Fetuin-A and Incident Diabetes Mellitus in Older Persons

ORIGINAL CONTRIBUTION

Joachim H. Ix, MD, MAS Context Fetuin-A is a hepatic secretory protein that binds the insulin receptor and Christina L. Wassel, MS inhibits insulin action in vitro. In prior cross-sectional studies in humans, higher fe- Alka M. Kanaya, MD tuin-A levels were associated with insulin resistance. However, the longitudinal association of fetuin-A with incident type 2 diabetes mellitus is unknown. Eric Vittinghoff, PhD Objective To determine whether fetuin-A levels are associated with incident diabe- Karen C. Johnson, MD, MPH tes in older persons. Annemarie Koster, PhD Design, Setting, and Participants Observational study among 3075 well- Jane A. Cauley, DrPh functioning persons aged 70 to 79 years. In this case-cohort study, we retrospectively measured fetuin-A levels in baseline serum among 406 randomly selected partici- Tamara B. Harris, MD pants without prevalent diabetes, and all participants who developed incident diabe- Steven R. Cummings, MD tes mellitus during a 6-year follow-up (to August 31, 2005). Michael G. Shlipak, MD, MPH Main Outcome Measure Incident diabetes mellitus. for the Health ABC Study Results Incident diabetes developed in 135 participants (10.1 cases/1000 person- years). Participants with fetuin-A levels within the highest tertile (Ͼ0.97 g/L) had an increased risk of incident diabetes (13.3 cases/1000 person-years) compared with par- YPE 2 DIABETES MELLITUS HAS ticipants in the lowest tertile (Յ0.76 g/L) (6.5 cases/1000 person-years) in models become a global epidemic and adjusted for age, sex, race, waist circumference, body weight, physical activity, blood the increased prevalence of pressure level, fasting glucose level, high-density lipoprotein cholesterol concentra- obesity1 is a major contribut- tion, triglyceride concentration, and C-reactive protein level (adjusted hazard ratio, ingT factor.2-4 However, diabetes does 2.41; 95% confidence interval, 1.28-4.53; P=.007). The association was not affected not develop in all obese individuals by adipocytokine levels but was moderately attenuated by adjustment for visceral adiposity (adjusted hazard ratio of highest vs lowest tertile 1.72; 95% confidence inter- and there is a strong genetic contribu- val, 0.98-3.05; P=.06). tion to risk.5 Despite significant recent advances,6 mechanisms respon- Conclusion Among well-functioning older persons, serum fetuin-A is associated with sible for individual differences in incident diabetes, independent of other markers of insulin resistance. clinical phenotype remain largely un- JAMA. 2008;300(2):182-188 www.jama.com known. Recent research has identified proteins secreted from adipose tissue In cross-sectional studies in humans, In this study, we measured baseline that regulate glucose metabolism, higher fetuin-A levels were associated serum fetuin-A levels among partici- termed adipocytokines.7-9 Study of these with insulin resistance.15-17 However, to pants in the Health, Aging, and Body proteins has provided new insights to our knowledge the temporal relation- Composition (Health ABC) Study who the biology of glucose regulation and ship of fetuin-A levels with incident dia- were diabetes-free. Participants were has identified novel candidate thera- betes has not been evaluated. followed longitudinally for the occur- peutic targets. In contrast to adipocytokines, fe- Author Affiliations: Division of Nephrology and Hy- Biometry, National Institute on Aging, Bethesda, Mary- ␣ pertension, Department of Medicine, and Division land (Drs Koster and Harris); Department of Epide- tuin-A ( 2–Heremans-Schmid glyco- of Preventive Medicine, Department of Family and miology, University of Pittsburgh, Pittsburgh, Penn- protein [Ahsg]) is produced in hepa- Preventive Medicine, University of California, San sylvania (Dr Cauley); Department of Medicine, tocytes and secreted into serum, where Diego, and San Diego Veterans Affairs Healthcare California Pacific Medical Center and San Francisco 10 System, San Diego, California (Dr Ix); Department of Coordinating Center, San Francisco (Dr Cummings); it is found in high concentrations. In Epidemiology, University of Minnesota, Minneapolis and San Francisco Veterans Affairs Medical Center, vitro, fetuin-A reversibly binds the in- (Ms Wassel); Department of Epidemiology and Bio- San Francisco (Dr Shlipak). statistics (Ms Wassel and Drs Vittinghoff and Shlipak) Corresponding Author: Joachim H. Ix, MD, MAS, Di- sulin receptor tyrosine kinase in muscle and Department of Medicine (Drs Kanaya, Cum- vision of Nephrology and Hypertension, Department and fat and decreases downstream sig- mings, and Shlipak), University of California, San Fran- of Medicine, University of California, San Diego, and cisco; Department of Medicine, University of Tennes- San Diego Veterans Affairs Healthcare System, 3350 nal cascades, which results in insulin see Health Science Center, Memphis (Dr Johnson); La Jolla Village Dr, Mail Code 111-H, San Diego, CA resistance in these target tissues.11-14 Laboratory for Epidemiology, Demography, and 92161 ([email protected]).

Downloaded From: https://jamanetwork.com/ on 09/30/2021 FETUIN-A AND INCIDENT DIABETES

rence of incident diabetes. We hypoth- Figure 1. Case-Cohort Study Design for This Health ABC Ancillary Study esized that higher fetuin-A levels would

be associated with incident diabetes and 3075 Participants in Health ABC cohort that the associations would be inde- 939 White men 855 White women pendent of previously recognized dia- 552 Black men betes risk factors that are commonly as- 729 Black women certained in clinical practice. METHODS 500 Randomly selected (125 from 2575 Not selected by random Participants each race/sex stratum) sampling At the baseline study visit (April 1997 to June 1998), Health ABC enrolled 3075 94 Excluded (prevalent diabetes) well-functioning men and women aged 70 to 79 years, recruited from a sample of Medicare beneficiaries at 2 clinical sites 406 Included in subcohort (Pittsburgh, Pennsylvania, and Mem- 23 Incident cases of diabetes 112 Incident cases of diabetes phis, Tennessee). Participant eligibility during follow-up during follow-up required self-reported ability in walking a quarter mile, climbing 10 steps, and performing basic activities of daily liv- 519 Total study sample ing. Venous blood samples were ob- 406 Subcohort (including 23 tained after overnight (8-hour) fasts and incident cases of diabetes) 112 Incident cases in cohort stored at −70°C. Participants under- not randomly sampled went a 1-day evaluation that included medical history, physical activity assess- ment, physical examination, and radio- pants had prevalent diabetes at base- a 2-hour postchallenge plasma glu- graphic tests. All participants provided line, we took a random sample of 500 cose level 200 mg/dL or greater (to con- written informed consent, and the study participants, assumed that approxi- vert glucose to mmol/L, multiply by was approved by the institutional re- mately 100 individuals (20%) would be 0.0555). These individuals were ex- view boards at the University of Pitts- excluded due to prevalent disease, and cluded and the resulting sample of 406 burgh and the University of Tennessee thereby derived the required number participants constituted the subco- Health Science Center. In addition, the of nondiabetic subcohort participants hort for this study. present study was approved by the Hu- required for comparison. All Health ABC participants were man Research Protection Program at the Thus, we randomly selected 500 par- contacted by telephone every 6 months University of California, San Diego. ticipants from the parent Health ABC and assessed at annual clinic visits at Study who were stratified equally in 4 which they were asked about interval Selection of Subcohort Participants sex and race strata (FIGURE 1). Ran- diagnosis of diabetes. Medication use and Incident Diabetes Cases dom numbers were generated on a con- was recorded at each annual visit. Fast- We used a case-cohort design for this tinuous standard uniform distribu- ing blood samples were obtained for study.18 Preliminary data in a separate tion (U [0,1]) and were assigned to each glucose measurement at the second, cohort of participants of similar age participant within each of the 4 race/ fourth, and sixth annual visits. Inci- demonstrated that individuals with sex strata. The participants were then dent diabetes was defined by new dia- prevalent diabetes had fetuin-A levels sorted in ascending order by that as- betes diagnosis between baseline and of 0.04 g/L higher on average, com- signed random number. One hundred study closeout (August 31, 2005), de- pared with individuals without diabe- twenty-five participants were chosen in fined by self-report, new use of diabetes (28% standardized effect size).17 As- order from each stratum until we tes medications, or a fasting glucose suming a similar strength of association reached the total subcohort sample size level 126 mg/dL or greater. Any par- for incident diabetes, and knowing a of 500. ticipant who was free of diabetes at priori that 135 incident diabetes cases From these 500 participants, 94 baseline and who developed diabetes accrued during follow-up in the Health (19%) had prevalent diabetes at base- during follow-up represented a case for ABC cohort, we calculated that a sub- line, which was defined as any 1 or this study (Figure 1). cohort of 384 participants without more of the following: (1) self-report Cases could arise from either the ran- prevalent diabetes was required to pro- of diabetes diagnosis; (2) use of hypo- domly selected subcohort or the re- vide 80% power with a 2-sided ␣=.05. glycemic medications; (3) a fasting glu- mainder of the Health ABC Study (as Because 19% of Health ABC partici- cose level 126 mg/dL or greater; or (4) per typical case-cohort study design).18

Downloaded From: https://jamanetwork.com/ on 09/30/2021 FETUIN-A AND INCIDENT DIABETES

We observed 135 cases of incident dia- Visceral and subcutaneous adiposity Statistical Analyses betes over the 6-year observation pe- were measured at the midpoint be- Because age-, sex-, and race-specific nor- riod (incidence rate 10.1 cases/1000 tween the fourth and fifth lumbar ver- mal ranges of fetuin-A are unknown, and person-years). Among these cases, 23 tebrae with participants in the supine because the distribution of fetuin-A was individuals (17%) were also sampled position. Fat area was calculated by positively skewed, we categorized par- within the subcohort. Therefore, the multiplying the number of pixels of fat ticipants into tertiles based on the dis- total study sample for this analysis con- tissue by pixel area using interactive tribution of fetuin-A in the subcohort. In sisted of 519 individuals. data language development software companion analysis, we also evaluated (RSI, Boulder, Colorado). Visceral adi- fetuin-A (natural log-transformed) as a Measurements posity was planimetrically distin- continuous predictor variable. Baseline Fetuin-A was measured in baseline se- guished from subcutaneous adiposity differences in demographic and clinical rum among the 519 participants se- using the internal abdominal wall fas- variables were compared across fe- lected for this study in 2007 (7-9 years cial plane. tuin-A tertiles by analysis of variance for after the Health ABC Study enroll- Seated systolic and diastolic blood continuous variables and the ␹2 test or ment) using a human fetuin-A en- pressure levels were measured using Fisher exact test for categorical variables. zyme linked immunosorbent assay kit manual sphygmomanometers by trained To accommodate the stratified sam- (Epitope Diagnostics, San Diego, Cali- research technicians. Fasting and 2-hour plingdesign,diabetesincidencerateswere fornia). The assay used a 2-site “sand- postchallengeplasmaglucoselevelswere estimated using all incident cases in the wich” technique with 2 polyclonal an- measured by an automated glucose oxi- Health ABC cohort. To estimate the cor- tibodies that bind to different epitopes dasereaction(YSI2300glucoseanalyzer, responding person-years at risk, we of human fetuin-A. Measurements were Yellow Springs Instruments, Yellow weighted follow-up times for the subco- performed at the Laboratory for Clini- Springs, Ohio). High-density lipopro- hort by the inverse of their probabilities cal Biochemistry Research, University tein cholesterol and triglyceride concen- of selection, which were known exactly of Vermont, Colchester. Fetuin-A was trations were measured using a Vitros and differed slightly by sex and race. The measured twice in the same sample for 950 analyzer (Johnson & Johnson, New association of fetuin-A with incident dia- each participant and results were av- Brunswick,NewJersey).High-sensitivity betes was evaluated by a modified Cox re- eraged. Intra-assay and interassay co- C-reactive protein was measured by gressionthataccountedforthecase-cohort efficients of variation were less than 5%. enzyme-linked immunosorbent assay study design.21,22 Subcohort noncases and Among a 5% blind duplicate assess- (Calbiochem, San Diego) and was stan- subcohort cases before their failure were ment at the Laboratory for Clinical Bio- dardized to the World Health Organi- weighted with the inverse of the sampling chemistry Research, the intraindi- zation’s First International Reference fraction (1/␣). Cases outside the subco- vidual coefficients of variation were Standard. Interleukin 6 was measured hort were not weighted before failure. All 13.5% to 20%. with the HS600 Quantikine kit (R&D cases, either outside or inside the subco- Age, sex, and race were determined by Systems, Minneapolis, Minnesota), and hort, were assigned a weight of 1 at the self-report. Physical activity was as- the HSTA50 kit was used to measure tu- time of failure, as described by Barlow sessed using self-report of walking and mor necrosis factor. Reliability, deter- etal.21 Jackknifeestimatesofvariancewere exercise and assigned kilocalories per minedbyblindduplicates,revealedmean used, which are equivalent to robust week to activities. Height and weight coefficients of variation of 8.0%, 10.3%, variance estimators from standard Cox were measured with participants wear- and 15.8%, respectively. models.23 ing light-weight clothing and no shoes, Adiponectin and leptin were mea- Initialmodelswerestratifiedonsexand and body mass index was calculated as sured in duplicate by radioimmunoas- race and age adjusted. Subsequent mod- weight in kilograms divided by height in say (Linco Research Inc, St Charles, els were adjusted for measures of insu- meters squared. Waist circumference was Missouri) with intra-assay coeffi- lin resistance that are commonly avail- assessed using a flexible measuring tape cients of variation of less than 3.6% and able in clinical practice (age, sex, race, on bare skin at the level of maximal cir- less than 7.5%, respectively. Plasmino- physical activity, systolic and diastolic cumference, midway between the lower gen activator inhibitor 1 was mea- bloodpressurelevels,fastingglucoselevel, ribs and anterior superior iliac spine. sured by 2-site enzyme-linked immu- waist circumference, body weight, high- Regional adiposity was measured by nosorbent assay according to previously density lipoprotein cholesterol concen- computed tomography using scan- published methods19 and had coeffi- tration, triglyceride concentration, and ners and standardized protocols of the cients of variation of less than 3.5%. C-reactive protein level). Subsequent Somatom Plus 4 (Siemens, Erlanger, Creatinine was measured by colorim- models evaluated candidate mediators of Germany), the Picker PQ 2000S (Mar- etry (Johnson & Johnson) and esti- the observed association that were se- coni Medical Systems, Cleveland, mated glomerular filtration rate was cal- lected a priori (visceral adiposity, plas- Ohio), or the 9800 Advantage (Gen- culated by the 4-variable Modification minogen activator inhibitor 1, adiponec- eral Electric, Milwaukee, Wisconsin). of Diet in Renal Disease equation.20 tin, leptin, tumor necrosis factor, and

Downloaded From: https://jamanetwork.com/ on 09/30/2021 FETUIN-A AND INCIDENT DIABETES

IL-6). Multiplicative interaction terms Figure 2. Distribution of Fetuin-A Among a Random Sample of Health ABC Study were created to determine whether the Participants (n=406) association was similar by race, sex, and obesity status (body mass index Ն30).24 20 Proportional hazards assumptions were 18 evaluatedbyformalhypothesistestingand 16 byvisualinspectionoflog-minus-logplots 14 andSchoenfeldresidualsvssurvivaltime. 12 No violations were observed. Two-tailed 10 Ͻ P values .05 were considered statisti- 8

callysignificant.Analyseswereperformed 6

with SAS version 9.1 (SAS Institute, Cary, Subcohort Participants, % 4 NorthCarolina)andSPlusversion6.1(In- 2 sightful Corp, Seattle, Washington). 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 RESULTS Fetuin-A Level, g/L Among the randomly selected subcohort of 406 participants without dia- self-reported new diagnosis by a physi- cant attenuation on the basis of avail- betes, the mean (SD) age was 73 (3) cian. We observed a graded increase in able measures of adipocytokines but years. The sample was 50% female and the incidence of diabetes with in- observed moderate attenuation by vis- black, reflecting stratified sampling. The creased fetuin-A levels (TABLE 2). Par- ceral adiposity, which diminished the as- mean (SD) body mass index was 27 (5). ticipants in the highest tertile had more sociation by approximately one-third. The distribution of fetuin-A was posi- than twice the incidence rate compared Whereas the point estimate for hazards tively skewed (FIGURE 2)withame- with the lowest tertile in unadjusted among the highest tertile remained 1.7- dian of 0.87 g/L (interquartile range, analysis (13.3 vs 6.5 cases/1000 person- fold higher than the lowest tertile, the 0.71-1.04 g/L). When compared with years, P=.005). We observed minimal association was no longer statistically participants with fetuin-A levels in the change in this estimate after adjust- significant (P=.06) with adjustment for lowest third of the subcohort, partici- ment for demographic and other clini- visceral adiposity (TABLE 3). pants with higher fetuin-A levels were cal predictors (adjusted hazard ratio, more often white, had higher serum tri- 2.41; 95% confidence interval, 1.28- COMMENT glyceride levels, and had more vis- 4.53). Results were similar when body In this study, we observed that higher ceral adiposity. Other measures of body mass index replaced waist circumfer- serum fetuin-A levels are associated composition, blood pressure levels, lipid ence and body weight as covariates in the with incident diabetes mellitus in hu- parameters, and adipocytokine levels model. Results were also similar be- mans. The association was indepen- were similar across tertile groups. Bi- tween men and women, between black dent of physical activity, inflamma- variate associations were similar among and white individuals, and among indi- tory biomarkers, and other commonly participants who developed incident viduals with or without obesity (P val- available measures of insulin resis- diabetes during follow-up (TABLE 1). ues for interaction all Ͼ.27). When tance and was similar irrespective of sex, Between baseline and the end of the treated as a continuous predictor, each race, and obesity status. Approxi- study, 60 of 406 subcohort partici- doubling in fetuin-A was associated with mately one-third of this association ap- pants died. Fetuin-A levels did not dif- a 57% increased risk of incident diabe- peared to be mediated by the quantity fer significantly between survivors and tes (hazard ratio, 1.57; 95% confidence of visceral adiposity. Yet fetuin-A re- nonsurvivors (mean [SD] levels, 0.93 interval, 0.99-2.49; P=.05) in the final mained associated with a 1.7-fold risk [0.40] and 0.96 [0.38 g/L], respec- multivariate model (adjusted for age, sex, of diabetes after adjustment for vis- tively; P=.56). Follow-up data were race, physical activity, body weight, waist ceral adiposity, an association that was available for more than 98% of the re- circumference, systolic and diastolic not statistically significant (P=.06). maining participants, and only 1% of blood pressure levels, fasting glucose In 1989, fetuin-A was reported as an visits were missed each year. level, high-density lipoprotein choles- endogenous inhibitor of insulin recep- Among survivors, we observed 135 terol concentration, triglyceride concen- tors through binding insulin receptor ty- cases of incident diabetes (10.1 cases/ tration, and C-reactive protein level). rosine kinase in adipocytes and skeletal 1000 person-years). Of these, 71 cases In additional analysis, candidate me- muscle, which resulted in decreased rates (53%) were due to a fasting glucose level diators were added individually to the of autophosphorylation and down- of 126 mg/dL or greater, 22 cases (16%) model to determine if they attenuated stream insulin signaling cascades.11,12 were due to new use of antidiabetic medi- the association of fetuin-A with inci- This function was conserved across all cation, and 42 cases (31%) were due to dent diabetes. We observed no signifi- species homologs evaluated to date in

Downloaded From: https://jamanetwork.com/ on 09/30/2021 FETUIN-A AND INCIDENT DIABETES

vitro25-28 and has been confirmed in vivo cus.29,30 Fetuin-A knockout mice are in- We17 and others15,16 have previously dem- in rats.28 The human fetuin-A gene re- sulin-sensitive; are resistant to weight onstrated that higher fetuin-A levels are sides on chromosome 3q27, which was gain; and have less adiposity, lower free associated with insulin resistance/ previously identified as a metabolic syn- fatty acid, and lower triglyceride levels metabolic syndrome in cross-sectional drome and diabetes susceptibility lo- compared with wild-type controls.13,14 studies. However, despite compelling in

Table 1. Baseline Measures of Subcohort and Incident Diabetes Case Participants by Fetuin-A Tertilesa Random Subcohort Incident Diabetes Fetuin-A, g/L Cases Fetuin-A, g/L

Tertile 1 Tertile 2 Tertile 3 Tertile 1 Tertile 2 Tertile 3 (Յ 0.76) (0.77-0.97) (Ͼ 0.97) P (Յ 0.76) (0.77-0.97) (Ͼ 0.97) P Characteristics (n = 134) (n = 138) (n = 134) Value (n = 23) (n = 37) (n = 52) Value Age, y 73 (3) 74 (3) 74 (3) .33 73 (3) 73 (3) 73 (3) .35 Female, No. (%) 64 (48) 64 (46) 73 (55) .36 14 (61) 21 (57) 26 (50) .65 Black, No. (%) 80 (60) 68 (49) 55 (41) .009 14 (61) 21 (57) 18 (35) .04 Physical activity, median (IQR), 536 (129-1588) 515 (141-1351) 408 (61-1206) .30 311 (43-1141) 449 (32-1468) 495 (105-1274) .39 kcal/wkb Body weight, kg 73 (14) 76 (14) 75 (15) .36 81 (14) 84 (15) 83 (16) .12 Waist circumference, cm 96 (13) 98 (13) 99 (12) .21 102 (12) 107 (12) 106 (12) .03 BMIc 27 (5) 27 (5) 27 (5) .52 30 (5) 30 (4) 30 (5) .25 Visceral adiposity, cm2 114 (53) 134 (58) 136 (57) .003 161 (60) 175 (61) 179 (68) Ͻ.001 Subcutaneous adiposity, cm2 269 (126) 280 (118) 290 (125) .41 328 (118) 346 (123) 330 (126) .25 SBP, mm Hg 136 (22) 136 (23) 136 (23) .98 137 (20) 139 (22) 135 (18) .99 DBP, mm Hg 72 (14) 73 (12) 72 (12) .69 76 (8) 72 (14) 71 (11) .81 Fasting glucose, median (IQR), 93 (87-97) 92 (86-98) 92 (86-98) .77 98 (94-105) 104 (94-113) 101 (96-110) .63 mg/dLb 2-Hour postchallenge glucose, 118 (96-133) 119 (98-145) 117 (101-147) .28 143 (129-175) 148 (116-174) 147 (133-172) .07 median (IQR), mg/dLb HDL-C, median (IQR), mg/dLb 54 (45-69) 56 (46-64) 53 (44-65) .43 53 (41-61) 48 (42-57) 48 (41-60) .18 Triglycerides, median (IQR), 102 (76-132) 110 (81-149) 120 (86-168) .003 106 (99-178) 139 (113-169) 139 (111-180) Ͻ.001 mg/dLb CRP, median (IQR), mg/Lb 1.6 (1.0-3.0) 1.7 (1.0-2.9) 1.3 (1.0-2.3) .10 2.3 (1.1-4.5) 2.4 (1.2-3.4) 2.1 (1.1-3.2) .17 IL-6, median (IQR), pg/mLb 1.9 (1.2-2.7) 1.9 (1.2-2.8) 1.7 (1.2-2.6) .36 1.6 (1.3-2.8) 1.9 (1.3-3.1) 1.9 (1.4-2.5) .92 TNF, median (IQR), pg/mLb 2.8 (2.2-3.6) 3.2 (2.5-4.1) 3.1 (2.3-4.0) .11 3.1 (2.4-3.8) 3.2 (2.5-4.1) 3.3 (2.3-3.9) .13 Adiponectin, median (IQR), 10 (7-15) 11 (7-15) 11 (7-15) .93 7 (5-12) 8 (5-10) 8 (6-14) .93 µg/mLb Leptin, median (IQR), ng/mLb 10 (5-18) 10 (4-22) 11 (5-22) .68 20 (8-27) 20 (13-42) 12 (7-24) .66 PAI-1, median (IQR), ng/mLb 18 (11-31) 19 (13-32) 21 (12-39) .20 24 (17-49) 31 (25-43) 29 (15-52) .07 eGFR, mL/min/1.73 m2 75 (16) 74 (15) 73 (16) .55 73 (12) 71 (22) 70 (14) .22 Abbreviations: BMI, body mass index; CRP, C-reactive protein; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; HDL-C, high-density lipoprotein cholesterol; IQR, interquartile range; PAI-1, plasminogen activator inhibitor; SBP, systolic blood pressure; TNF, tumor necrosis factor. SI conversion factors: to convert glucose to mmol/L, multiply by 0.0555; high-density lipoprotein cholesterol to mmol/L, multiply by 0.0259; triglycerides to mmol/L, multiply by 0.0113; C-reactive protein to nmol/L, multiply by 9.524; plasminogen activator inhibitor-1 to pmol/L, multiply by 19.231. a Data are presented as mean (SD) unless otherwise specified. b P values obtained after natural log-transformation. c Calculated as weight in kilograms divided by height in meters squared.

Table 2. Incident Diabetes Events Among Older Individuals by Fetuin-A Tertiles Fetuin-A, HR (95% CI), g/L

Tertile 1 Tertile 2 Tertile 3 (Յ 0.76) (0.77-0.97) (Ͼ 0.97) Tertile 2 vs 1 Tertile 3 vs 1 P Value (n = 28) (n = 47) (n = 60) P Value P Value for Trend Incidence (rate per 1000 person-years) 6.5 10.3 13.3 Unadjusted [Reference] 1.60 (0.94-2.73) 2.19 (1.31-3.65) .08 .002 .002 Demographic adjusteda [Reference] 1.64 (0.96-2.80) 2.23 (1.34-3.73) .07 .002 .001 Multivariate adjustedb [Reference] 1.82 (0.94-3.53) 2.41 (1.28-4.53) .07 .007 .006 Abbreviations: CI, confidence interval; HR, hazard ratio. a Adjusted for age, sex, and race. b Adjusted for demographic variables: physical activity score, body weight, waist circumference, systolic blood pressure, diastolic blood pressure, fasting glucose level, high-density lipoprotein cholesterol concentration, triglyceride concentration, and C-reactive protein level.

Downloaded From: https://jamanetwork.com/ on 09/30/2021 FETUIN-A AND INCIDENT DIABETES

Table 3. Evaluation of Candidate Mediators of the Association of Fetuin-A With Incident Diabetes Fetuin-A, HR (95% CI), g/L

Tertile 1 Tertile 2 Tertile 3 Tertile 2 vs 1 Tertile 3 vs 1 P Value (Յ 0.76) (0.77-0.97) (Ͼ 0.97) P Value P Value for Trend Unadjusted [Reference] 1.60 (0.94-2.73) 2.19 (1.31-3.65) .08 .002 .002 Demographic adjusteda [Reference] 1.64 (0.96-2.80) 2.23 (1.34-3.73) .07 .002 .001 Demographic adjusted ϩ visceral [Reference] 1.32 (0.73-2.36) 1.72 (0.98-3.05) .36 .06 .05 adiposityb Demographic adjusted ϩ PAI-1, [Reference] 1.71 (0.95-3.10) 1.94 (1.09-3.45) .08 .03 .02 adiponectin, and leptinb Demographic adjusted ϩ TNF and IL-6b [Reference] 1.61 (0.90-2.89) 2.38 (1.37-4.15) .11 .001 .001 Fully adjusted for all candidate mediatorsc [Reference] 1.83 (0.80-4.17) 2.44 (1.14-5.22) .15 .02 .02 Abbreviations: CI, confidence interval; HR, hazard ratio; PAI-1, plasminogen activator inhibitor; TNF, tumor necrosis factor. a Adjusted for age, sex, and race. b Denotes adjusted demographic plus other variables as specified. c Adjusted for age, sex, race, visceral adiposity, PAI-1, adiponectin, leptin, TNF, and IL-6.

vitro and animal data, to our knowl- fetuin-A levels have an inverse associa- time point. Whether or not longitudi- edge the association of fetuin-A with in- tion with vascular calcification. Beyond nal trajectories of fetuin-A provide cident diabetes has not previously been the insulin-sensitive phenotype, the fe- additional or more specific information evaluated. The data presented here are tuin-A knockout mouse develops wide- regarding future diabetes risk is an im- novel in demonstrating the longitudi- spread soft-tissue calcification.32,33 Our portant topic for future research. nal link between fetuin-A and incident group34 and others35-37 have demon- In conclusion, fetuin-A is indepen- diabetes in humans. Moreover, the data strated that lower fetuin-A levels are as- dently associated with incident diabe- suggest that not only adipocyte-derived sociated cross-sectionally with vascular tes in older individuals. Future stud- factors and pancreatic hormones, but also and cardiac valvular calcification in hu- ies should evaluate whether the results hepatocyte-derived proteins, such as fe- mans and are associated with mortality may generalize to middle-aged indi- tuin-A, may regulate glucose metabo- after myocardial infarction38 and in end- viduals in whom the incidence rate is lism in humans. stage renal disease.37-41 Therefore, any highest.46 If confirmed in future stud- The association of fetuin-A with in- novel therapeutic that blocks fetuin-A ies, fetuin-A may ultimately prove use- cident diabetes was partially attenuated should be closely evaluated for safety, ful as a target for therapeutics, and its with adjustment for visceral adiposity. particularly with respect to cardiovas- study may provide novel insights to glu- Previous studies have demonstrated that cular consequences. cose metabolism in humans. fetuin-A stimulates adipogenesis in cell Our study has limitations. Participa- 31 Author Contributions: Ms Wassel had full access to culture models, and conversely, fe- tion required that individuals be aged 70 all of the data in the study and takes responsibility for tuin-A knockout mice have less adipos- to 79 years and of black or white race. the integrity of the data and the accuracy of the data 14 analysis. ity than wild-type controls. We hy- The findings may not generalize to Study concept and design: Ix, Kanaya, Vittinghoff, pothesize that the direct correlation of younger individuals or to other races or Cummings, Shlipak. Acquisition of data: Ix, Johnson, Cauley, Harris, Shlipak. fetuin-A with visceral adiposity ob- ethnicities. Two-hour oral glucose tol- Analysis and interpretation of data: Ix, Wassel, Kanaya, served in this study is a consequence of erance tests are more sensitive than fast- Vittinghoff, Johnson, Koster, Shlipak. higher fetuin-A levels and that accumu- ing glucose for diagnosis of diabetes.42-44 Drafting of the manuscript: Ix. Critical revision of the manuscript for important in- lation of visceral adiposity may lie on a This measure was available at baseline, tellectual content: Ix, Wassel, Kanaya, Vittinghoff, causal pathway between fetuin-A and in- which provided accurate classification Johnson, Koster, Cauley, Harris, Cummings, Shlipak. Statistical analysis: Wassel, Vittinghoff. cident diabetes. However, despite statis- of diabetes status at entry, but not at Obtained funding: Ix, Johnson, Cauley, Harris, tical adjustment for visceral adiposity, the follow-up. Unavailability of this measure Cummings. Financial Disclosures: None reported. association of fetuin-A with incident dia- at follow-up visits could have resulted Funding/Support: This study was supported by an betes was only partially attenuated, which in some incident diabetes cases being ADA-ASP Young Investigator Innovation Award in Geriatric Endocrinology sponsored by the Atlantic Phi- suggests that mechanisms other than ac- misclassified as normal and therefore lanthropies, American Diabetes Association, John A. cumulated visceral adiposity may likely should have biased our results toward Hartford Foundation, and Association of Subspe- also contribute to the link between fe- the null hypothesis. Blind duplicate in- cialty Professors ( J.H.I.); an American Heart Associa- tion Fellow to Faculty Transition Award ( J.H.I.); and tuin-A and incident diabetes. traindividual coefficients of variation of contracts N01-AG-6-2101, N01-AG-6-2103, and N01- On the basis of these data, blockade fetuin-A were higher in this study than AG-6-2106 from the National Institute on Aging. The 34,45 research was also supported in part by the Intramural of fetuin-A binding to the insulin recep- in previous articles. Again, measure- Research Program of the National Institutes of Health. tor might be considered a novel thera- ment error misclassification should also Role of the Sponsors: The funding sources played no role in the design and conduct of the study; collec- peutic target for prevention or treat- have biased our results toward the null. tion, management, analysis, or interpretation of the ment of insulin-resistant states. However, Last, fetuin-A was measured at only 1 data, nor in the preparation of the manuscript. The

Downloaded From: https://jamanetwork.com/ on 09/30/2021 FETUIN-A AND INCIDENT DIABETES

National Institute on Aging reviewed and approved proved insulin sensitivity and resistance to weight gain Quantitative trait loci on chromosomes 3 and 17 in- this manuscript prior to submission. in mice null for the Ahsg gene. Diabetes. 2002; fluence phenotypes of the metabolic syndrome. Proc Additional Contributions: We thank the other inves- 51(8):2450-2458. Natl Acad Sci U S A. 2000;97(26):14478-14483. tigators, the staff, and the participants of the Health 14. Mathews ST, Rakhade S, Zhou X, et al. Fetuin- 31. Cayatte AJ, Kumbla L, Subbiah MT. Marked ac- ABC Study for their valuable contributions and Frances null mice are protected against obesity and insulin re- celeration of exogenous fatty acid incorporation into A. Tylavsky, DrPH, Department of Preventive Medi- sistance associated with aging. Biochem Biophys Res cellular triglycerides by fetuin. J Biol Chem. 1990; cine, University of Tennessee Health Science Center, Commun. 2006;350(2):437-443. 265(10):5883-5888. Memphis, for her critical review of the manuscript. 15. Stefan N, Hennige AM, Staiger H, et al. Alpha2- 32. Schafer C, Heiss A, Schwarz A, et al. The serum We also thank the following Health ABC core inves- Heremans-Schmid glycoprotein/fetuin-A is associ- protein alpha 2-Heremans-Schmid glycoprotein tigators: Anne B. Newman, MD, MPH, and Piera Kost, ated with insulin resistance and fat accumulation in /fetuin-A is a systemically acting inhibitor of ectopic University of Pittsburgh, Pittsburgh, Pennsylvania; Su- the liver in humans. Diabetes Care. 2006;29(4): calcification. J Clin Invest. 2003;112(3):357-366. zanne Satterfield, MD, DrPH, and Susan Thomas, Uni- 853-857. 33. Westenfeld R, Schafer C, Smeets R, et al. Fetuin-A versity of Tennessee, Memphis; Stephen B. Kritchev- 16. Mori K, Emoto M, Yokoyama H, et al. Associa- (Ahsg) prevents extraosseous calcification induced by sky, PhD, Wake Forest University, Winston-Salem, tion of serum fetuin-A with insulin resistance in type uraemia and phosphate challenge in mice. Nephrol Dial North Carolina; Michael C. Nevitt, PhD, and Susan M. 2 diabetic and nondiabetic subjects [letter]. Diabetes Transplant. 2007;22(6):1537-1546. Rubin, MPH, University of California, San Francisco; Care. 2006;29(2):468. 34. Ix JH, Chertow GM, Shlipak MG, et al. Associa- and Melissa E. Garcia, MPH, National Institute on Ag- 17. Ix JH, Shlipak MG, Brandenburg VM, et al. As- tion of fetuin-A with mitral annular calcification and ing, Bethesda, Maryland. sociation between human fetuin-A and the meta- aortic stenosis among persons with coronary heart dis- bolic syndrome: data from the Heart and Soul Study. ease: data from the Heart and Soul Study. Circulation. Circulation. 2006;113(14):1760-1767. 2007;115(19):2533-2539. REFERENCES 18. Prentice RL. A case-cohort design for epidemiologic 35. Kaden JJ, Reinohl JO, Blesch B, et al. Systemic and cohort studies and disease prevention trials. Biometrika. local levels of fetuin-A in calcific aortic valve stenosis. 1. Flegal KM, Carroll MD, Ogden CL, et al. Preva- 1986;73:1-11. doi:10.1093/biomet/73.1.1. Int J Mol Med. 2007;20(2):193-197. lence and trends in obesity among US adults, 19. Declerck PJ, Alessi MC, Verstreken M, et al. Mea- 36. Moe SM, Reslerova M, Ketteler M, et al. Role of 1999-2000. JAMA. 2002;288(14):1723-1727. surement of plasminogen activator inhibitor 1 in bio- calcification inhibitors in the pathogenesis of vascu- 2. Mokdad AH, Bowman BA, Ford ES, et al. The con- logic fluids with a murine monoclonal antibody- lar calcification in chronic kidney disease (CKD). Kid- tinuing epidemics of obesity and diabetes in the United based enzyme-linked immunosorbent assay. Blood. ney Int. 2005;67(6):2295-2304. States. JAMA. 2001;286(10):1195-1200. 1988;71(1):220-225. 37. Wang AY, Woo J, Lam CW, et al. Associations 3. King H, Aubert RE, Herman WH. Global burden 20. Levey AS, Bosch JP, Lewis JB, et al. A more ac- of serum fetuin-A with malnutrition, inflammation, ath- of diabetes, 1995-2025: prevalence, numerical esti- curate method to estimate glomerular filtration rate erosclerosis and valvular calcification syndrome and mates, and projections. Diabetes Care. 1998;21 from serum creatinine: a new prediction equation: outcome in peritoneal dialysis patients. Nephrol Dial (9):1414-1431. Modification of Diet in Renal Disease Study Group. Transplant. 2005;20(8):1676-1685. 4. Howard BV, Rodriguez BL, Bennett PH, et al. Pre- Ann Intern Med. 1999;130(6):461-470. 38. Lim P, Collet JP, Moutereau S, et al. Fetuin-A is vention conference VI: diabetes and cardiovascular dis- 21. Barlow WE, Ichikawa L, Rosner D, et al. Analysis an independent predictor of death after ST-elevation ease: writing group I: epidemiology. Circulation. 2002; of case-cohort designs. J Clin Epidemiol. 1999; myocardial infarction. Clin Chem. 2007;53(10): 105(18):e132-e137. 52(12):1165-1172. 1835-1840. 5. Perseghin G, Ghosh S, Gerow K, et al. Metabolic 22. Therneau TM, Li H. Computing the Cox model 39. Stenvinkel P, Wang K, Qureshi AR, et al. Low defects in lean nondiabetic offspring of NIDDM par- for case cohort designs. Lifetime Data Anal. 1999; fetuin-A levels are associated with cardiovascular death: ents: a cross-sectional study. Diabetes. 1997;46 5(2):99-112. impact of variations in the gene encoding fetuin. Kid- (6):1001-1009. 23. Lin DYWL. The robust inference for the Cox pro- ney Int. 2005;67(6):2383-2392. 6. Saxena R, Voight BF, Lyssenko V, et al. Genome- portional hazards model. J Am Stat Assoc. 1989; 40. Ketteler M, Bongartz P, Westenfeld R, et al. As- wide association analysis identifies loci for type 2 dia- 84:1074-1078. sociation of low fetuin-A (Ahsg) concentrations in se- betes and triglyceride levels. Science. 2007;316 24. Clinical guidelines on the identification, evalua- rum with cardiovascular mortality in patients on di- (5829):1331-1336. tion, and treatment of overweight and obesity in adults: alysis: a cross-sectional study. Lancet. 2003;361 7. McNeely MJ, Boyko EJ, Weigle DS, et al. Associa- the Evidence Report: National Institutes of Health. Obes (9360):827-833. tion between baseline plasma leptin levels and sub- Res. 1998;6(suppl 2):51S-209S. 41. Hermans MM, Brandenburg V, Ketteler M, et al. sequent development of diabetes in Japanese 25. Srinivas PR, Wagner AS, Reddy LV, et al. Serum Association of serum fetuin-A levels with mortality in Americans. Diabetes Care. 1999;22(1):65-70. alpha 2-HS-glycoprotein is an inhibitor of the human dialysis patients. Kidney Int. 2007;72(2):202-207. 8. Festa A, D’Agostino R Jr, Tracy RP, et al. Elevated insulin receptor at the tyrosine kinase level. Mol 42. Wahl PW, Savage PJ, Psaty BM, et al. Diabetes levels of acute-phase proteins and plasminogen acti- Endocrinol. 1993;7(11):1445-1455. in older adults: comparison of 1997 American Diabe- vator inhibitor-1 predict the development of type 2 26. Mathews ST, Srinivas PR, Leon MA, et al. Bovine tes Association classification of diabetes mellitus with diabetes: the insulin resistance atherosclerosis study. fetuin is an inhibitor of insulin receptor tyrosine kinase. 1985 WHO classification. Lancet. 1998;352(9133): Diabetes. 2002;51(4):1131-1137. Life Sci. 1997;61(16):1583-1592. 1012-1015. 9. Lindsay RS, Funahashi T, Hanson RL, et al. Adipo- 27. Grunberger GMS, Mathews ST, Deutsch DD. Ty- 43. Resnick HE, Shorr RI, Kuller L, et al. Prevalence nectin and development of type 2 diabetes in the Pima rosine kinase inhibitors. In: Grunberger GMS, Zick Y, and clinical implications of American Diabetes Indian population. Lancet. 2002;360(9326):57- eds. Insulin Signaling: From Cultured Cells to Ani- Association-defined diabetes and other categories of 58. mal Models, Frontiers in Animal Diabetes Research. glucose dysregulation in older adults: the health, ag- 10. Ix JH, Chertow GM, Shlipak MG, et al. Fetuin-A New York, NY: CRC Press; 2002:281-298. ing and body composition study. J Clin Epidemiol. and kidney function in persons with coronary artery 28. Cintro´ n VJ, Ko MS, Chi KD, et al. Genetic map- 2001;54(9):869-876. disease: data from the Heart and Soul Study. Nephrol ping and functional studies of a natural inhibitor of 44. Willnewdiagnosticcriteriafordiabetesmellituschange Dial Transplant. 2006;21(8):2144-2151. the insulin receptor tyrosine kinase: the mouse ortho- phenotype of patients with diabetes? reanalysis of Eu- 11. Rauth G, Poschke O, Fink E, et al. The nucleo- log of human alpha2-HS glycoprotein. Int J Exp Dia- ropean epidemiological data: DECODE Study Group on tide and partial amino acid sequences of rat fetuin: iden- betes Res. 2001;1(4):249-263. behalf of the European Diabetes Epidemiology Study tity with the natural tyrosine kinase inhibitor of the 29. Vionnet N, Hani EH, Dupont S, et al. Genome- Group. BMJ. 1998;317(7155):371-375. rat insulin receptor. Eur J Biochem. 1992;204(2): wide search for type 2 diabetes-susceptibility genes 45. Ix JH, Shlipak MG, Sarnak MJ, et al. Fetuin-A is 523-529. in French whites: evidence for a novel susceptibility not associated with mortality in chronic kidney disease. 12. Auberger P, Falquerho L, Contreres JO, et al. Char- locus for early-onset diabetes on chromosome Kidney Int. 2007;72(11):1394-1399. acterization of a natural inhibitor of the insulin recep- 3q27-qter and independent replication of a type 46. Incidence of diabetes in the United States. Na- tor tyrosine kinase: cDNA cloning, purification, and 2-diabetes locus on chromosome 1q21-q24. Am J Hum tional Diabetes Information Clearing House. http: anti-mitogenic activity. Cell. 1989;58(4):631-640. Genet. 2000;67(6):1470-1480. //diabetes.niddk.nih.gov/dm/pubs/statistics/#11. Ac- 13. Mathews ST, Singh GP, Ranalletta M, et al. Im- 30. Kissebah AH, Sonnenberg GE, Myklebust J, et al. cessed May 22, 2007.

Downloaded From: https://jamanetwork.com/ on 09/30/2021