance test) during 7 years follow up on the other hand. For example, per 10μg/L difference in baseline ferritin between two subjects, the absolute change in HOMA2-IR during 7 years differs by -0.001 units. These analyses are stratified into a group that received lifestyle and/or medical interventions that influence glucose metabolism (“Treated group” (n=174), defined by the presence of</p><p>T2DM at baseline and/or the use of glucose-lowering and/or lipid-lowering medication at any time), and a group that received no interventions at all during the follow up (“Observational group” (n=212)). All analyses were adjusted for baseline covariates age, sex, glucose metabolism status (only for outcomes</p><p>HOMA2-IR, hepatic IR, and adipocyte IR), cardiovascular disease, estimated glomerular filtration rate, smoking status, alcohol consumption, dietary energy intake, physical activity, family history of T2DM, use of medication, waist circumference, low-grade inflammation score, and ferritin and transferrin adjusted for each other. 95%CI: 95% confidence interval.</p><p>*p<0.05; †p<0.01; ‡p<0.001. </p><p>7</p>