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Relation Between Serum Free Fatty Acids and Adiposity, Insulin Resistance, and Cardiovascular Risk Factors From Adolescence to Adulthood

Relation Between Serum Free Fatty Acids and Adiposity, Insulin Resistance, and Cardiovascular... ORIGINAL ARTICLE Relation Between Serum Free Fatty Acids and Adiposity, Insulin Resistance, and Cardiovascular Risk Factors From Adolescence to Adulthood 1 2 1 1 2 Brigitte I. Frohnert, David R. Jacobs Jr., Julia Steinberger, Antoinette Moran, Lyn M. Steffen, and Alan R. Sinaiko FFA production is altered in individuals with known ge- The objective of this study was to describe longitudinal relations netic risk for type 2 diabetes mellitus, regardless of weight. of serum total free fatty acids (FFAs) to insulin resistance (IR) Glucose-tolerant Mexican American adult children of two and cardiovascular (CV) risk factors from adolescence into adulthood. The cohort included participants in a longitudinal parents with type 2 diabetes mellitus were found to have study of obesity and IR with complete data, including anthropo- impaired glucose tolerance by insulin clamp and reduced metric measures, FFAs, IR measured by euglycemic clamp, blood insulin-mediated suppression of lipid oxidation and plasma pressure, fasting serum lipids, and insulin at mean 15 and 22 years FFA concentration compared with age-matched, sex- of age (n = 207) and their parents (n = 272). FFAs and IR were matched, and weight-matched control subjects with non- not significantly related at mean 15 years of age but were signif- diabetic parents (7,18). icantly related at mean age 22 years. FFA did not relate to BMI at Information is limited for FFA in childhood and for the either age. FFA at 15 years of age estimated IR at 22 years of age. natural history of changes in FFA during transition from In parents (mean age 51 years), FFA was significantly correlated childhood to adulthood. The goal of the current study was with BMI, percent body fat, systolic blood pressure, LDL, and IR. Associations with all risk factors except IR in parents were at- to determine relations of FFA with adiposity, IR, and car- tenuated by adjustment for BMI. Most 22 years of age correla- diovascular (CV) risk factors during late childhood/early tions with parents were higher than corresponding 15 years of adolescence and early adulthood. We examined cross- age correlations. This study finds that FFA is associated with IR sectional relations of these factors at mean ages 15 and 22 starting in young adulthood. The relation between FFA and CV years and longitudinal relations of factors between these risk factors does not become significant until later adulthood. age-groups. As an older adult comparison, relations be- The results support a significant impact of early metabolic dys- tween factors were examined in parents of subjects. function on later CV risk. Diabetes 62:3163–3169, 2013 RESEARCH DESIGN AND METHODS Subjects. This study was approved by the University of Minnesota Committee ree fatty acids (FFAs) are elevated in obese for the Use of Human Subjects in Research. At each time point, the study was individuals, primarily as a result of release from explained to participants and parents; parents and participants 18 years of age increased fat mass. This release is enhanced by and older signed informed consent, and children younger than 18 years of age Fthe resistance of obese adipose tissue to the gave informed oral assent. antilipolytic effect of insulin and inability of obese adipo- This longitudinal study of the influence of obesity and IR in childhood on development of adult CV risk has been described previously (19). Initial studies cytes to effectively recycle FFAs via re-esterification (1,2). were conducted at mean 13 years of age, with additional cohort studies at Elevated FFAs compete with glucose as an energy source mean 15, 19, and 22 years of age. The present report consists of data from the in both muscle and fat and are associated with decreased mean 15 years of age (range 12–18) and 22 years of age (range 18–25) visits, glucose oxidation and increased insulin resistance (IR) in the times during which samples were obtained and analyzed for FFA. Of 329 muscle tissue and liver (3–5). Further, increased FFAs in individuals seen at mean age 15 years and 280 individuals seen at mean age 22 adults have been associated with endothelial dysfunction, years, inclusion in this study was limited to those with complete datasets at both time points (n = 207). Of these, 187 had Tanner stage at mean 15 years of blood pressure, and pancreatic b-cell dysfunction (6–9). age recorded. There were no significant differences in clinical factors between Lipid infusion in lean, obese, and type 2 diabetic adults, those included and excluded at either age, except for slightly higher fasting a model of short-term elevation of FFA, results in periph- glucose in the included group at mean 15 years of age (4.82 vs. 4.71 mmol/L; eral IR and decreased peripheral glucose disposal (10–15). P = 0.024) and lower steady-state insulin during euglycemic-hyperinsulinemic The FFA effect on peripheral IR is dose dependent and clamp at age 22 years (394.7 vs. 463.8 pmol/L; P = 0.044). Among the subjects occurs at the level of insulin-stimulated glucose uptake, with complete datasets, there was no difference in any measure between those adversely affecting insulin signaling pathways (6,16,17). with or without Tanner stage recorded at mean 15 years of age. Parents of participants were recruited throughout the study (n = 379) and, at either 13 or 15 years of age visits of their children, underwent many of the same measurements as their offspring. Analysis was limited to parents with From the Department of Pediatrics, Medical School, University of Minnesota, complete datasets (n = 272). There were differences between those included Minneapolis, Minnesota; and the Division of Epidemiology and Commu- nity Health, School of Public Health, University of Minnesota, Minneapolis, and excluded in BMI (28.6 vs. 30.3 kg/m ; P , 0.05), waist circumference Minnesota. (95.5 vs. 100.2 cm; P , 0.05), systolic blood pressure (SBP) (114.6 vs. 119.2 Corresponding author: Brigitte I. Frohnert, frohn001@umn.edu. mmHg; P , 0.01), fasting glucose (5.13 vs. 5.58 mmol/L; P , 0.001), and Received 20 August 2012 and accepted 7 May 2013. steady-state insulin during euglycemic-hyperinsulinemic clamp (465.1 vs. DOI: 10.2337/db12-1122 685.8; P , 0.01). This article contains Supplementary Data online at http://diabetes Clinical measurements. Height, weight, and blood pressure were measured .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1122/-/DC1. as previously described (19). Waist circumference was measured in duplicate 2013 by the American Diabetes Association. Readers may use this article midway between anterior superior iliac spine and lower rib margin. Percent- as long as the work is properly cited, the use is educational and not for age of body fat and lean body mass (LBM) were determined by the skinfold profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. method of Slaughter et al. (20) at mean 15 years of age and by dual-energy diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3163 FATTY ACIDS FROM ADOLESCENCE TO ADULTHOOD X-ray absorptiometry at mean 22 years of age and in the parent group. LBM at mean 22 years of age from FFA level at mean 15 years of age, adjusting for values at mean 15 years of age were adjusted to dual-energy X-ray absorpti- age, sex, race, and BMI at initial visit. Multiple regression analyses also were ometry values according to previously derived equations (21). performed with the interaction term of sex and FFA; in no case was this term Euglycemic-hyperinsulinemic clamps were conducted at the University of significant. Natural log transformation of the skewed variable, triglycerides, Minnesota Clinical Research Center after a 10-h overnight fast as previously was evaluated and presented when different from the untransformed variable. described (19,22). Insulin sensitivity was determined from the amount of Three multivariable models were developed adjusting for baseline age, sex, glucose administered over the final 40 min of euglycemic-hyperinsulinemic and race (Caucasian vs. African American) as follows: model 1, baseline FFA clamp and expressed as milligrams of glucose utilization per kilogram of lean and BMI as independent variables; model 2, baseline FFA and change in FFA, LBM per minute (M ). A lower M indicates greater IR. baseline BMI, and change in BMI; and model 3, the same as model 2 with the LBM LBM Serum insulin samples were collected on ice, centrifuged within 20 min, and addition of baseline level of the outcome variable. Changes in BMI or FFA analyzed by radioimmunoassay using a double-antibody method (Equate RIA; were calculated as mean 22 years of age value minus mean 15 years of age Binax, Portland, ME). Blood samples for serum lipids were analyzed in the value. Analyses at mean 15 years of age also were adjusted with and without University of Minnesota laboratory with a Cobas Fara. Cholesterol was de- Tanner stage for each analysis and were not different, unless noted otherwise. termined by a standard enzymatic cholesterol oxidase-based method; HDL P , 0.05 was considered to be statistically significant. cholesterol was determined after precipitation of non-HDL lipoproteins with magnesium/dextran precipitating reagent. Triglycerides were determined with a standard glycerol-blanked, enzymatic triglyceride method. LDL cholesterol RESULTS was estimated by the Friedewald equation. Total mmol/L FFA in serum was Participant characteristics. Of 207 individuals studied at determined at mean 22 years of age and in the parent group by quantitative both average 15 and 22 years of age, 59% were male and spectrophotometry. At mean 15 years of age, individual FFA levels were de- 82% were Caucasian. Most of the 15-year-olds were in the termined by tandem mass spectrometry and converted from microgram of each fatty acid per milliliter (mg/mL) sample to mmol/L of total FFA using the late pubertal stage (Tanner 1, none; Tanner 2, 2%; Tanner 3, molar mass of each FFA identified. Supplementary Tables 1 and 2 show 5%; and Tanner 4–5, 93%). BMI increased from 24 to 26 a comparison of mean total and percentage of FFA before and after conver- kg/m between mean 15 and 22 years of age, but percent sion to mmol/L. body fat decreased from 34 to 28%. Both SBP and diastolic Statistical analysis. All analyses were performed using SAS version 9.3 (SAS blood pressure increased significantly from mean 15 to Institute, Cary, NC). Participant characteristics were expressed as means 6 mean 22 years of age. Lipids, including total cholesterol, SEM for continuous variables and as frequencies for categorical variables. Comparisons were made using Student t test and x analysis, respectively. LDL, HDL, and triglycerides, were significantly higher at Pearson partial correlation coefficients, adjusting for age, sex, and race, with mean 22 years of age than at mean 15 years of age. No or without baseline BMI, evaluated within-person tracking of clinical measures difference in fasting glucose was observed between mean between ages 15 and 22 years. Unadjusted Pearson correlations were used to 15 and 22 years of age. Fasting insulin decreased signifi- evaluate relations between clinical measures in subjects at mean 15 and 22 cantly from mean age 15 to mean 22 years of age, coincident years of age and their parents. Of 272 individuals in the parent group, only 196 with a significant increase in IR (M ). FFAs were signif- were parents of included participants. Of 139 child-parent sets, 25 included LBM fathers, 57 included mothers, and 57 included both parents, for whom mid- icantly lower at mean 22 years of age. parental value was used. Pearson partial correlation coefficients, adjusted for The parent group (n = 272, mean age 51 years) had sex, race, and baseline age, were used for cross-sectional analyses of FFA with a significantly smaller percentage of males and a higher other variables at mean 15 and 22 years of age and those of the parents. percentage of Caucasians (Table 1). All anthropometric, Multiple regression analysis evaluated relations of insulin sensitivity (M ) LBM blood pressure, lipid, and fasting glucose measures were with FFA level, adjusting for age, sex, and BMI for adolescents (mean 15 years significantly higher in parents. M , fasting insulin, and of age), young adults (mean 22 years of age), and their parents (mean 51 years LBM of age). Additionally, multiple regression analysis was used to estimate M FFA levels in parents were significantly lower than those LBM TABLE 1 Characteristics of repeated measures in study participants at mean ages 15 and 22 years and of a single measure of their parents Mean age 15 years Mean age 22 years Parents n 207 207 272 Age, years 15.1 6 0.1 21.5 6 0.1* 51.2 6 0.4*‡ Race African American (%) 37 (18) 28 (10) Caucasian (%) 170 (82) 244 (90)§ Male (%) 122 (59) 108 (40)‖ BMI, kg/m 23.6 6 0.4 26.1 6 0.4* 28.6 6 0.4*‡ Body fat, % 33.7 6 1.0 28.3 6 0.8* 36.4 6 0.6†‡ Waist circumference, cm 80.7 6 0.9 87.2 6 1.1* 95.5 6 1.0*‡ SBP, mmHg 109.0 6 0.6 110.4 6 0.7† 114.6 6 0.9*‡ DBP, mmHg 57.0 6 1.0 65.4 6 0.7* 71.4 6 0.6*‡ Total cholesterol, mmol/L 3.77 6 0.05 4.14 6 0.05* 5.23 6 0.06*‡ LDL cholesterol, mmol/L 2.19 6 0.05 2.45 6 0.05* 3.24 6 0.05*‡ HDL cholesterol, mmol/L 1.11 6 0.02 1.17 6 0.02* 1.32 6 0.02*‡ Triglycerides, mmol/L 1.00 6 0.04 1.14 6 0.05* 1.53 6 0.10*‡ Fasting glucose, mmol/L 4.82 6 0.03 4.76 6 0.03 5.13 6 0.05*‡ Fasting insulin, pmol/L 81.4 6 4.0 61.9 6 6.2* 67.3 6 2.7* Steady-state insulin, pmol/L 538.3 6 12.1 394.7 6 10.5* 465.1 6 10.1*‡ M , mg/kg/min 12.6 6 0.3 11.5 6 0.3* 11.3 6 0.2* LBM FFA, mmol/L 0.52 6 0.01 0.36 6 0.01* 0.37 6 0.01* Data presented as mean 6 SEM unless otherwise specified. Sex and race were compared using x analysis. All other variables were compared using an independent Student t test. DBP, diastolic blood pressure. *Significantly different from participant group at mean age 15 years (P , 0.01). †Significantly different from participant group at mean age 15 years (P , 0.05). ‡Significantly different from participant group at mean age 22 years (P , 0.01). §Significantly different from participant group (P , 0.05). ‖Significantly different from participant group (P , 0.01). 3164 DIABETES, VOL. 62, SEPTEMBER 2013 diabetes.diabetesjournals.org B.I. FROHNERT AND ASSOCIATES TABLE 3 in participants at 15 years of age (P , 0.01) but were not Correlations of CV risk factors between subjects at mean ages 15 significantly different from those in participants at 22 years and 22 years and midparental measures of age. Relations of clinical measures between 15 and 22 Mean age Mean age years of age. All clinical and laboratory measures were 15 years 22 years correlated significantly between the two ages, except for rP r P FFA (Table 2). When partial correlations were additionally adjusted for Tanner stage at mean 15 years of age, these BMI, kg/m 0.33 ,0.001 0.43 ,0.001 relations were unchanged. When partial correlations were Body fat, % 0.19 0.02 0.28 ,0.001 additionally adjusted for BMI at mean 15 years of age, Waist circumference, cm 0.30 ,0.001 0.37 ,0.001 relations did not change with the exception of insulin, SBP, mmHg 0.11 0.20 0.27 0.001 which was greatly attenuated (r = 0.07; P = 0.30). Rela- DBP, mmHg 0.24 0.004 0.25 0.003 tionships in males and females separately were similar to Total cholesterol, mmol/L 0.15 0.08 0.20 0.02 LDL cholesterol, mmol/L 0.06 0.48 0.13 0.14 those computed in the entire group, with the exception of HDL cholesterol, mmol/L 0.17 0.05 0.29 ,0.001 FFAs, which were significantly correlated between mean Triglycerides, mmol/L 0.05 0.54 0.09 0.27 15 and 22 years of age in females only (r = 0.27; P = 0.01). Ln(triglycerides), ln(mmol/L) 0.22 0.008 0.18 0.03 Relations of clinical measures between 15 and 22 Fasting glucose, mmol/L 20.09 0.29 0.08 0.33 years of age and midparental measure. For the 139 Fasting insulin, pmol/L 20.04 0.66 0.23 0.006 subjects with parental data, clinical measures were com- M , mg/kg/min 0.11 0.21 0.17 0.04 LBM pared between subjects and parents (Table 3). At 15 and FFA, mmol/L 20.01 0.94 0.14 0.11 22 years of age, there was a significant correlation between offspring and parents for BMI, percent body fat, waist n = 139 parent-child sets. DBP, diastolic blood pressure; Ln(triglycer- circumference, diastolic blood pressure, and natural log ides), natural log transformation of triglycerides. transformation of triglycerides. Correlations between off- spring and parents became stronger for virtually all CV risk factors at 22 years of age, and SBP, total cholesterol, HDL, age, FFAs were not significantly correlated with blood fasting insulin, and M were significantly related be- pressure, measures of adiposity, serum lipids, or fasting LBM tween parents and their children only at mean 22 years of insulin. There was a significant inverse relation with fast- age. When adjusted for parental contribution (i.e., mother, ing glucose and M . Adjustment for BMI did not appre- LBM father, or both), these correlations were not significantly ciably affect these relations at either age (data not shown). different. In separate analyses by sex, results were unchanged, ex- Relations of FFA to other measures. As shown in Table cept correlations in females between FFAs and both fast- 4, FFAs at mean 15 years of age, adjusted for age, sex, and ing glucose and insulin were not significant. race, were not significantly correlated with blood pressure, In contrast to 15- and 22-year-olds, FFAs in parents were measures of adiposity, lipids, fasting glucose, fasting in- significantly correlated with measures of adiposity, SBP, sulin, FFA, or IR (M ), with similar results when ana- LBM fasting glucose, fasting insulin, M , total cholesterol, LBM lyzed separately for males and females. When additionally natural log transformation of triglycerides, and LDL. After adjusted for Tanner stage (n = 183), only the relation with adjustment for BMI in the parent group, there was no longer fasting insulin and natural log transformation of triglyc- asignificant relation with waist circumference (r =0.09; erides became significant (r = 20.17 and P = 0.02 and P =0.16), SBP (r =0.091; P = 0.14), fasting glucose (r =0.09; r = 20.16 and P = 0.03, respectively). At mean 22 years of P = 0.14), natural log transformation of triglycerides (r = 0.09; P = 0.14), or insulin (r =0.09; P = 0.13), but relations with percent body fat, blood lipids, and IR continued to be significant. Separate analyses by sex showed some differ- TABLE 2 ences between mothers and fathers. Significant correlations Tracking correlations of CV disease risk factors between mean between FFA and BMI, waist circumference, and fasting ages 15 and 22 years insulin were found only in mothers; a significant relation r* Pr† P between FFA and SBP was seen only in fathers. The relation between FFA and IR (M ) by age and 2 LBM BMI, kg/m 0.86 ,0.001 NA NA sex, adjusted for BMI and race, is shown in Fig. 1. Linear Body fat, % 0.64 ,0.001 0.15 0.03 regression of M on FFA with age, sex, race, and BMI in LBM Waist circumference, cm 0.81 ,0.001 0.27 ,0.001 the model showed a nonsignificant inverse trend at mean SBP, mmHg 0.49 ,0.001 0.50 ,0.001 age 15 years (b = 21.35; P = 0.37), a significant inverse DBP, mmHg 0.30 ,0.001 0.30 ,0.001 relation at mean 22 years of age (b = 25.22; P , 0.001), Total cholesterol, mmol/L 0.59 ,0.001 0.56 ,0.001 and the strongest inverse relation in parents (b = 26.12; LDL cholesterol, mmol/L 0.66 ,0.001 0.63 ,0.001 P , 0.0001). Further adjustment of IR at 15 years of age for HDL cholesterol, mmol/L 0.63 ,0.001 0.60 ,0.001 Tanner stage did not change results (data not shown). Triglycerides, mmol/L 0.32 ,0.001 0.26 ,0.001 Ln(triglycerides), ln(mmol/L) 0.41 ,0.001 0.35 ,0.001 Estimation of CV risk factors and IR at mean 22 years Fasting glucose, mmol/L 0.39 ,0.001 0.38 ,0.001 of age from mean 15 years of age, FFAs, and changes Fasting insulin, pmol/L 0.22 0.002 0.07 0.30 in FFAs from mean 15 to 22 years of age. In multivar- M , mg/kg/min 0.33 ,0.001 0.32 ,0.001 iable model 1, with baseline (mean 15 years of age) FFAs LBM FFA, mmol/L 0.11 0.11 0.11 0.12 and BMI as independent variables and age, sex, and race as covariates, baseline FFA was associated only with M LBM n = 207. DBP, diastolic blood pressure; Ln(triglycerides), natural log (P = 0.03) and not with later FFA (Table 5 and Fig. 1). In transformation of triglycerides; NA, not applicable. *Pearson partial model 2, which included change in both FFA and BMI from correlations adjusted for age at baseline, sex, and race. †Additionally adjusted for BMI. mean 15 years of age to mean 22 years of age in addition to diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3165 FATTY ACIDS FROM ADOLESCENCE TO ADULTHOOD TABLE 4 Cross-sectional correlation of serum FFA with measures of adiposity and CV risk factors, adjusted for age, sex, and race, at participant mean ages 15 and 22 years and in their parents Mean age 15 years Mean age 22 years Parents (n = 207) (n = 207) (n = 272) r P rPrP BMI, kg/m 20.05 0.51 0.01 0.92 0.19 0.002 Body fat, % 20.01 0.94 0.01 0.93 0.23 ,0.001 Waist, cm 20.07 0.31 0.00 0.97 0.20 ,0.001 SBP, mmHg 20.02 0.75 20.02 0.79 0.12 0.05 DBP, mmHg 0.07 0.35 20.02 0.76 0.09 0.15 Total cholesterol, mmol/L 0.03 0.67 0.11 0.12 0.14 0.02 LDL cholesterol, mmol/L 0.10 0.16 0.10 0.16 0.13 0.03 HDL cholesterol, mmol/L 20.04 0.53 0.07 0.30 20.05 0.41 Triglycerides, mmol/L 20.12 0.08 0.03 0.70 0.07 0.23 Ln(triglycerides), ln(mmol/L) 20.12 0.09 0.00 0.99 0.15 0.02 Fasting glucose, mmol/L 0.01 0.91 20.19 0.01 0.16 0.01 Fasting insulin, pmol/L 20.14 0.05 20.08 0.23 0.18 0.003 M , mg/kg/min 20.06 0.40 20.25 ,0.001 20.31 ,0.001 LBM DBP, diastolic blood pressure; Ln(triglycerides), natural log transformation of triglycerides. baseline FFA and BMI, fasting glucose was estimated by by showing longitudinal developmental changes during the change in FFA (P = 0.004) and M was estimated by transition from adolescence to young adulthood, the in- LBM baseline FFA (P = 0.0002) and change in FFA (P = 0.002). creasing strength of relations between FFA and IR with In model 3 (data not shown), in which the baseline CV risk aging, and the increasing strength of the relations between factor under consideration was included in addition to the parents and children from mean 15 years of age to mean 22 independent variables in model 2, there was little change years of age. At mean 15 years of age, FFA levels were not from model 2; M was again estimated by baseline FFA associated with any of the measures of adiposity, CV risk LBM (P = 0.0003) and change in FFA (P = 0.002), and fasting factors, or IR. By mean 22 years of age, FFA levels began glucose was estimated by change in FFA (P = 0.002); in to show a significant relation to IR but not to adiposity or addition, change in FFA now estimated LDL (P = 0.02). CV risk factors. Only in the older adult group (parents; Adjustment for Tanner stage at mean 15 years of age did mean 51 years of age) was there a significant relation of not change these results (data not shown). FFA to a broad group of cardiometabolic risk factors, in- Linear regression analyses were used to assess the re- cluding adiposity, systolic blood pressure, lipids, fasting lation between FFAs at mean 15 years of age and M at insulin, and glucose, and in particular, there was a more LBM mean 22 years of age, adjusting for sex, baseline age, and pronounced significant relation to IR. BMI (Fig. 2). FFAs at mean 15 years of age was a signifi- Although there were significant correlations between cant estimator of M at mean 22 years of age (b = parents and children for a number of CV risk factors, LBM 23.03; P = 0.03). After adjustment for Tanner stage at 15 relation with FFA was not significant. To our knowl- years of age, this relationship persisted (b = 23.29; P =0.03). edge, this is the first time FFA and IR by euglycemic- hyperinsulinemic clamp have been compared in a large parent-child cohort. Similar to previous studies, we also DISCUSSION found significant relations between parents and children This study provides new information on relations between for BMI, blood pressure, and lipids, with the exception of FFA and both CV risk factors and IR in children and adults LDL (23). Although LDL also was significantly related FIG. 1. Cross-sectional multiple regression of IR (M ) on FFA level, adjusted for age, race, BMI, and sex in adolescents (mean 15 years of age), LBM young adults (mean 22 years of age), and their parents (mean 51 years of age). At mean 15 years of age, b = 21.35 mg/kg/min per mmol/L (P = 0.37); at mean 22 years of age, b = 25.22 mg/kg/min per mmol/L (P < 0.001); and in parents, b = 26.12 mg/kg/min per mmol/L (P < 0.0001). Sex-specific mean M quartiles are included for goodness-of-fit assessment. ○, females; ■, males. LBM 3166 DIABETES, VOL. 62, SEPTEMBER 2013 diabetes.diabetesjournals.org B.I. FROHNERT AND ASSOCIATES TABLE 5 Results of multivariable models using baseline FFA at mean age 15 years to estimate CV risk factor levels at mean age 22 years Model 1 Model 2 Dependent variable Baseline FFA Baseline FFA DFFA BMI, kg/m 20.21 6 1.15 1.48 6 1.58 1.89 6 1.21 SBP, mmHg 24.35 6 3.59 25.89 6 4.88 21.87 6 3.76 Total cholesterol, mmol/L 20.01 6 0.27 0.45 6 0.37 0.50 6 0.28 LDL cholesterol, mmol/L 0.09 6 0.23 0.45 6 0.32 0.39 6 0.24 HDL cholesterol, mmol/L 20.19 6 0.10 20.05 6 0.13 0.16 6 0.10 Fasting glucose, mmol/L 0.23 6 0.17 20.23 6 0.23 20.52 6 0.18† Fasting insulin, pmol/L 216.8 6 30.9 251.4 6 42.1 239.6 6 32.4 M , mg/kg/min 23.03 6 1.40‡ 27.07 6 1.86* 24.45 6 1.43† LBM FFA, mmol/L 0.11 6 0.07 0.11 6 0.07 NA Data presented as b-coefficient 6 SEM unless otherwise specified. Models are adjusted for corresponding BMI levels (n = 207). Models adjusted for age, sex, race, and BMI. Model 2 is further adjusted for change in BMI from baseline to adult follow-up. DBP, diastolic blood pressure; DFFA, FFA at adult follow-up minus FFA at baseline. NA, not applicable. *P , 0.001. †P , 0.01. ‡P , 0.05. between parents and children in the Bogalusa Heart of CV disease. This is supported by studies in a rat model Study, the correlation was similarly fairly weak and that system, which showed an increased FFA-induced in- cohort was significantly larger, with greater power to flammatory response and IR with aging (36). Together, detect such associations (23). these findings support the hypothesis that fat metabolism Previous studies have reported relations between FFA and distribution are early pathogenic factors in develop- and both adiposity and IR; however, studies in the pedi- ment of IR. atric population are limited. In contrast to adults, fasting The relations between FFAs and IR did not completely FFAs in prepubertal children were not associated with IR parallel relations of FFAs with fasting insulin, probably, in as measured by the euglycemic-hyperinsulinemic clamp part, because of developmental changes associated with (24). Although an inverse relation between FFAs and transition from adolescence to young adulthood. Both fasting insulin has been reported in prepubertal children, fasting insulin and insulin sensitivity decreased from 15 to other studies showed no relation of FFAs with fasting 22 years of age. Previous studies of this normal cohort, insulin in both prepubertal and pubertal children (24–28). with a wide range of BMI and IR, have shown during ad- Lipid infusion, which results in elevated FFA levels, in- olescence a poor correlation between fasting insulin and creased IR in pubertal but not in prepubertal children IR (37), a significant increase in IR without a significant (29). There was no difference in FFA-mediated changes in change in fasting insulin during puberty (19), and separate IR after lipid infusion in African American adolescents patterns of change in fasting insulin and IR from 11 to 19 when compared with Caucasian adolescents (30). It is years of age (19). Thus, these studies provide evidence to unclear whether increased basal levels of FFAs seen in support the complex relation between fasting insulin and obese adolescents relate to increased fat mass or im- IR, particularly during the second decade of life. The paired antilipolytic effect of insulin (31,32). Whereas finding that levels of both were similar at 22 and 51 years adefinite pattern cannot be established from those studies, of age (parents) suggests that the relations become more they suggest a potential age-dependent or development- stable during adulthood. dependent change in the relation between FFAs and both FFA levels were overall higher, independent of BMI, at fasting insulin and IR. mean 15 years of age compared with later measures in Longitudinal studies in adults have shown higher FFA young adulthood and in their parents. This observation levels were associated with increased risk of impaired glucose tolerance or type 2 diabetes (33); in Pima Indians with normal glucose tolerance, there is an association of baseline FFA levels with later IR by euglycemic clamp (34). In contrast, another study showed no association of baseline FFAs with fasting insulin and glucose on follow- up (35). The current study expands on previous studies of children or adults by exploring longitudinal changes be- tween adolescence and adulthood and relating FFA levels between adolescents and their adult parents. The relation between FFA levels and IR at mean 22 years of age is independent of degree of adiposity, existing before any association with adiposity or FFA levels develops, as shown in the older adults. Together with the observation that higher FFA levels at mean 15 years of age are associated with higher IR at mean 22 years of age, this suggests that FFA-related metabolic abnormalities develop FIG. 2. Multiple regression of IR (M ) at mean 22 years of age on with increasing age, exhibiting a causal relation in the LBM FFA level at mean 15 years of age, adjusted for age, race, BMI, and sex development of IR. Association of FFA levels with other (b = 23.03 mg/kg/min per mmol/L; P = 0.03). Sex-specific mean M LBM CV risk factors does not appear until later adulthood, quartiles are included for goodness-of-fit assessment. ○, females; ■, suggesting a progressive effect of FFAs on the pathogenesis males. diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3167 FATTY ACIDS FROM ADOLESCENCE TO ADULTHOOD may relate to differences in FFA assays; however, it is parental group was limited by availability of only one consistent with a previous study in which higher FFA parent in some cases, potentially affecting relation of levels in children were associated with higher rates of midparental measures to offspring. The sex of the parent basal lipolysis than in adults (38). Moreover, increased li- or availability of both parents was not significant when polytic activity was correlated with IGF-I levels, which are added to the analysis. Whereas comparison of parents substantially higher in children and in adolescents than andchildrenallowssomeevaluation of genetic contri- bution, this is confounded by environment, which also during adulthood (38,39). FFA levels in circulation reflect the balance between may be shared. Measures in which genetics and envi- lipolysis, formation of triglyceride droplets in adipo- ronment have opposite effects may make these relations cytes, and utilization of fatty acids by muscle. Lipolysis more difficult to detect. is regulated hormonally, stimulated by catecholamines, This study also had some specific strengths. It is unique and inhibited by insulin. In the insulin-sensitive state, in its analysis of relations between FFAs and both CV risk triglyceride storage is promoted via the peroxisome factors and IR in a longitudinal cohort, using the gold proliferator–activated receptor-g signaling pathway, and standard euglycemic-hyperinsulinemic clamp to measure peroxisome proliferator–activated receptor-g agonists IR. The large size of the cohort is a further strength. This is decrease plasma FFA levels (40). In obesity, lipolysis is the first time FFA levels during childhood have been re- driven, in part, by increased adipocyte size but also lated to later IR in adulthood. Finally, inclusion of the parental group allows for studying developmental changes results from adipocyte resistance to the antilipolytic effect of insulin. The latter appears to be related to local in FFA relations, suggesting a gradually increasing asso- ciation with CV risk factors and IR from adolescence to adipose tissue inflammation, which is associated with macrophage infiltration and increased local production young adulthood. In summary, this study shows that the relation between of tumor necrosis factor-a (40). Tumor necrosis factor-a promotes lipolysis via inhibition of the insulin signaling FFAs and IR is not significant in early adolescence; how- ever, FFA estimates future IR, the relation becomes sig- pathway, modulation of G-protein signaling, and down- regulation of perilipin, a lipid droplet-associated protein nificant during the transition to young adulthood, and it that plays a critical role in lipolysis (41). Beyond inflam- continues to increase, along with development of signifi- matory changes in adipose tissue, there is some evidence cant relations to adiposity and CV risk factors, during ag- that dysfunction of other adipocyte processes may play ing. The observation that FFA in the parental group was a role in lipolysis. Oxidative stress in the adipocyte has significantly associated with BMI and most of the CV risk been shown to result in protein carbonylation, a cova- factors, unlike at younger ages, may suggest that elevated lent modification of proteins by oxidized lipids. Protein FFA and adipose dysfunction have cumulative effects on carbonylation has been linked to mitochondrial dys- metabolic disease that are not fully expressed until later adulthood. Better understanding of mechanisms underlying function and increased lipolysis (42). It is also increased in human obesity and positively associated with serum serum FFA levels may help define development of IR and interactions with adiposity. FFA (43). Reducing FFA levels early in life may be protective against development of adult CV disease. FFAs are among ACKNOWLEDGMENTS a group of circulating lipids shown to induce chronic in- flammation and cellular dysfunction in nonadipose tissues, The work presented in this article was supported by a process termed lipotoxicity (44). Macrophages and other National Institutes of Health grants HL-52851 and M01- tissues take up excess FFA, resulting in steatotic liver in- RR-00400 (General Clinic Research Centers). jury, IR, atherosclerosis, and chronic kidney disease (44). No potential conflicts of interest relevant to this article Lipid infusion causes increased oxidative stress and IR in were reported. both liver and muscle and is associated with increased B.I.F. researched and analyzed data and wrote the serum levels of malondialdehyde, a product of adipose manuscript. D.R.J. researched and analyzed data and oxidative stress (45,46). Lipid infusion also impairs mi- reviewed and edited the manuscript. J.S., A.M., L.M.S., crovascular recruitment in skeletal and cardiac muscle, and A.R.S. researched data and reviewed and edited the suggesting a potential role for FFAs in CV disease (47). manuscript. A.R.S. is the guarantor of this work and, as Among the limitations of this study, the predominately such, had full access to all the data in the study and takes Caucasian cohort, reflecting the local population, reduces responsibility for the integrity of the data and the accuracy the generalizability to more diverse populations. 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Fasting nonesterified fatty acid lesteryl ester fatty acids and estimated desaturase activities are related to profiles in childhood and their relationship with adiposity, insulin sensi- overweight and cardiovascular risk factors in adolescents. Int J Obes tivity, and lipid levels. Pediatrics 2007;120:e1426–e1433 (Lond) 2008;32:1297–1304 diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3169 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Pubmed Central

Relation Between Serum Free Fatty Acids and Adiposity, Insulin Resistance, and Cardiovascular Risk Factors From Adolescence to Adulthood

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Pubmed Central
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© 2013 by the American Diabetes Association.
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0012-1797
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1939-327X
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10.2337/db12-1122
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Abstract

ORIGINAL ARTICLE Relation Between Serum Free Fatty Acids and Adiposity, Insulin Resistance, and Cardiovascular Risk Factors From Adolescence to Adulthood 1 2 1 1 2 Brigitte I. Frohnert, David R. Jacobs Jr., Julia Steinberger, Antoinette Moran, Lyn M. Steffen, and Alan R. Sinaiko FFA production is altered in individuals with known ge- The objective of this study was to describe longitudinal relations netic risk for type 2 diabetes mellitus, regardless of weight. of serum total free fatty acids (FFAs) to insulin resistance (IR) Glucose-tolerant Mexican American adult children of two and cardiovascular (CV) risk factors from adolescence into adulthood. The cohort included participants in a longitudinal parents with type 2 diabetes mellitus were found to have study of obesity and IR with complete data, including anthropo- impaired glucose tolerance by insulin clamp and reduced metric measures, FFAs, IR measured by euglycemic clamp, blood insulin-mediated suppression of lipid oxidation and plasma pressure, fasting serum lipids, and insulin at mean 15 and 22 years FFA concentration compared with age-matched, sex- of age (n = 207) and their parents (n = 272). FFAs and IR were matched, and weight-matched control subjects with non- not significantly related at mean 15 years of age but were signif- diabetic parents (7,18). icantly related at mean age 22 years. FFA did not relate to BMI at Information is limited for FFA in childhood and for the either age. FFA at 15 years of age estimated IR at 22 years of age. natural history of changes in FFA during transition from In parents (mean age 51 years), FFA was significantly correlated childhood to adulthood. The goal of the current study was with BMI, percent body fat, systolic blood pressure, LDL, and IR. Associations with all risk factors except IR in parents were at- to determine relations of FFA with adiposity, IR, and car- tenuated by adjustment for BMI. Most 22 years of age correla- diovascular (CV) risk factors during late childhood/early tions with parents were higher than corresponding 15 years of adolescence and early adulthood. We examined cross- age correlations. This study finds that FFA is associated with IR sectional relations of these factors at mean ages 15 and 22 starting in young adulthood. The relation between FFA and CV years and longitudinal relations of factors between these risk factors does not become significant until later adulthood. age-groups. As an older adult comparison, relations be- The results support a significant impact of early metabolic dys- tween factors were examined in parents of subjects. function on later CV risk. Diabetes 62:3163–3169, 2013 RESEARCH DESIGN AND METHODS Subjects. This study was approved by the University of Minnesota Committee ree fatty acids (FFAs) are elevated in obese for the Use of Human Subjects in Research. At each time point, the study was individuals, primarily as a result of release from explained to participants and parents; parents and participants 18 years of age increased fat mass. This release is enhanced by and older signed informed consent, and children younger than 18 years of age Fthe resistance of obese adipose tissue to the gave informed oral assent. antilipolytic effect of insulin and inability of obese adipo- This longitudinal study of the influence of obesity and IR in childhood on development of adult CV risk has been described previously (19). Initial studies cytes to effectively recycle FFAs via re-esterification (1,2). were conducted at mean 13 years of age, with additional cohort studies at Elevated FFAs compete with glucose as an energy source mean 15, 19, and 22 years of age. The present report consists of data from the in both muscle and fat and are associated with decreased mean 15 years of age (range 12–18) and 22 years of age (range 18–25) visits, glucose oxidation and increased insulin resistance (IR) in the times during which samples were obtained and analyzed for FFA. Of 329 muscle tissue and liver (3–5). Further, increased FFAs in individuals seen at mean age 15 years and 280 individuals seen at mean age 22 adults have been associated with endothelial dysfunction, years, inclusion in this study was limited to those with complete datasets at both time points (n = 207). Of these, 187 had Tanner stage at mean 15 years of blood pressure, and pancreatic b-cell dysfunction (6–9). age recorded. There were no significant differences in clinical factors between Lipid infusion in lean, obese, and type 2 diabetic adults, those included and excluded at either age, except for slightly higher fasting a model of short-term elevation of FFA, results in periph- glucose in the included group at mean 15 years of age (4.82 vs. 4.71 mmol/L; eral IR and decreased peripheral glucose disposal (10–15). P = 0.024) and lower steady-state insulin during euglycemic-hyperinsulinemic The FFA effect on peripheral IR is dose dependent and clamp at age 22 years (394.7 vs. 463.8 pmol/L; P = 0.044). Among the subjects occurs at the level of insulin-stimulated glucose uptake, with complete datasets, there was no difference in any measure between those adversely affecting insulin signaling pathways (6,16,17). with or without Tanner stage recorded at mean 15 years of age. Parents of participants were recruited throughout the study (n = 379) and, at either 13 or 15 years of age visits of their children, underwent many of the same measurements as their offspring. Analysis was limited to parents with From the Department of Pediatrics, Medical School, University of Minnesota, complete datasets (n = 272). There were differences between those included Minneapolis, Minnesota; and the Division of Epidemiology and Commu- nity Health, School of Public Health, University of Minnesota, Minneapolis, and excluded in BMI (28.6 vs. 30.3 kg/m ; P , 0.05), waist circumference Minnesota. (95.5 vs. 100.2 cm; P , 0.05), systolic blood pressure (SBP) (114.6 vs. 119.2 Corresponding author: Brigitte I. Frohnert, frohn001@umn.edu. mmHg; P , 0.01), fasting glucose (5.13 vs. 5.58 mmol/L; P , 0.001), and Received 20 August 2012 and accepted 7 May 2013. steady-state insulin during euglycemic-hyperinsulinemic clamp (465.1 vs. DOI: 10.2337/db12-1122 685.8; P , 0.01). This article contains Supplementary Data online at http://diabetes Clinical measurements. Height, weight, and blood pressure were measured .diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1122/-/DC1. as previously described (19). Waist circumference was measured in duplicate 2013 by the American Diabetes Association. Readers may use this article midway between anterior superior iliac spine and lower rib margin. Percent- as long as the work is properly cited, the use is educational and not for age of body fat and lean body mass (LBM) were determined by the skinfold profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. method of Slaughter et al. (20) at mean 15 years of age and by dual-energy diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3163 FATTY ACIDS FROM ADOLESCENCE TO ADULTHOOD X-ray absorptiometry at mean 22 years of age and in the parent group. LBM at mean 22 years of age from FFA level at mean 15 years of age, adjusting for values at mean 15 years of age were adjusted to dual-energy X-ray absorpti- age, sex, race, and BMI at initial visit. Multiple regression analyses also were ometry values according to previously derived equations (21). performed with the interaction term of sex and FFA; in no case was this term Euglycemic-hyperinsulinemic clamps were conducted at the University of significant. Natural log transformation of the skewed variable, triglycerides, Minnesota Clinical Research Center after a 10-h overnight fast as previously was evaluated and presented when different from the untransformed variable. described (19,22). Insulin sensitivity was determined from the amount of Three multivariable models were developed adjusting for baseline age, sex, glucose administered over the final 40 min of euglycemic-hyperinsulinemic and race (Caucasian vs. African American) as follows: model 1, baseline FFA clamp and expressed as milligrams of glucose utilization per kilogram of lean and BMI as independent variables; model 2, baseline FFA and change in FFA, LBM per minute (M ). A lower M indicates greater IR. baseline BMI, and change in BMI; and model 3, the same as model 2 with the LBM LBM Serum insulin samples were collected on ice, centrifuged within 20 min, and addition of baseline level of the outcome variable. Changes in BMI or FFA analyzed by radioimmunoassay using a double-antibody method (Equate RIA; were calculated as mean 22 years of age value minus mean 15 years of age Binax, Portland, ME). Blood samples for serum lipids were analyzed in the value. Analyses at mean 15 years of age also were adjusted with and without University of Minnesota laboratory with a Cobas Fara. Cholesterol was de- Tanner stage for each analysis and were not different, unless noted otherwise. termined by a standard enzymatic cholesterol oxidase-based method; HDL P , 0.05 was considered to be statistically significant. cholesterol was determined after precipitation of non-HDL lipoproteins with magnesium/dextran precipitating reagent. Triglycerides were determined with a standard glycerol-blanked, enzymatic triglyceride method. LDL cholesterol RESULTS was estimated by the Friedewald equation. Total mmol/L FFA in serum was Participant characteristics. Of 207 individuals studied at determined at mean 22 years of age and in the parent group by quantitative both average 15 and 22 years of age, 59% were male and spectrophotometry. At mean 15 years of age, individual FFA levels were de- 82% were Caucasian. Most of the 15-year-olds were in the termined by tandem mass spectrometry and converted from microgram of each fatty acid per milliliter (mg/mL) sample to mmol/L of total FFA using the late pubertal stage (Tanner 1, none; Tanner 2, 2%; Tanner 3, molar mass of each FFA identified. Supplementary Tables 1 and 2 show 5%; and Tanner 4–5, 93%). BMI increased from 24 to 26 a comparison of mean total and percentage of FFA before and after conver- kg/m between mean 15 and 22 years of age, but percent sion to mmol/L. body fat decreased from 34 to 28%. Both SBP and diastolic Statistical analysis. All analyses were performed using SAS version 9.3 (SAS blood pressure increased significantly from mean 15 to Institute, Cary, NC). Participant characteristics were expressed as means 6 mean 22 years of age. Lipids, including total cholesterol, SEM for continuous variables and as frequencies for categorical variables. Comparisons were made using Student t test and x analysis, respectively. LDL, HDL, and triglycerides, were significantly higher at Pearson partial correlation coefficients, adjusting for age, sex, and race, with mean 22 years of age than at mean 15 years of age. No or without baseline BMI, evaluated within-person tracking of clinical measures difference in fasting glucose was observed between mean between ages 15 and 22 years. Unadjusted Pearson correlations were used to 15 and 22 years of age. Fasting insulin decreased signifi- evaluate relations between clinical measures in subjects at mean 15 and 22 cantly from mean age 15 to mean 22 years of age, coincident years of age and their parents. Of 272 individuals in the parent group, only 196 with a significant increase in IR (M ). FFAs were signif- were parents of included participants. Of 139 child-parent sets, 25 included LBM fathers, 57 included mothers, and 57 included both parents, for whom mid- icantly lower at mean 22 years of age. parental value was used. Pearson partial correlation coefficients, adjusted for The parent group (n = 272, mean age 51 years) had sex, race, and baseline age, were used for cross-sectional analyses of FFA with a significantly smaller percentage of males and a higher other variables at mean 15 and 22 years of age and those of the parents. percentage of Caucasians (Table 1). All anthropometric, Multiple regression analysis evaluated relations of insulin sensitivity (M ) LBM blood pressure, lipid, and fasting glucose measures were with FFA level, adjusting for age, sex, and BMI for adolescents (mean 15 years significantly higher in parents. M , fasting insulin, and of age), young adults (mean 22 years of age), and their parents (mean 51 years LBM of age). Additionally, multiple regression analysis was used to estimate M FFA levels in parents were significantly lower than those LBM TABLE 1 Characteristics of repeated measures in study participants at mean ages 15 and 22 years and of a single measure of their parents Mean age 15 years Mean age 22 years Parents n 207 207 272 Age, years 15.1 6 0.1 21.5 6 0.1* 51.2 6 0.4*‡ Race African American (%) 37 (18) 28 (10) Caucasian (%) 170 (82) 244 (90)§ Male (%) 122 (59) 108 (40)‖ BMI, kg/m 23.6 6 0.4 26.1 6 0.4* 28.6 6 0.4*‡ Body fat, % 33.7 6 1.0 28.3 6 0.8* 36.4 6 0.6†‡ Waist circumference, cm 80.7 6 0.9 87.2 6 1.1* 95.5 6 1.0*‡ SBP, mmHg 109.0 6 0.6 110.4 6 0.7† 114.6 6 0.9*‡ DBP, mmHg 57.0 6 1.0 65.4 6 0.7* 71.4 6 0.6*‡ Total cholesterol, mmol/L 3.77 6 0.05 4.14 6 0.05* 5.23 6 0.06*‡ LDL cholesterol, mmol/L 2.19 6 0.05 2.45 6 0.05* 3.24 6 0.05*‡ HDL cholesterol, mmol/L 1.11 6 0.02 1.17 6 0.02* 1.32 6 0.02*‡ Triglycerides, mmol/L 1.00 6 0.04 1.14 6 0.05* 1.53 6 0.10*‡ Fasting glucose, mmol/L 4.82 6 0.03 4.76 6 0.03 5.13 6 0.05*‡ Fasting insulin, pmol/L 81.4 6 4.0 61.9 6 6.2* 67.3 6 2.7* Steady-state insulin, pmol/L 538.3 6 12.1 394.7 6 10.5* 465.1 6 10.1*‡ M , mg/kg/min 12.6 6 0.3 11.5 6 0.3* 11.3 6 0.2* LBM FFA, mmol/L 0.52 6 0.01 0.36 6 0.01* 0.37 6 0.01* Data presented as mean 6 SEM unless otherwise specified. Sex and race were compared using x analysis. All other variables were compared using an independent Student t test. DBP, diastolic blood pressure. *Significantly different from participant group at mean age 15 years (P , 0.01). †Significantly different from participant group at mean age 15 years (P , 0.05). ‡Significantly different from participant group at mean age 22 years (P , 0.01). §Significantly different from participant group (P , 0.05). ‖Significantly different from participant group (P , 0.01). 3164 DIABETES, VOL. 62, SEPTEMBER 2013 diabetes.diabetesjournals.org B.I. FROHNERT AND ASSOCIATES TABLE 3 in participants at 15 years of age (P , 0.01) but were not Correlations of CV risk factors between subjects at mean ages 15 significantly different from those in participants at 22 years and 22 years and midparental measures of age. Relations of clinical measures between 15 and 22 Mean age Mean age years of age. All clinical and laboratory measures were 15 years 22 years correlated significantly between the two ages, except for rP r P FFA (Table 2). When partial correlations were additionally adjusted for Tanner stage at mean 15 years of age, these BMI, kg/m 0.33 ,0.001 0.43 ,0.001 relations were unchanged. When partial correlations were Body fat, % 0.19 0.02 0.28 ,0.001 additionally adjusted for BMI at mean 15 years of age, Waist circumference, cm 0.30 ,0.001 0.37 ,0.001 relations did not change with the exception of insulin, SBP, mmHg 0.11 0.20 0.27 0.001 which was greatly attenuated (r = 0.07; P = 0.30). Rela- DBP, mmHg 0.24 0.004 0.25 0.003 tionships in males and females separately were similar to Total cholesterol, mmol/L 0.15 0.08 0.20 0.02 LDL cholesterol, mmol/L 0.06 0.48 0.13 0.14 those computed in the entire group, with the exception of HDL cholesterol, mmol/L 0.17 0.05 0.29 ,0.001 FFAs, which were significantly correlated between mean Triglycerides, mmol/L 0.05 0.54 0.09 0.27 15 and 22 years of age in females only (r = 0.27; P = 0.01). Ln(triglycerides), ln(mmol/L) 0.22 0.008 0.18 0.03 Relations of clinical measures between 15 and 22 Fasting glucose, mmol/L 20.09 0.29 0.08 0.33 years of age and midparental measure. For the 139 Fasting insulin, pmol/L 20.04 0.66 0.23 0.006 subjects with parental data, clinical measures were com- M , mg/kg/min 0.11 0.21 0.17 0.04 LBM pared between subjects and parents (Table 3). At 15 and FFA, mmol/L 20.01 0.94 0.14 0.11 22 years of age, there was a significant correlation between offspring and parents for BMI, percent body fat, waist n = 139 parent-child sets. DBP, diastolic blood pressure; Ln(triglycer- circumference, diastolic blood pressure, and natural log ides), natural log transformation of triglycerides. transformation of triglycerides. Correlations between off- spring and parents became stronger for virtually all CV risk factors at 22 years of age, and SBP, total cholesterol, HDL, age, FFAs were not significantly correlated with blood fasting insulin, and M were significantly related be- pressure, measures of adiposity, serum lipids, or fasting LBM tween parents and their children only at mean 22 years of insulin. There was a significant inverse relation with fast- age. When adjusted for parental contribution (i.e., mother, ing glucose and M . Adjustment for BMI did not appre- LBM father, or both), these correlations were not significantly ciably affect these relations at either age (data not shown). different. In separate analyses by sex, results were unchanged, ex- Relations of FFA to other measures. As shown in Table cept correlations in females between FFAs and both fast- 4, FFAs at mean 15 years of age, adjusted for age, sex, and ing glucose and insulin were not significant. race, were not significantly correlated with blood pressure, In contrast to 15- and 22-year-olds, FFAs in parents were measures of adiposity, lipids, fasting glucose, fasting in- significantly correlated with measures of adiposity, SBP, sulin, FFA, or IR (M ), with similar results when ana- LBM fasting glucose, fasting insulin, M , total cholesterol, LBM lyzed separately for males and females. When additionally natural log transformation of triglycerides, and LDL. After adjusted for Tanner stage (n = 183), only the relation with adjustment for BMI in the parent group, there was no longer fasting insulin and natural log transformation of triglyc- asignificant relation with waist circumference (r =0.09; erides became significant (r = 20.17 and P = 0.02 and P =0.16), SBP (r =0.091; P = 0.14), fasting glucose (r =0.09; r = 20.16 and P = 0.03, respectively). At mean 22 years of P = 0.14), natural log transformation of triglycerides (r = 0.09; P = 0.14), or insulin (r =0.09; P = 0.13), but relations with percent body fat, blood lipids, and IR continued to be significant. Separate analyses by sex showed some differ- TABLE 2 ences between mothers and fathers. Significant correlations Tracking correlations of CV disease risk factors between mean between FFA and BMI, waist circumference, and fasting ages 15 and 22 years insulin were found only in mothers; a significant relation r* Pr† P between FFA and SBP was seen only in fathers. The relation between FFA and IR (M ) by age and 2 LBM BMI, kg/m 0.86 ,0.001 NA NA sex, adjusted for BMI and race, is shown in Fig. 1. Linear Body fat, % 0.64 ,0.001 0.15 0.03 regression of M on FFA with age, sex, race, and BMI in LBM Waist circumference, cm 0.81 ,0.001 0.27 ,0.001 the model showed a nonsignificant inverse trend at mean SBP, mmHg 0.49 ,0.001 0.50 ,0.001 age 15 years (b = 21.35; P = 0.37), a significant inverse DBP, mmHg 0.30 ,0.001 0.30 ,0.001 relation at mean 22 years of age (b = 25.22; P , 0.001), Total cholesterol, mmol/L 0.59 ,0.001 0.56 ,0.001 and the strongest inverse relation in parents (b = 26.12; LDL cholesterol, mmol/L 0.66 ,0.001 0.63 ,0.001 P , 0.0001). Further adjustment of IR at 15 years of age for HDL cholesterol, mmol/L 0.63 ,0.001 0.60 ,0.001 Tanner stage did not change results (data not shown). Triglycerides, mmol/L 0.32 ,0.001 0.26 ,0.001 Ln(triglycerides), ln(mmol/L) 0.41 ,0.001 0.35 ,0.001 Estimation of CV risk factors and IR at mean 22 years Fasting glucose, mmol/L 0.39 ,0.001 0.38 ,0.001 of age from mean 15 years of age, FFAs, and changes Fasting insulin, pmol/L 0.22 0.002 0.07 0.30 in FFAs from mean 15 to 22 years of age. In multivar- M , mg/kg/min 0.33 ,0.001 0.32 ,0.001 iable model 1, with baseline (mean 15 years of age) FFAs LBM FFA, mmol/L 0.11 0.11 0.11 0.12 and BMI as independent variables and age, sex, and race as covariates, baseline FFA was associated only with M LBM n = 207. DBP, diastolic blood pressure; Ln(triglycerides), natural log (P = 0.03) and not with later FFA (Table 5 and Fig. 1). In transformation of triglycerides; NA, not applicable. *Pearson partial model 2, which included change in both FFA and BMI from correlations adjusted for age at baseline, sex, and race. †Additionally adjusted for BMI. mean 15 years of age to mean 22 years of age in addition to diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3165 FATTY ACIDS FROM ADOLESCENCE TO ADULTHOOD TABLE 4 Cross-sectional correlation of serum FFA with measures of adiposity and CV risk factors, adjusted for age, sex, and race, at participant mean ages 15 and 22 years and in their parents Mean age 15 years Mean age 22 years Parents (n = 207) (n = 207) (n = 272) r P rPrP BMI, kg/m 20.05 0.51 0.01 0.92 0.19 0.002 Body fat, % 20.01 0.94 0.01 0.93 0.23 ,0.001 Waist, cm 20.07 0.31 0.00 0.97 0.20 ,0.001 SBP, mmHg 20.02 0.75 20.02 0.79 0.12 0.05 DBP, mmHg 0.07 0.35 20.02 0.76 0.09 0.15 Total cholesterol, mmol/L 0.03 0.67 0.11 0.12 0.14 0.02 LDL cholesterol, mmol/L 0.10 0.16 0.10 0.16 0.13 0.03 HDL cholesterol, mmol/L 20.04 0.53 0.07 0.30 20.05 0.41 Triglycerides, mmol/L 20.12 0.08 0.03 0.70 0.07 0.23 Ln(triglycerides), ln(mmol/L) 20.12 0.09 0.00 0.99 0.15 0.02 Fasting glucose, mmol/L 0.01 0.91 20.19 0.01 0.16 0.01 Fasting insulin, pmol/L 20.14 0.05 20.08 0.23 0.18 0.003 M , mg/kg/min 20.06 0.40 20.25 ,0.001 20.31 ,0.001 LBM DBP, diastolic blood pressure; Ln(triglycerides), natural log transformation of triglycerides. baseline FFA and BMI, fasting glucose was estimated by by showing longitudinal developmental changes during the change in FFA (P = 0.004) and M was estimated by transition from adolescence to young adulthood, the in- LBM baseline FFA (P = 0.0002) and change in FFA (P = 0.002). creasing strength of relations between FFA and IR with In model 3 (data not shown), in which the baseline CV risk aging, and the increasing strength of the relations between factor under consideration was included in addition to the parents and children from mean 15 years of age to mean 22 independent variables in model 2, there was little change years of age. At mean 15 years of age, FFA levels were not from model 2; M was again estimated by baseline FFA associated with any of the measures of adiposity, CV risk LBM (P = 0.0003) and change in FFA (P = 0.002), and fasting factors, or IR. By mean 22 years of age, FFA levels began glucose was estimated by change in FFA (P = 0.002); in to show a significant relation to IR but not to adiposity or addition, change in FFA now estimated LDL (P = 0.02). CV risk factors. Only in the older adult group (parents; Adjustment for Tanner stage at mean 15 years of age did mean 51 years of age) was there a significant relation of not change these results (data not shown). FFA to a broad group of cardiometabolic risk factors, in- Linear regression analyses were used to assess the re- cluding adiposity, systolic blood pressure, lipids, fasting lation between FFAs at mean 15 years of age and M at insulin, and glucose, and in particular, there was a more LBM mean 22 years of age, adjusting for sex, baseline age, and pronounced significant relation to IR. BMI (Fig. 2). FFAs at mean 15 years of age was a signifi- Although there were significant correlations between cant estimator of M at mean 22 years of age (b = parents and children for a number of CV risk factors, LBM 23.03; P = 0.03). After adjustment for Tanner stage at 15 relation with FFA was not significant. To our knowl- years of age, this relationship persisted (b = 23.29; P =0.03). edge, this is the first time FFA and IR by euglycemic- hyperinsulinemic clamp have been compared in a large parent-child cohort. Similar to previous studies, we also DISCUSSION found significant relations between parents and children This study provides new information on relations between for BMI, blood pressure, and lipids, with the exception of FFA and both CV risk factors and IR in children and adults LDL (23). Although LDL also was significantly related FIG. 1. Cross-sectional multiple regression of IR (M ) on FFA level, adjusted for age, race, BMI, and sex in adolescents (mean 15 years of age), LBM young adults (mean 22 years of age), and their parents (mean 51 years of age). At mean 15 years of age, b = 21.35 mg/kg/min per mmol/L (P = 0.37); at mean 22 years of age, b = 25.22 mg/kg/min per mmol/L (P < 0.001); and in parents, b = 26.12 mg/kg/min per mmol/L (P < 0.0001). Sex-specific mean M quartiles are included for goodness-of-fit assessment. ○, females; ■, males. LBM 3166 DIABETES, VOL. 62, SEPTEMBER 2013 diabetes.diabetesjournals.org B.I. FROHNERT AND ASSOCIATES TABLE 5 Results of multivariable models using baseline FFA at mean age 15 years to estimate CV risk factor levels at mean age 22 years Model 1 Model 2 Dependent variable Baseline FFA Baseline FFA DFFA BMI, kg/m 20.21 6 1.15 1.48 6 1.58 1.89 6 1.21 SBP, mmHg 24.35 6 3.59 25.89 6 4.88 21.87 6 3.76 Total cholesterol, mmol/L 20.01 6 0.27 0.45 6 0.37 0.50 6 0.28 LDL cholesterol, mmol/L 0.09 6 0.23 0.45 6 0.32 0.39 6 0.24 HDL cholesterol, mmol/L 20.19 6 0.10 20.05 6 0.13 0.16 6 0.10 Fasting glucose, mmol/L 0.23 6 0.17 20.23 6 0.23 20.52 6 0.18† Fasting insulin, pmol/L 216.8 6 30.9 251.4 6 42.1 239.6 6 32.4 M , mg/kg/min 23.03 6 1.40‡ 27.07 6 1.86* 24.45 6 1.43† LBM FFA, mmol/L 0.11 6 0.07 0.11 6 0.07 NA Data presented as b-coefficient 6 SEM unless otherwise specified. Models are adjusted for corresponding BMI levels (n = 207). Models adjusted for age, sex, race, and BMI. Model 2 is further adjusted for change in BMI from baseline to adult follow-up. DBP, diastolic blood pressure; DFFA, FFA at adult follow-up minus FFA at baseline. NA, not applicable. *P , 0.001. †P , 0.01. ‡P , 0.05. between parents and children in the Bogalusa Heart of CV disease. This is supported by studies in a rat model Study, the correlation was similarly fairly weak and that system, which showed an increased FFA-induced in- cohort was significantly larger, with greater power to flammatory response and IR with aging (36). Together, detect such associations (23). these findings support the hypothesis that fat metabolism Previous studies have reported relations between FFA and distribution are early pathogenic factors in develop- and both adiposity and IR; however, studies in the pedi- ment of IR. atric population are limited. In contrast to adults, fasting The relations between FFAs and IR did not completely FFAs in prepubertal children were not associated with IR parallel relations of FFAs with fasting insulin, probably, in as measured by the euglycemic-hyperinsulinemic clamp part, because of developmental changes associated with (24). Although an inverse relation between FFAs and transition from adolescence to young adulthood. Both fasting insulin has been reported in prepubertal children, fasting insulin and insulin sensitivity decreased from 15 to other studies showed no relation of FFAs with fasting 22 years of age. Previous studies of this normal cohort, insulin in both prepubertal and pubertal children (24–28). with a wide range of BMI and IR, have shown during ad- Lipid infusion, which results in elevated FFA levels, in- olescence a poor correlation between fasting insulin and creased IR in pubertal but not in prepubertal children IR (37), a significant increase in IR without a significant (29). There was no difference in FFA-mediated changes in change in fasting insulin during puberty (19), and separate IR after lipid infusion in African American adolescents patterns of change in fasting insulin and IR from 11 to 19 when compared with Caucasian adolescents (30). It is years of age (19). Thus, these studies provide evidence to unclear whether increased basal levels of FFAs seen in support the complex relation between fasting insulin and obese adolescents relate to increased fat mass or im- IR, particularly during the second decade of life. The paired antilipolytic effect of insulin (31,32). Whereas finding that levels of both were similar at 22 and 51 years adefinite pattern cannot be established from those studies, of age (parents) suggests that the relations become more they suggest a potential age-dependent or development- stable during adulthood. dependent change in the relation between FFAs and both FFA levels were overall higher, independent of BMI, at fasting insulin and IR. mean 15 years of age compared with later measures in Longitudinal studies in adults have shown higher FFA young adulthood and in their parents. This observation levels were associated with increased risk of impaired glucose tolerance or type 2 diabetes (33); in Pima Indians with normal glucose tolerance, there is an association of baseline FFA levels with later IR by euglycemic clamp (34). In contrast, another study showed no association of baseline FFAs with fasting insulin and glucose on follow- up (35). The current study expands on previous studies of children or adults by exploring longitudinal changes be- tween adolescence and adulthood and relating FFA levels between adolescents and their adult parents. The relation between FFA levels and IR at mean 22 years of age is independent of degree of adiposity, existing before any association with adiposity or FFA levels develops, as shown in the older adults. Together with the observation that higher FFA levels at mean 15 years of age are associated with higher IR at mean 22 years of age, this suggests that FFA-related metabolic abnormalities develop FIG. 2. Multiple regression of IR (M ) at mean 22 years of age on with increasing age, exhibiting a causal relation in the LBM FFA level at mean 15 years of age, adjusted for age, race, BMI, and sex development of IR. Association of FFA levels with other (b = 23.03 mg/kg/min per mmol/L; P = 0.03). Sex-specific mean M LBM CV risk factors does not appear until later adulthood, quartiles are included for goodness-of-fit assessment. ○, females; ■, suggesting a progressive effect of FFAs on the pathogenesis males. diabetes.diabetesjournals.org DIABETES, VOL. 62, SEPTEMBER 2013 3167 FATTY ACIDS FROM ADOLESCENCE TO ADULTHOOD may relate to differences in FFA assays; however, it is parental group was limited by availability of only one consistent with a previous study in which higher FFA parent in some cases, potentially affecting relation of levels in children were associated with higher rates of midparental measures to offspring. The sex of the parent basal lipolysis than in adults (38). Moreover, increased li- or availability of both parents was not significant when polytic activity was correlated with IGF-I levels, which are added to the analysis. Whereas comparison of parents substantially higher in children and in adolescents than andchildrenallowssomeevaluation of genetic contri- bution, this is confounded by environment, which also during adulthood (38,39). FFA levels in circulation reflect the balance between may be shared. Measures in which genetics and envi- lipolysis, formation of triglyceride droplets in adipo- ronment have opposite effects may make these relations cytes, and utilization of fatty acids by muscle. Lipolysis more difficult to detect. is regulated hormonally, stimulated by catecholamines, This study also had some specific strengths. It is unique and inhibited by insulin. In the insulin-sensitive state, in its analysis of relations between FFAs and both CV risk triglyceride storage is promoted via the peroxisome factors and IR in a longitudinal cohort, using the gold proliferator–activated receptor-g signaling pathway, and standard euglycemic-hyperinsulinemic clamp to measure peroxisome proliferator–activated receptor-g agonists IR. The large size of the cohort is a further strength. This is decrease plasma FFA levels (40). In obesity, lipolysis is the first time FFA levels during childhood have been re- driven, in part, by increased adipocyte size but also lated to later IR in adulthood. Finally, inclusion of the parental group allows for studying developmental changes results from adipocyte resistance to the antilipolytic effect of insulin. The latter appears to be related to local in FFA relations, suggesting a gradually increasing asso- ciation with CV risk factors and IR from adolescence to adipose tissue inflammation, which is associated with macrophage infiltration and increased local production young adulthood. In summary, this study shows that the relation between of tumor necrosis factor-a (40). Tumor necrosis factor-a promotes lipolysis via inhibition of the insulin signaling FFAs and IR is not significant in early adolescence; how- ever, FFA estimates future IR, the relation becomes sig- pathway, modulation of G-protein signaling, and down- regulation of perilipin, a lipid droplet-associated protein nificant during the transition to young adulthood, and it that plays a critical role in lipolysis (41). Beyond inflam- continues to increase, along with development of signifi- matory changes in adipose tissue, there is some evidence cant relations to adiposity and CV risk factors, during ag- that dysfunction of other adipocyte processes may play ing. The observation that FFA in the parental group was a role in lipolysis. Oxidative stress in the adipocyte has significantly associated with BMI and most of the CV risk been shown to result in protein carbonylation, a cova- factors, unlike at younger ages, may suggest that elevated lent modification of proteins by oxidized lipids. Protein FFA and adipose dysfunction have cumulative effects on carbonylation has been linked to mitochondrial dys- metabolic disease that are not fully expressed until later adulthood. Better understanding of mechanisms underlying function and increased lipolysis (42). It is also increased in human obesity and positively associated with serum serum FFA levels may help define development of IR and interactions with adiposity. FFA (43). Reducing FFA levels early in life may be protective against development of adult CV disease. FFAs are among ACKNOWLEDGMENTS a group of circulating lipids shown to induce chronic in- flammation and cellular dysfunction in nonadipose tissues, The work presented in this article was supported by a process termed lipotoxicity (44). Macrophages and other National Institutes of Health grants HL-52851 and M01- tissues take up excess FFA, resulting in steatotic liver in- RR-00400 (General Clinic Research Centers). jury, IR, atherosclerosis, and chronic kidney disease (44). No potential conflicts of interest relevant to this article Lipid infusion causes increased oxidative stress and IR in were reported. both liver and muscle and is associated with increased B.I.F. researched and analyzed data and wrote the serum levels of malondialdehyde, a product of adipose manuscript. D.R.J. researched and analyzed data and oxidative stress (45,46). Lipid infusion also impairs mi- reviewed and edited the manuscript. J.S., A.M., L.M.S., crovascular recruitment in skeletal and cardiac muscle, and A.R.S. researched data and reviewed and edited the suggesting a potential role for FFAs in CV disease (47). manuscript. A.R.S. is the guarantor of this work and, as Among the limitations of this study, the predominately such, had full access to all the data in the study and takes Caucasian cohort, reflecting the local population, reduces responsibility for the integrity of the data and the accuracy the generalizability to more diverse populations. 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DiabetesPubmed Central

Published: Aug 15, 2013

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