- Email: [email protected]
Irisin levels correlate with energy expenditure in a subgroup of humans with energy expenditure greater than predicted by fat free mass
M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 2 (2 0 1 3) 1 07 0–1 07 3
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Clinical Science
Irisin levels correlate with energy expenditure in a subgroup of humans with energy expenditure greater than predicted by fat free mass Andrew G. Swick⁎, Stephen Orena, Annalouise O’Connor UNC Nutrition Research Institute, University of North Carolina Chapel Hill, North Carolina Research Campus, Kannapolis, North Carolina, USA
A R T I C LE I N FO Article history:
AB S T R A C T Objective. Obesity is a result of chronic overconsumption of calories relative to the amount of
Received 7 December 2012
energy expended. While fat free mass can account for ~80% of the variance in energy expenditure,
Accepted 27 February 2013
there is still considerable variability in energy requirements between individuals that cannot be
Keywords:
which has been touted to increase energy expenditure via activation of brown adipocytes in
FNDC5
rodents and possibly humans, may explain some of the variability in energy expenditure.
explained. We hypothesized that responsiveness to the recently discovered myokine, irisin,
Materials/methods. Post-menopausal women (n = 17) spent 24-h in a whole room indirect
BAT Indirect calorimetry
calorimeter. During the study day, subjects remained sedentary and consumed meals
RQ
tailored to their energy requirements. Plasma irisin, leptin and adiponectin were measured in samples taken from each subject. Results. Our results suggest that in general, irisin levels do not correlate with 24-h energy expenditure, however, for a subpopulation irisin levels and energy expenditure are highly correlative. Conclusion. Irisin may help explain some of the observed variability in individual energy requirements that cannot be accounted for by fat free mass. Therefore, interventions designed to increase irisin action may prove to be promising avenues for the treatment of obesity. © 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Obesity is an ever-increasing, worldwide health concern associated with a significant economic burden. Obesity results from chronic overconsumption of calories (kcal) relative to the amount of calories expended. While fat free mass (FFM) can explain approximately 80% of the variance in 24 h energy expenditure (EE) [1], there is still considerable variability in
energy requirements between individuals. In adults, EE is generally correlated with FFM, but there is significant variability between individuals with similar FFM, that cannot be explained by age, BMI, sex or any other phenotypic characteristic. The 20% variation in EE has a potentially tremendous impact on the prevalence and severity of obesity in the general population. In spite of this, understanding the underlying biology of this difference, identification of novel genes
Abbreviations: BAT, brown adipose tissue; BMI, body mass index; DXA, dual x-ray absorptiometry; EE, energy expenditure; FFM, fat free mass; FM, fat mass; FNDC5, fibronectin type III domain containing 5; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; RQ, respiratory quotient; VO2, volume oxygen consumed (liters/min); VCO2, volume CO2 expired (liters/min). Clinical Trials Registration Number: NCT01729143. ⁎ Corresponding author. Tel.: +1 704 250 5015; fax: + 1 704 250 5001. E-mail address: [email protected] (A.G. Swick). 0026-0495/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.metabol.2013.02.012
M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 2 (2 0 1 3) 1 07 0 –1 0 73
regulating EE and development of effective therapies to modulate EE have been elusive. One potential biological source of variable EE is brown adipose tissue (BAT) which contributes to EE in some animals, including small rodents such as mice and rats. Although BAT is present in newborn humans, its existence and function in adult humans have been debated [2]. Recent reports using positron emission tomography suggest the presence of active BAT in some, but not all adults [3,4]. Furthermore, there appears to be a correlation between the presence of active BAT and increased EE in response to stimuli such as cold exposure and consumption of capsaicin [5,6]. The regulation of BAT in humans is poorly understood, however Bostrom et al. recently reported the discovery of irisin, a myokine that stimulates the development of BAT and beige adipose tissue in mice and in cell culture [7,8]. Levels of circulating irisin increase in response to chronic exercise in mice [9] and humans [10] and administration of irisin to mice results in increased EE [9]. Furthermore, irisin levels are reported to be decreased in patients with type 2 diabetes, while positively correlated with BMI, fat mass and muscle mass across a very broad spectrum of body weight [11,12] We hypothesized that irisin action could contribute to and account for the differential in EE in individuals whose EE is greater than predicted by FFM, since the BAT component of EE is not accounted for by calculations using FFM.
2.
Methods and procedures
2.1.
Study subjects
The study subjects were post-menopausal women with BMI between 24 and 45. Other inclusion criteria included age between 50 and 70 years; no illness or medical condition that may affect the results (e.g. diabetes); does not exercise heavily (defined as > 150 min/week for greater than 3 months).
2.2.
Fat free mass and predicted energy expenditure
Body composition (fat mass and fat free mass (FFM)) was determined via dual energy x-ray absorptiometry (DXA) (GE Lunar iDXA; Milwaukee, WI). Resting Metabolic Rate (RMR) was calculated using an FFM-based equation [418 + (20.3*FFM)] [13]. Predicted 24 h EE was calculated as RMRX 1.3 (physical activity level (PAL)) whilst in the metabolic chamber.
2.3.
Indirect calorimetry
Indirect calorimetry was conducted using the metabolic chamber located at the UNC Nutrition Research Institute, Kannapolis, NC. The chamber was modelled on chambers at the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD [14], and is an open-circuit, pull-type, whole room indirect calorimeter built with walk-in cooler panels. This chamber and the reproducibility of the data generated have been previously described [15].
2.4.
Metabolic chamber study day protocol
At approximately 0730 h subjects reported to the metabolic chamber following an overnight fast (no food or beverage from
1071
2200 h). Subjects were instructed to avoid exercise the day before entering the chamber, and to not consume alcohol or caffeine during the two days prior to each study day. Before entering the chamber, subjects were weighed. At 0800 h, subjects were sealed in the chamber. Except for a 2-min interval each hour during which subjects were requested to stand and stretch, subjects were asked to remain seated or reclined, but awake throughout the study day. Subjects were asked to perform necessary daily activities such as using the restroom during these 2-min intervals where possible. Breakfast (at 0900 h), lunch (at 1330 h), and dinner (at 1700 h), were served through an air-lock passage. Subjects were instructed to finish each meal within 30 min. At 1400 h subjects were asked to place one of their arms through an iris port for blood sampling. Subjects were requested to prepare for bed at 2200 h, and at 2230 h, lights were turned off. Subjects were woken at 0630 h and at 0715 h, subjects exited the chamber and were weighed.
2.5.
Irisin immunoprecipitations
Plasma samples (500 μg) were immunoprecipitated with one microgram anti-FNDC5 antibody (Acris Antibodies, San Diego CA) and 15 μL of a 50% slurry of protein A agarose CL-4B (Sigma, St. Louis MO) overnight at 4 °C. Agarose pellets were washed and irisin eluted with 25 μL Laemmli sample buffer. Irisin was resolved by SDS-PAGE (Bio-Rad, Hercules CA), transferred to nitrocellulose, blocked for 1 h and immunoblotted overnight at 4 °C with the same anti-FNDC5 antibody diluted 1:200 in Odyssey block buffer. The blot was washed and incubated for one hour at room temperature with goat anti-rabbit IgG IRDye 700DX antibody (Rockland Immunochemicals, Gilbertsville PA) diluted 1:10,000 in Odyssey block. After washing, bands were visualized on an Odyssey fluorescence imaging system (LI-COR, Lincoln NE). Irisin bands were quantitated using Odyssey software and integrated intensities were normalized to millilitres plasma (II/ml). Linear regressions, goodness of fit (R2) and p-values were determined with GraphPad Prism software. Plasma leptin levels were determined by a Luminex assay (EMD Millipore, Billerica, MA) and plasma adiponectin was assessed by an ELISA (Mercodia, Uppsala, Sweden).
3.
Results
There was significant correlation between irisin levels and EE/ kg FFM/h for subjects whose EE was greater than predicted by the FFM-based equation [418 + (20.3*FFM)] [13]. (p = 0.0016) (Fig. 1, open circles). In contrast, subjects whose EE was equal to or less than predicted by FFM exhibited no correlation with irisin levels (Fig. 1, closed circles). Furthermore, mean total 24 h measured EE was significantly higher (208 kcal) in the correlated (+C) group as compared with group for which there was no correlation (−C) (mean ± sd: 1808 ± 244 vs. 1600 ± 57; p = 0.033). There was no significant correlation between EE/kg/ FFM and plasma levels of leptin (Fig. 2A) or adiponectin (Fig. 2B). No significant difference existed in age (60.9 ± 4.7 vs. 59.9 ± 5.9; p = 0.68) between the two sub-groups respectively. Additionally, groups were comparable for indices of body composition, with no significant differences observed in BMI (kg/m2) (32.9 ± 5.9 vs.
1072
M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 2 (2 0 1 3) 1 07 0–1 07 3
significant difference was observed in % activity during the metabolic chamber study day between the subgroup that exhibited a correlation between irisin levels and energy expenditure (8.3 ± 4.2) or the other subgroup (7.9 ± 3.5) (p = 0.84).
4.
Fig. 1 – Irisin sensitivity is associated with increased EE in humans. Irisin levels correlated with EE (as measured in the indirect room calorimeter) in subjects whose EE was greater than predicted by the FFM equation (open circles) but not in subjects whose EE was predicted by the FFM equation (filled circles). Circles represent individual subjects (postmenopausal overweight women). Irisin (expressed as integrated intensity/mL plasma) was measured by western blot. P-values less than 0.05 suggest the slopes are significantly non-zero (GraphPad Prism). EE, energy expenditure; FFM, fat free mass; II, integrated intensity; kcal, Calorie.
28.4 ± 3.3; p = 0.075), FFM (%) (51.3 ± 3.4 vs. 53.5 ± 4.0; p = 0.24), fat mass (%) (48.6 ± 3.4 vs. 46.4 ± 4.1; p = 0.24) or volume (cm3) of visceral adipose tissue (280.2 ± 138.6 vs. 231.4 ± 70.6; p = 0.385) between the subgroups, respectively. Circulating irisin concentrations (ll/mg) were comparable between both sub-groups (42.4 ± 8.9 vs. 48.0 ± 10.9; respectively; p = 0.265). Lastly, no
Discussion
We measured plasma irisin levels in similar subjects with a range of EE (kcal) as determined by 24 h indirect room calorimetry. There was a significant correlation between irisin levels and EE/kg/FFM for subjects whose EE was greater than predicted (p = 0.0016). For subjects whose EE was equal to or less than predicted by FFM there was not a significant correlation with irisin levels (Fig. 1). The mean and range of plasma irisin levels were not different between the two subgroups (42.4 ± 8.9 vs. 48.0 ± 10 II/ml of plasma; p = 0.265). Furthermore, total 24 h measured EE was significantly higher in the subgroup with correlated irisin levels and EE versus the other subgroup, despite no significant differences in FFM, fat mass, age etc. Interestingly, there was no correlation between EE and plasma leptin which is known to increase EE [16] or adiponectin which has been reported to suppress [17] or increase EE [18,19]. The lack of correlation with leptin may be attributable to decreased leptin responsiveness in obesity [20]. Therefore maximal effects of leptin on EE may occur at a low level of leptin and additional leptin may not further drive EE. Since irisin stimulates BAT, we propose that BAT thermogenesis may explain the differences in EE between the two groups and thus contribute to the differential in EE that is not accounted for by FFM. It was recently shown that muscle expression of FNDC5 correlated with PGC-1α expression and peak VO2 consumption in a ‘high aerobic performance’ group of heart failure (HF) patients while no such correlation was observed in a ‘low aerobic performance’ group of HF patients [21]. This suggests that peak VO2 might correlate to plasma irisin levels in a sub-group (i.e. high performance) of HF patients; however plasma irisin levels were not measured. It may turn out that plasma irisin levels are the same, and high versus low aerobic performance was due to differential irisin responsiveness. Although additional studies will be required to determine the extent and significance of irisin “responsiveness”, it will be important to understand whether irisin “resistance” exists and contributes to obesity. Increasing irisin responsiveness may be a promising avenue for the prevention and treatment of obesity.
Author contributions Andrew G. Swick designed the study and was the primary author of the manuscript. Annalouise O’Connor and Stephen Orena conducted the experiment and contributed to the writing of the manuscript. Fig. 2 – Neither leptin nor adiponectin correlates with energy expenditure. Energy expenditure (per kg FFM/h) did not correlate with plasma leptin (A) or adiponectin (B). P-values less than 0.05 suggest the slopes are significantly non-zero (GraphPad Prism). EE, energy expenditure; FFM, fat free mass; II, integrated intensity; kcal, Calorie.
Conflict of interest There are no disclosures to reveal with respect to this manuscript.
M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 2 (2 0 1 3) 1 07 0 –1 0 73
REFERENCES [11] [1] Ravussin E, Lillioja S, Anderson TE, et al. Determinants of 24hour energy expenditure in man. Methods and results using a respiratory chamber. J Clin Invest 1986;78(6):1568–78. [2] Enerbäck S. Human brown adipose tissue. Cell Metab 2010;11(4):248–52. [3] Yoneshiro T, Aita S, Matsushita M, et al. Brown adipose tissue, whole-body energy expenditure, and thermogenesis in healthy adult men. Obesity 2011;19(1):13–6. [4] van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM, et al. Cold-activated brown adipose tissue in healthy men. N Engl J Med 2009;360(15):1500–8. [5] Yoneshiro T, Aita S, Kawai Y, et al. Nonpungent capsaicin analogs (capsinoids) increase energy expenditure through the activation of brown adipose tissue in humans. Am J Clin Nutr 2012;95(4):845–50. [6] Garofalo C, Surmacz E. Leptin and cancer. J Cell Physiol 2006;207(1):12–22. [7] Qin B, Dawson H, Anderson RA. Elevation of tumor necrosis factor-α induces the overproduction of postprandial intestinal apolipoprotein b48-containing very low-density lipoprotein particles: evidence for related gene expression of inflammatory, insulin and lipoprotein signaling in enterocytes. Exp Biol Med 2010;235(2):199–205. [8] Wu J, Boström P, Sparks Lauren M, et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 2012;150(2):366–76. [9] Bostrom P, Wu J, Jedrychowski MP, et al. A pgc1-[agr]dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 2012 [advance online publication]. [10] Huh JY, Panagiotou G, Mougios V, et al. Fndc5 and irisin in humans: I. Predictors of circulating concentrations in serum and plasma and ii. Mrna expression and circulating concen-
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trations in response to weight loss and exercise. Metabolism 2012. Choi Y-K, Kim M-K, Bae KH, et al. Serum irisin levels in newonset type 2 diabetes. Diabetes Research and Clinical Practice (0). Stengel A, Hofmann T, Goebel-Stengel M, et al. Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity — correlation with body mass index. Peptides 2013;39:125–30. Arciero PJ, Goran MI, Poehlman ET. Resting metabolic rate is lower in women than in men. J Appl Physiol 1993;75(6): 2514–20. Brychta RJ, Rothney MP, Skarulis MC, et al. Optimizing energy expenditure detection in human metabolic chambers. Conf Proc IEEE Eng Med Biol Soc 2009;2009:6864–8. Knab AM, Shanely RA, Corbin KD, et al. A 45-minute vigorous exercise bout increases metabolic rate for 14 hours. Med Sci Sports Exerc 2011;43(9):1643–8. Leibel RL. Molecular physiology of weight regulation in mice and humans. Int J Obes (Lond) 2008;32(Suppl 7):S98–S108. Kubota N, Yano W, Kubota T, et al. Adiponectin stimulates amp-activated protein kinase in the hypothalamus and increases food intake. Cell Metab 2007;6(1):55–68. Qi Y, Takahashi N, Hileman SM, et al. Adiponectin acts in the brain to decrease body weight. Nat Med 2004;10(5):524–9. Park S, Kim DS, Kwon DY, et al. Long-term central infusion of adiponectin improves energy and glucose homeostasis by decreasing fat storage and suppressing hepatic gluconeogenesis without changing food intake. J Neuroendocrinol 2011;23(8):687–98. Myers Jr Martin G, Heymsfield Steven B, Haft C, et al. Challenges and opportunities of defining clinical leptin resistance. Cell Metab 2012;15(2):150–6. Lecker SH, Zavin A, Cao P, et al. Expression of the irisin precursor fndc5 in skeletal muscle correlates with aerobic exercise performance in patients with heart failure. Circ Heart Fail 2012.
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Clinical Science
Irisin levels correlate with energy expenditure in a subgroup of humans with energy expenditure greater than predicted by fat free mass Andrew G. Swick⁎, Stephen Orena, Annalouise O’Connor UNC Nutrition Research Institute, University of North Carolina Chapel Hill, North Carolina Research Campus, Kannapolis, North Carolina, USA
A R T I C LE I N FO Article history:
AB S T R A C T Objective. Obesity is a result of chronic overconsumption of calories relative to the amount of
Received 7 December 2012
energy expended. While fat free mass can account for ~80% of the variance in energy expenditure,
Accepted 27 February 2013
there is still considerable variability in energy requirements between individuals that cannot be
Keywords:
which has been touted to increase energy expenditure via activation of brown adipocytes in
FNDC5
rodents and possibly humans, may explain some of the variability in energy expenditure.
explained. We hypothesized that responsiveness to the recently discovered myokine, irisin,
Materials/methods. Post-menopausal women (n = 17) spent 24-h in a whole room indirect
BAT Indirect calorimetry
calorimeter. During the study day, subjects remained sedentary and consumed meals
RQ
tailored to their energy requirements. Plasma irisin, leptin and adiponectin were measured in samples taken from each subject. Results. Our results suggest that in general, irisin levels do not correlate with 24-h energy expenditure, however, for a subpopulation irisin levels and energy expenditure are highly correlative. Conclusion. Irisin may help explain some of the observed variability in individual energy requirements that cannot be accounted for by fat free mass. Therefore, interventions designed to increase irisin action may prove to be promising avenues for the treatment of obesity. © 2013 Elsevier Inc. All rights reserved.
1.
Introduction
Obesity is an ever-increasing, worldwide health concern associated with a significant economic burden. Obesity results from chronic overconsumption of calories (kcal) relative to the amount of calories expended. While fat free mass (FFM) can explain approximately 80% of the variance in 24 h energy expenditure (EE) [1], there is still considerable variability in
energy requirements between individuals. In adults, EE is generally correlated with FFM, but there is significant variability between individuals with similar FFM, that cannot be explained by age, BMI, sex or any other phenotypic characteristic. The 20% variation in EE has a potentially tremendous impact on the prevalence and severity of obesity in the general population. In spite of this, understanding the underlying biology of this difference, identification of novel genes
Abbreviations: BAT, brown adipose tissue; BMI, body mass index; DXA, dual x-ray absorptiometry; EE, energy expenditure; FFM, fat free mass; FM, fat mass; FNDC5, fibronectin type III domain containing 5; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; RQ, respiratory quotient; VO2, volume oxygen consumed (liters/min); VCO2, volume CO2 expired (liters/min). Clinical Trials Registration Number: NCT01729143. ⁎ Corresponding author. Tel.: +1 704 250 5015; fax: + 1 704 250 5001. E-mail address: [email protected] (A.G. Swick). 0026-0495/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.metabol.2013.02.012
M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 2 (2 0 1 3) 1 07 0 –1 0 73
regulating EE and development of effective therapies to modulate EE have been elusive. One potential biological source of variable EE is brown adipose tissue (BAT) which contributes to EE in some animals, including small rodents such as mice and rats. Although BAT is present in newborn humans, its existence and function in adult humans have been debated [2]. Recent reports using positron emission tomography suggest the presence of active BAT in some, but not all adults [3,4]. Furthermore, there appears to be a correlation between the presence of active BAT and increased EE in response to stimuli such as cold exposure and consumption of capsaicin [5,6]. The regulation of BAT in humans is poorly understood, however Bostrom et al. recently reported the discovery of irisin, a myokine that stimulates the development of BAT and beige adipose tissue in mice and in cell culture [7,8]. Levels of circulating irisin increase in response to chronic exercise in mice [9] and humans [10] and administration of irisin to mice results in increased EE [9]. Furthermore, irisin levels are reported to be decreased in patients with type 2 diabetes, while positively correlated with BMI, fat mass and muscle mass across a very broad spectrum of body weight [11,12] We hypothesized that irisin action could contribute to and account for the differential in EE in individuals whose EE is greater than predicted by FFM, since the BAT component of EE is not accounted for by calculations using FFM.
2.
Methods and procedures
2.1.
Study subjects
The study subjects were post-menopausal women with BMI between 24 and 45. Other inclusion criteria included age between 50 and 70 years; no illness or medical condition that may affect the results (e.g. diabetes); does not exercise heavily (defined as > 150 min/week for greater than 3 months).
2.2.
Fat free mass and predicted energy expenditure
Body composition (fat mass and fat free mass (FFM)) was determined via dual energy x-ray absorptiometry (DXA) (GE Lunar iDXA; Milwaukee, WI). Resting Metabolic Rate (RMR) was calculated using an FFM-based equation [418 + (20.3*FFM)] [13]. Predicted 24 h EE was calculated as RMRX 1.3 (physical activity level (PAL)) whilst in the metabolic chamber.
2.3.
Indirect calorimetry
Indirect calorimetry was conducted using the metabolic chamber located at the UNC Nutrition Research Institute, Kannapolis, NC. The chamber was modelled on chambers at the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD [14], and is an open-circuit, pull-type, whole room indirect calorimeter built with walk-in cooler panels. This chamber and the reproducibility of the data generated have been previously described [15].
2.4.
Metabolic chamber study day protocol
At approximately 0730 h subjects reported to the metabolic chamber following an overnight fast (no food or beverage from
1071
2200 h). Subjects were instructed to avoid exercise the day before entering the chamber, and to not consume alcohol or caffeine during the two days prior to each study day. Before entering the chamber, subjects were weighed. At 0800 h, subjects were sealed in the chamber. Except for a 2-min interval each hour during which subjects were requested to stand and stretch, subjects were asked to remain seated or reclined, but awake throughout the study day. Subjects were asked to perform necessary daily activities such as using the restroom during these 2-min intervals where possible. Breakfast (at 0900 h), lunch (at 1330 h), and dinner (at 1700 h), were served through an air-lock passage. Subjects were instructed to finish each meal within 30 min. At 1400 h subjects were asked to place one of their arms through an iris port for blood sampling. Subjects were requested to prepare for bed at 2200 h, and at 2230 h, lights were turned off. Subjects were woken at 0630 h and at 0715 h, subjects exited the chamber and were weighed.
2.5.
Irisin immunoprecipitations
Plasma samples (500 μg) were immunoprecipitated with one microgram anti-FNDC5 antibody (Acris Antibodies, San Diego CA) and 15 μL of a 50% slurry of protein A agarose CL-4B (Sigma, St. Louis MO) overnight at 4 °C. Agarose pellets were washed and irisin eluted with 25 μL Laemmli sample buffer. Irisin was resolved by SDS-PAGE (Bio-Rad, Hercules CA), transferred to nitrocellulose, blocked for 1 h and immunoblotted overnight at 4 °C with the same anti-FNDC5 antibody diluted 1:200 in Odyssey block buffer. The blot was washed and incubated for one hour at room temperature with goat anti-rabbit IgG IRDye 700DX antibody (Rockland Immunochemicals, Gilbertsville PA) diluted 1:10,000 in Odyssey block. After washing, bands were visualized on an Odyssey fluorescence imaging system (LI-COR, Lincoln NE). Irisin bands were quantitated using Odyssey software and integrated intensities were normalized to millilitres plasma (II/ml). Linear regressions, goodness of fit (R2) and p-values were determined with GraphPad Prism software. Plasma leptin levels were determined by a Luminex assay (EMD Millipore, Billerica, MA) and plasma adiponectin was assessed by an ELISA (Mercodia, Uppsala, Sweden).
3.
Results
There was significant correlation between irisin levels and EE/ kg FFM/h for subjects whose EE was greater than predicted by the FFM-based equation [418 + (20.3*FFM)] [13]. (p = 0.0016) (Fig. 1, open circles). In contrast, subjects whose EE was equal to or less than predicted by FFM exhibited no correlation with irisin levels (Fig. 1, closed circles). Furthermore, mean total 24 h measured EE was significantly higher (208 kcal) in the correlated (+C) group as compared with group for which there was no correlation (−C) (mean ± sd: 1808 ± 244 vs. 1600 ± 57; p = 0.033). There was no significant correlation between EE/kg/ FFM and plasma levels of leptin (Fig. 2A) or adiponectin (Fig. 2B). No significant difference existed in age (60.9 ± 4.7 vs. 59.9 ± 5.9; p = 0.68) between the two sub-groups respectively. Additionally, groups were comparable for indices of body composition, with no significant differences observed in BMI (kg/m2) (32.9 ± 5.9 vs.
1072
M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 2 (2 0 1 3) 1 07 0–1 07 3
significant difference was observed in % activity during the metabolic chamber study day between the subgroup that exhibited a correlation between irisin levels and energy expenditure (8.3 ± 4.2) or the other subgroup (7.9 ± 3.5) (p = 0.84).
4.
Fig. 1 – Irisin sensitivity is associated with increased EE in humans. Irisin levels correlated with EE (as measured in the indirect room calorimeter) in subjects whose EE was greater than predicted by the FFM equation (open circles) but not in subjects whose EE was predicted by the FFM equation (filled circles). Circles represent individual subjects (postmenopausal overweight women). Irisin (expressed as integrated intensity/mL plasma) was measured by western blot. P-values less than 0.05 suggest the slopes are significantly non-zero (GraphPad Prism). EE, energy expenditure; FFM, fat free mass; II, integrated intensity; kcal, Calorie.
28.4 ± 3.3; p = 0.075), FFM (%) (51.3 ± 3.4 vs. 53.5 ± 4.0; p = 0.24), fat mass (%) (48.6 ± 3.4 vs. 46.4 ± 4.1; p = 0.24) or volume (cm3) of visceral adipose tissue (280.2 ± 138.6 vs. 231.4 ± 70.6; p = 0.385) between the subgroups, respectively. Circulating irisin concentrations (ll/mg) were comparable between both sub-groups (42.4 ± 8.9 vs. 48.0 ± 10.9; respectively; p = 0.265). Lastly, no
Discussion
We measured plasma irisin levels in similar subjects with a range of EE (kcal) as determined by 24 h indirect room calorimetry. There was a significant correlation between irisin levels and EE/kg/FFM for subjects whose EE was greater than predicted (p = 0.0016). For subjects whose EE was equal to or less than predicted by FFM there was not a significant correlation with irisin levels (Fig. 1). The mean and range of plasma irisin levels were not different between the two subgroups (42.4 ± 8.9 vs. 48.0 ± 10 II/ml of plasma; p = 0.265). Furthermore, total 24 h measured EE was significantly higher in the subgroup with correlated irisin levels and EE versus the other subgroup, despite no significant differences in FFM, fat mass, age etc. Interestingly, there was no correlation between EE and plasma leptin which is known to increase EE [16] or adiponectin which has been reported to suppress [17] or increase EE [18,19]. The lack of correlation with leptin may be attributable to decreased leptin responsiveness in obesity [20]. Therefore maximal effects of leptin on EE may occur at a low level of leptin and additional leptin may not further drive EE. Since irisin stimulates BAT, we propose that BAT thermogenesis may explain the differences in EE between the two groups and thus contribute to the differential in EE that is not accounted for by FFM. It was recently shown that muscle expression of FNDC5 correlated with PGC-1α expression and peak VO2 consumption in a ‘high aerobic performance’ group of heart failure (HF) patients while no such correlation was observed in a ‘low aerobic performance’ group of HF patients [21]. This suggests that peak VO2 might correlate to plasma irisin levels in a sub-group (i.e. high performance) of HF patients; however plasma irisin levels were not measured. It may turn out that plasma irisin levels are the same, and high versus low aerobic performance was due to differential irisin responsiveness. Although additional studies will be required to determine the extent and significance of irisin “responsiveness”, it will be important to understand whether irisin “resistance” exists and contributes to obesity. Increasing irisin responsiveness may be a promising avenue for the prevention and treatment of obesity.
Author contributions Andrew G. Swick designed the study and was the primary author of the manuscript. Annalouise O’Connor and Stephen Orena conducted the experiment and contributed to the writing of the manuscript. Fig. 2 – Neither leptin nor adiponectin correlates with energy expenditure. Energy expenditure (per kg FFM/h) did not correlate with plasma leptin (A) or adiponectin (B). P-values less than 0.05 suggest the slopes are significantly non-zero (GraphPad Prism). EE, energy expenditure; FFM, fat free mass; II, integrated intensity; kcal, Calorie.
Conflict of interest There are no disclosures to reveal with respect to this manuscript.
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