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Adiponectin, an adipokine secreted by the adipocyte, is inversely related to adiposity and directly related to insulin sensitivity. In T1DM, however, data thus far are contradictory. We investigated the relationship between adiponectin and exercise in type 1 diabetes.
49 children (14.5 ± 2.0yrs, range 8–17) with T1DM on an insulin pump were studied during two 75min exercise sessions with and without continuation of the basal rate within 4w. Adiponectin and epinephrine concentrations were measured before and during exercise.
Mean pre-exercise adiponectin concentration was 11.2 ± 4.7 mg/L (range 2.7–23.0) with a mean absolute difference of 1.7 mg/L between the 2 days. Adiponectin concentrations did not change meaningfully during exercise (mean change: −0.1 ± 1.2; P=0.17). Adiponectin correlated inversely with BMI percentile (p=0.02) but not with age, gender, duration of diabetes, HbA1c or pre-exercise glucose. However, those with higher baseline adiponectin were less likely to become hypoglycemic during exercise, 36% becoming hypoglycemic when baseline adiponectin was <10 mg/L, 42% when 10-<15, 15% when ≥15 (p=0.02). Baseline epinephrine concentrations were not associated with adiponectin, and in those whose nadir glucose was ≤100mg/dL, there was no correlation between epinephrine response and adiponectin (p=0.16).
Adiponectin concentrations are stable from day to day, are not affected by acute exercise or metabolic control, and vary inversely with adiposity. Higher adiponectin appears to be associated with a decrease in hypoglycemia risk during exercise. Further studies are needed to examine whether adiponectin protects against exercise-induced hypoglycemia by directly enhancing the oxidation of alternate fuels.
Adiponectin is an adipokine secreted exclusively by adipocytes, which is inversely related to degree of adiposity and directly related to insulin sensitivity. It has structural homology with TNFα and its expression regulated by PPAR-β. It binds to 2 different receptors, AdipoR1/R2 in liver and skeletal muscle. Several isoforms of adiponectin have been identified but the biological relevance of these isoforms has not been fully characterized (1). The role of adiponectin in improving insulin sensitivity has made this peptide a potential therapeutic target in obesity and diabetes.
The relationship between adiponectin and insulin sensitivity has been well studied in healthy adults and in type 2 diabetes mellitus (T2DM), as well as in healthy children. A large cohort (N=1632 Canadian children) studied at ages 9, 13 and 16 showed that boys have lower adiponectin than girls, that puberty is associated with a decrease in adiponectin, and that adiponectin is negatively associated with BMI (2). Using percutaneous muscle biopsies in either healthy adults or those with impaired glucose tolerance (IGT) or T2DM, investigators showed that prolonged physical training for 4w increased adiponectin and the expression of its 2 receptors, whereas acutely, 3 hrs of exercise only increased adiponectin receptors expression, but not the circulating levels, suggesting that the improvement in insulin resistance observed with physical training is mediated in part via adiponectin concentrations or increased receptor sensitivity (3).
However, the relation between adiponectin and insulin sensitivity in T1DM may be more complex, and the data thus far reported are somewhat contradictory. Reports have shown that patients with T1DM who have higher adiponectin concentrations had an increased risk of microvascular complications (4, 5), whereas others have found the opposite, the lower the adiponectin in T1DM, the greater the risk of and progression of coronary artery disease (6, 7). Progression of diabetic nephropathy has been shown to be associated with higher adiponectin in T1DM subjects, but whether this is a compensatory response to nephropathy is not clear (8). Even though previous studies have shown that pre-pubertal and pubertal children with poorly controlled type 1 diabetes are more insulin resistant than non-diabetic children and adolescents (9), two pediatric studies, show no difference in adiponectin concentrations between healthy children and those with T1DM after correction for BMI (10, 11). Another recent pediatric study of 91 children with T1DM vs. 91 non-diabetic controls showed higher adiponectin concentrations in the prepubertal children with T1DM as compared to the prepubertal controls, but no differences between the pubertal children with and without diabetes (12). These same investigators however found adiponectin concentrations directly related to HbA1c.
Because of its role enhancing insulin sensitivity in normal individuals, we sought to investigate further the relationship between adiponectin and exercise, with and without insulin infusions in diabetic children on pump therapy. We hypothesized that those with more severe hypoglycemia during exercise would have overall higher adiponectin concentrations, hence were more insulin sensitive. Furthermore, we aimed to better understand the relation between adiponectin and the adreno-medullary/parasympathetic hormone responses to exercise as the latter are the first line of defense against hypoglycemia. We recently reported our data in a group of children with diabetes on insulin pump therapy who were exercised twice within approximately one month, once during continuation of the basal rates during exercise, and the other with the basal discontinued. We showed a higher incidence of hypoglycemia in those children whose basal rates were continued (13). We targeted to study changes in adiponectin concentrations during exercise in all the children who participated and catecholamine concentrations in a subset of those patients.
Details of the protocol have been described elsewhere (13) and are only briefly summarized here. Forty-nine children (8-<18 years) with type 1 diabetes of duration ≥18 months were studied after informed written consent and IRB approval at the 5 participating diabetes centers. Subjects were required to have been on a stable insulin regimen using an insulin pump for at least one month and to have an HbA1c ≤10.0%.
On both days, the subjects arrived at the clinical research centers (CRC) before 12:00 P.M. and remained until ~6:30 P.M. Lunch was served at approximately noon using the prelunch bolus and correction factor the subject usually used at home. Glucose was checked using a home glucose meter at 1:00, 2:00, and 3:00 P.M., with the goal of having the subject’s glucose concentration between 120 and 200 mg/dl just before the start of exercise (~4:00 P.M.). An intravenous bolus of regular insulin (0.05–0.1 units/kg) was given because of its short half-life and duration of action if the investigator felt the subject’s glucose level would likely be >200 mg/dL at 4:00 P.M., or 15–30 g carbohydrate was given orally if the investigator felt the subject’s 4:00 P.M. glucose would likely be <120 mg/dl. If at 4:00 P.M. the subject’s glucose was not within 120–200 mg/dL or the subject had been given intravenous insulin within the previous hour, then the exercise session was delayed.
The study consisted of two 75-minute exercise sessions in the late afternoon, separated by 6 to 36 days. For one of the visits (chosen at random), the insulin pump was turned off at the beginning of the exercise session and re-started 45 minutes post-exercise (approximately 2 hours total). For the other visit, the basal rate was continued throughout exercise. The exercise session consisted of 15 minutes of brisk walking on a treadmill at a heart rate of approximately 140 beats/minute followed by a 5-minute rest period. This cycle was repeated 3 more times for a total of four 15-minute exercise periods with 5-minute rest periods in between (75 minutes total).
Venous samples were used for glucose measurements using a FreeStyle® Flash™ glucose meter (14), “Freestyle”, Abbott Diabetes Care, Alameda, CA) and by the DirecNet Central Biochemistry Laboratory at the University of Minnesota using a hexokinase enzymatic method (15, 16). Glucose concentrations were measured prior to starting exercise, during each of the 3 rest periods, immediately following exercise, and at 15, 30 and 45 minutes following completion of exercise. If during exercise the bedside Freestyle meter glucose dropped ≤65 mg/dL, the subject was treated with 15–30g CHO and further exercise was delayed until the glucose was >70 mg/dL.
Blood samples for adiponectin were taken immediately pre- and post-exercise on both visits for all 49 subjects. Samples were processed and sera immediately frozen. Adiponectin was measured on serum using the quantitative sandwich enzyme technique of the enzyme-linked immunosorbent assay (ELISA) Quantikine kit from R & D Systems, Inc. (Minneapolis, MN) at the University of Minnesota laboratory. Duplicate blood draws were taken pre-exercise at each visit. The median relative absolute difference between the two concurrent measurements was 5% matching the inter-assay CV reported by the laboratory. The duplicate measurements were within ± 1.0 mg/L 70% of the time.
A subset of 32 out of the 49 subjects was selected for catecholamine measurements based on whether or not they became hypoglycemic during exercise. Twenty-one “discordant” subjects were selected because they became hypoglycemic on one of their visits, but not the other. Additionally, six subjects who became hypoglycemic during both visits were matched to five subjects with similar pre-exercise glucose concentrations who did not become hypoglycemic on either visit (collectively, 11 “concordant” subjects).
Blood samples for catecholamines were collected in EDTA tubes, with plasma separated soon after the blood draw and immediately frozen. Plasma epinephrine and norepinephrine concentrations were measured at the Mayo Clinic Laboratory (Rochester, MN) using a reverse phase (C18) HPLC column to separate norepinephrine and epinephrine, which were detected coulometrically, using an ESA Coulochem II instrument. The lower limit of detection in this assay is 10 pg/mL (0.06 nmol/L and 55 pmol/L, respectively). CVs were 7–11% and 6–7% on 3 controls.
The pre-exercise adiponectin value was taken as the average of the duplicate measurements. Since there was little change in adiponectin during exercise (see Results), the post-exercise value was imputed for one visit where both pre-exercise measurements were missing. The paired t-test was used to compare the first vs. second visit and a repeated measures least squares regression was used to compare pre- vs. post-exercise values. Results were similar when three potential outliers (adiponectin concentration fell more than 3.0 mg/L during exercise) were excluded.
Adiponectin concentrations were evaluated according to levels of various demographic and clinical characteristics (Table 1) using the mean pre-exercise concentration over both visits. A t-test was used for the discrete factor (gender) and least squares regression for the continuous factors.
Hypoglycemia was defined as either a laboratory glucose concentration ≤70 mg/dL and/or treatment for low glucose (based on the Freestyle meter value available at the time) during or following exercise. Repeated measures logistic regression with generalized estimating equations (GEE) was used to associate hypoglycemia with pre-exercise adiponectin adjusting for age, gender, BMI percentile, HbA1c, basal-insulin stopped vs. continued, period effect (1st vs. 2nd visit) and pre-exercise glucose. BMI percentile was calculated adjusting for age and gender (17).
A value of 10 pg/mL was imputed for the 14% of epinephrine measurements below the detection limit. An epinephrine outlier of 604 pg/mL was truncated to 350 pg/mL (the next largest value). A square root transformation was used to account for the skewed distribution of epinephrine and norepinephrine values. Repeated measures least squares regression was used to associate catecholamine concentrations with the pre-exercise adiponectin adjusting for discordant vs. concordant (see above), basal-insulin stopped vs. continued, period effect (1st vs. 2nd visit), age, gender, BMI percentile, HbA1c, pre-exercise epinephrine, and the pre-exercise and nadir glucose values.
Residual values were confirmed to have an approximate normal distribution for each regression model except as noted above for the catecholamines where a transformation was used.
The mean (± SD) age of the 49 subjects was 14.5 ± 2.0 years (range 8 to 17 years); 43% were female; 94% were Caucasian, 2% Hispanic, 2% Asian, and 2% reported more than one race. The mean body mass index (BMI) of the subjects was 22.3 ± 3.0 kg/m2 (range 15.8 to 30.1) and the mean BMI percentile adjusting for age and gender was 73% ± 21% (range 4% to 98%). The mean duration of diabetes was 7.2 ± 3.8 years and the mean HbA1c was 7.5 ± 0.9%. A severe episode of hypoglycemia (resulting in seizure or loss of consciousness) in the 6 months prior to the study was reported by 3 subjects (6%). Twenty-six subjects completed the basal-continued visit first and 23 completed the basal-stopped visit first. The median time between the two visits was 14 days (25th, 75th percentiles 8, 20 days; range 6–36 days).
As previously reported (13), baseline glucose concentrations before the start of the exercise measured at the central laboratory ranged from 115 to 230 mg/dL (all but one of the Freestyle values were within the specified range of 120–200 mg/dL). Baseline values were quite similar on basal-continued and basal-stopped visits (mean ±SD 156 ± 27 mg/dL vs. 161 ± 24 mg/dL, respectively; P = 0.30). The absolute change in glucose concentrations from baseline during exercise was less during the basal-stopped visit than during the basal-continued visit (absolute change 44 ± 38 mg/dL vs. 63 ± 33 mg/dL, P < 0.001; relative change 28% ± 23% vs. 41% ± 19%, P < 0.001), as was the frequency of hypoglycemia (16% vs. 43%, P = 0.003) (13).
The mean adiponectin concentration pre-exercise was 11.2 ± 4.7 mg/L with median (25th, 75th percentiles) 10.3 (8.0, 13.6) ranging from 2.7 to 23.0 mg/L. Pre-exercise adiponectin concentrations were similar for the first and second visits (mean difference: −0.5 ± 2.4; P=0.19; mean absolute difference: 1.7 ± 1.7). There was a negative association between adiponectin concentrations and BMI percentile (P=0.02). Although adiponectin levels tended to decrease with age and higher HbA1c levels and increase with longer duration of diabetes, these small differences were not statistically significant (Table 1).
As shown in Figure 1, adiponectin concentrations did not change meaningfully during exercise (mean change: −0.1 ± 1.2; P=0.17) with similar results whether the basal insulin was stopped or continued. Pre-exercise glucose concentrations were not associated with adiponectin. Subjects with higher baseline adiponectin concentrations were less likely to become hypoglycemic during or following exercise. The percentage of subjects becoming hypoglycemic was 36% when the pre-exercise adiponectin was <10 mg/L, 42% when the adiponectin was 10-<15 mg/L and 15% when the adiponectin was ≥15 mg/L (P=0.02) with similar trends whether the basal-insulin was stopped or continued (Table 2). The percentage of subjects becoming hypoglycemic on both visits was 17% vs. 13% vs. 0% when the average adiponectin concentration was <10, 10-<15 and ≥15 mg/L, respectively. Corresponding percentages becoming hypoglycemic on neither visit were 38%, 40% and 70%, respectively.
Pre-exercise epinephrine concentrations were not associated with adiponectin or baseline glucose concentrations. Epinephrine increased by ≥50 pg/mL over the pre-exercise concentration for 57% (median increase from baseline to peak: 66 pg/mL) of visits where the glucose dropped ≤70 mg/dL (Figure 2). Corresponding percentages (median increases) were 53% (51 pg/mL) and 0% (18 pg/mL) when the nadir glucose was 71–100 and >100 mg/dL, respectively.
Among 47 visits from 30 subjects where the glucose dropped to ≤100 mg/dL, there was no significant association of catecholamine response with baseline adiponectin (p=0.16). Epinephrine increased by ≥50 pg/mL for 50% (median increase: 46 pg/mL), 61% (67 pg/mL) and 57% (70 pg/mL) when the pre-exercise adiponectin was <10, 10-<15 and ≥15 mg/L, respectively.
Norepinephrine increased by ≥200 pg/mL over the pre-exercise concentration for 57% (median increase: 244 pg/mL) of visits where the glucose dropped ≤70 mg/dL, 41% (170 pg/mL) when the nadir glucose was 71–100 and 59% (216 pg/mL) when the glucose remained above100 mg/dL. The norepinephrine response was not associated with adiponectin concentrations.
Plasma adiponectin concentrations have become a useful marker for insulin sensitivity, with higher levels associated with lower BMI and higher insulin sensitivity in subjects without diabetics. The well- reported observation that adiponectin is inversely correlated to BMI percentiles was also observed in our subjects. Moreover, in this population there was no significant correlation between adiponectin levels and other putative mediators of adiponectin regulation, like age, gender and glycemic control.
This study was undertaken to examine the hypothesis that children with higher adiponectin concentrations would be more insulin sensitive and, in turn, more susceptible to exercise-induced hypoglycemia, especially when the basal insulin infusion was continued during the exercise session. Overall, acute exercise did not alter adiponectin concentrations (Figure 1), congruent with data just published in adults with T2DM (3). However, contrary to our stated hypothesis, higher adiponectin concentrations appear to be associated with a decrease in hypoglycemia risk whether there is continuation of basal insulin therapy or not.
Despite the fact that the pre-exercise BGs in all subjects were not exactly the same, the narrow range in which they were kept (essentially within 80mg/dl among all the visits) and in the near-normoglycemic range, with relative glycemic homogeneity allowing for reasonable comparisons of the changes in adiponectin with exercise in this cohort. Although the insulin concentrations were not measured here (all the study subjects were using insulin analogs) we know a priori, by design, that the insulin concentrations were higher in the basal continued group.
As illustrated by the subjects whose nadir glucose remained above 100mg/dL, exercise itself stimulated a slight rise in epinephrine concentrations that was ≤50 pg/mL. As expected, subjects who became hypoglycemic during exercise had more robust plasma epinephrine and norepinephrine responses with hypoglycemic thresholds for augmented release of catecholamines between 60–100 mg/dL, as has been previously reported in youth with T1DM (18). However the catecholamine responses were not related to the baseline adiponectin concentrations. This may reflect in part the large variability in adrenomedullary responses, especially in a set up of exercise where plasma glucose was not clamped. These data support the emerging concept that adiponectin may be a form of counterregulation and hypoglycemia prevention during exercise, independent of catecholamines. These results are however preliminary and should be confirmed in larger studies measuring a broader array of metabolic parameters during exercise during clamped studies.
We acknowledge the fact that the amount of abdominal fat and cardiovascular fitness, neither of which were measured here, are important variables when examining adiponectin levels. Despite similar BMIs in our study, someone with lower visceral adiposity and higher muscle mass may be better conditioned and have higher adiponectin and be protected from hypoglycemia in relation to better cardiovascular fitness and better substrate utilization. However, accumulating evidence suggest that a certain level of circulating adiponectin is required to maintain energy homeostasis and prevent metabolic diseases. For example, the administration of adiponectin increases fatty acid oxidation in skeletal muscle cells, and data recently published demonstrate that adiponectin stimulates free fatty acid oxidation in skeletal muscle directly by sequential activation of AMP-activated protein kinase (AMPK), p38 MAPK and PPARα (19). Although our studies are largely observational, if confirmed, they suggest that the increase in adiponectin in the diabetic state may be a compensatory response to enhance alternate fuel oxidation. Epinephrine facilitates lipolysis and adiponectin enhances lipid oxidation, hence increased lipid mobilization and increased fatty acid oxidation may be both operative in supplying the energy needs of skeletal muscle. The latter may decrease the need for available glucose substrate, perhaps protecting from hypoglycemia. In aggregate, these data would support the emerging hypothesis that higher adiponectins may represent a higher counterregulatory response in diabetes (4). This hypothesis can be tested in future metabolic studies.
Appreciation is expressed for the work performed by the CRC Nurses at the five clinical centers. This research has been supported by the following NIH/NICHD Grants: HD041919-01; HD041915-01; HD041890; HD041918-01; HD041908-01; and HD041906-01. Clinical Centers also received funding through the following GCRC Grant Numbers M01 RR00069; RR00059; RR 06022 and RR00070-41. This publication was made possible/the project described was supported by a Mayo Clinic Grant Number UL1 RR024150-01 from the National Center for Research Resources (NCRR), a component of the National Institute of Health (NIH), and NIH Roadmap for Medical Research.
Abbott Diabetes Care, Alameda, CA, provided the Freestyle® Blood Glucose Monitoring Systems and test strips and the Precision Xtra™ Ketone Monitoring Systems and test strips.
Nelly Mauras, MD-Writing committee chair; Craig Kollman, PhD; Michael W. Steffes, MD, PhD; Ravinder Singh, PhD; Rosanna Fiallo-Scharer, MD; Eva Tsalikian, MD; Stuart A. Weinzimer, MD; Bruce Buckingham, MD; Roy W. Beck, MD, PhD; Katrina J. Ruedy, MSPH; Dongyuan Xing MPH; William V. Tamborlane, MD
Clinical Centers: (Listed in alphabetical order with clinical center name, city, and state. Personnel are listed as (PI) for Principal Investigator, (I) for co-Investigator and (C) for Coordinators.) (1) Barbara Davis Center for Childhood Diabetes, University of Colorado, Denver, CO: H. Peter Chase, MD (PI); Rosanna Fiallo-Scharer, MD (I); Laurel Messer, RN (C); Barbara Tallant, RN, MA (C); (2) Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA: Eva Tsalikian, MD (PI); Michael J. Tansey, MD (I); Linda F. Larson, RN (C); Julie Coffey, MSN (C); Joanne Cabbage (C); (3) Nemours Children’s Clinic, Jacksonville, FL: Tim Wysocki, PhD, ABPP (PI); Nelly Mauras, MD (I); Larry A. Fox, MD (I); Keisha Bird, MSN (C); Kim Englert, RN (C); (4) Division of Pediatric Endocrinology and Diabetes, Stanford University, Stanford, CA: Bruce A. Buckingham, MD (PI); Darrell M. Wilson, MD (I); Jennifer M. Block, RN, CDE (C); Paula Clinton, RD, CDE (C); Kimberly Caswell, APRN; (5) Department of Pediatrics, Yale University School of Medicine, New Haven, CT: Stuart A. Weinzimer, MD (PI); William V. Tamborlane, MD (I); Elizabeth A. Doyle, MSN (C); Heather Mokotoff, MSN (C); Amy Steffen (C); Coordinating Center: Jaeb Center for Health Research, Tampa, FL: Roy W. Beck, MD, PhD; Katrina J. Ruedy, MSPH; Craig Kollman, PhD; Dongyuan Xing, MPH; Mariya Dontchev , MPH; Cynthia R. Stockdale; Judy Jackson; University of Minnesota Central Laboratory: Michael W. Steffes, MD, PhD; Jean M. Bucksa, CLS; Maren L. Nowicki, CLS; Carol A. Van Hale, CLS; Vicky Makky, CLS;
Department of Laboratory Medicine and Pathology, Mayo Clinic: Ravinder Jit Singh, PhD; National Institutes of Health: Gilman D. Grave, MD; Mary Horlick, PhD; Karen Teff, PhD; Karen K. Winer, MD; Data and Safety Monitoring Board: Dorothy M. Becker, MBBCh; Patricia Cleary, MS; Christopher M. Ryan, PhD; Neil H. White, MD, CDE; Perrin C. White, MD