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The energy balance positively regulates the levels of circulating TNF-related apoptosis inducing ligand in humans
Clinical Nutrition 31 (2012) 1018e1021
Contents lists available at SciVerse ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Short communication
The energy balance positively regulates the levels of circulating TNF-related apoptosis inducing ligand in humans Gianni Biolo a, d, Paola Secchiero b, d, Sara De Giorgi a, Veronica Tisato b, Giorgio Zauli c, * a
Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy Department of Morphology and Embryology and LTTA Centre, University of Ferrara, Ferrara, Italy c Institute for Maternal and Child Health IRCCS “Burlo Garofolo”, Via dell’Istria 65/1, 34137 Trieste, Italy b
a r t i c l e i n f o
s u m m a r y
Article history: Received 3 February 2012 Accepted 27 April 2012
Background & aims: Although decreased levels of circulating TRAIL have been associated to cardiovascular risk and overall mortality, the mechanisms controlling TRAIL levels in physiopathological conditions are currently unknown. The aim of the present study was to investigate whether changes in the energy intake and insulin sensitivity may influence circulating TRAIL, and to analyze potential relationships between circulating TRAIL and changes in fat mass in healthy subjects receiving hypocaloric or hypercaloric diets. Methods: Three distinct groups of participants were studied, at the end of a 14-day (n ¼ 9), 35-day (n ¼ 30) or 60-day (n ¼ 16) period of experimental bed rest to induce insulin resistance and during controlled ambulation, after receiving eucaloric, hypocaloric or hypercaloric diets. Results: After bed rest conditions, energy restriction significantly decreased circulating TRAIL, while overfeeding significantly increased TRAIL levels with respect to eucaloric control subjects. Moreover, a positive correlation was found between levels of circulating TRAIL and energy intake as well as between circulating TRAIL and energy balance, as determined by changes in fat mass in these subjects. Conclusions: Circulating levels of TRAIL exhibit a clear-cut positive correlation with the energy intake and balance in healthy subjects during experimental physical inactivity. Ó 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: TRAIL Energy balance Bed rest
1. Introduction TNF-related apoptosis inducing ligand (TRAIL) is a TNF family member either expressed by several cell types as a type II transmembrane protein or, similarly to other membrane-bound ligands of the TNF superfamily, as a soluble protein, which is detectable in the plasma/serum under physiological conditions.1 Although the best characterized biological activity of TRAIL, also known as Apo2 ligand, is represented by a potent induction of apoptosis in a variety of cancer cell types, accumulating evidence has shown that TRAIL plays multiple non-apoptotic functions in a variety of tissues, including the vascular system.1 It is also noteworthy that while the induction of apoptosis in cancer cells usually requires high concentrations of recombinant soluble
Abbreviations: TRAIL, TNF-related apoptosis inducing ligand. * Corresponding author. Tel.: þ39 0 40 378478. E-mail address: [email protected] (G. Zauli). d The first two authors equally contributed to this work.
TRAIL (>10 ng/ml), TRAIL activates intracellular signal transduction pathways involved in cell survival, migration and proliferation in a variety of normal cells at lower concentrations (10e100 pg/ml),1 comparable to those found physiologically in serum or plasma.2e5 Although it has been shown that circulating TRAIL levels are inversely related to the risk of mortality in patients affected by cardiovascular disease,2,3 only few studies have attempted to correlate circulating TRAIL with body adiposity and serum lipid levels.4,5 In one of these studies,4 increased levels of TRAIL were associated with greater body fat and LDL cholesterol as well as with diminished lean body mass. It is presently unknown, however, whether changes in energy balance, adipose tissue and skeletal muscle mass may affect the levels of circulating TRAIL. On these bases, the aim of the present study was to evaluate whether the levels of soluble TRAIL in humans can be modulated in response to specific interventions that affect the energy balance in healthy volunteers, kept for different time periods under overfeeding or energy restriction conditions combined with experimental bed rest to induce changes in muscle mass.
0261-5614/$ e see front matter Ó 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2012.04.016
G. Biolo et al. / Clinical Nutrition 31 (2012) 1018e1021
2. Materials and methods 2.1. Subjects The study populations comprise three different groups of healthy volunteers who have been previously characterized in studies aimed to investigate the effect of different periods of either calorie restriction or overfeeding coupled to experimental bed rest on the lean and fatty body mass.6e9 All subjects had a normal body mass index and underwent a series of clinical and biochemical evaluations, which have been previously described.6e9 Briefly, group 1 was composed by 16 healthy females aged 31 4 years, body mass index 21 2 kg/m2, who were enrolled at MEDES Clinical Research Facility of the Rangueil University Hospital (Toulouse, France).6 Group 2 was composed by 30 healthy males aged 23 0.4 years, body mass index 24 0.4 kg/m2, who were enrolled for the present study at the Valdoltra Hospital, University of Primorska (Ankaran-Capodistria, Slovenia).7,8 Group 3 was composed by 9 healthy males aged 24 1 years, body mass index 23 1 kg/m2, who were enrolled for the present study at the Clinical Research Center of the German Aerospace Institute (Cologne, Germany).9 All subjects were physically active before the study. Each subject signed an informed consent form upon admission. The study was performed in accordance with the Declaration of Helsinki for human studies and relative amendments. 2.2. Experimental design In order to investigate the potential contribution of body fat, energy balance, muscle mass and insulin sensitivity in regulating the levels of circulating TRAIL, we have used a well-characterized bed rest protocol as a model of experimental muscle atrophy and insulin resistance in humans. In particular, we took advantage of previous studies performed on healthy volunteers, who underwent experimental bed rest for 60 days (group 1, n ¼ 16), 35 days (group 2, n ¼ 30) and 14 days (group 3, n ¼ 9) at different levels of energy intake (calorie restriction, near-neutral energy balance, overfeeding) according to different experimental protocols.6e9 Energy balance was calculated according to changes in fat mass during the experimental periods. Fat mass was determined by dual-emission X-ray absorptiometry (DXA) in group 1 and 3 and by bioimpedance in group 2. Subjects of group 1 were randomized to complete 60 days of strict bed rest (n ¼ 8) or to combine bed rest with daily resistance or aerobic exercise routines in supine position (n ¼ 8), energy balance resulted near-neutral and slightly negative in the two sub-groups, respectively.6 Subjects of group 2 completed 35 days of strict bed rest at different levels of energy intakes.7 Nineteen out of the 30 subjects of group 2 were randomized to combine strict bed rest with overfeeding (n ¼ 10) or with near-adequate energy intake (n ¼ 9) according to a parallel design.8 Subjects of group 3 have been studied 4 times for 14-day periods over 2 years in bed rest or in ambulatory condition in combination with eucaloric or hypocaloric diets using a crossover experimental design. Energy intakes were individually tailored to account for the decrease in requirement during bed rest and then decreased by about 20% during the hypocaloric periods.9 2.3. Analyses Insulin, glucose and leptin concentrations were measured by laboratory analyses as referenced.6e9 Body fat mass was determined by DEXA or bioimpedance, as previously described.6e9 Insulin sensitivity was calculated according to the formula of the
1019
homeostasis model assessment (HOMA-IR index) method: insulin resistance ¼ fasting plasma insulin (mUI/ml) fasting plasma glucose (mmol/l)/22.5. The index is highly correlated with the insulin resistance index assessed by the euglycemichyperinsulinemic clamp, which is the gold standard of insulin resistance measurement and is widely adopted in clinical studies for individuals with various degree of insulin sensitivity. Plasma TRAIL was measured in duplicate by using a specific, commercially available ELISA kit, which detect total TRAIL levels (both free and bound to other proteins, such as osteoprotegerin) in accordance with the manufacturer’s instructions (R&D Systems, Minneapolis, MN) in frozen (80 C) aliquots of plasma, obtained at the time of the original studies.6e9 It is important to point out that the present study was possible thanks to the good stability of circulating TRAIL in plasma samples correctly frozen 80 C at over periods of many years.1 Sensitivity of the assay was 2.86 pg/ml and the intra- and inter-assay coefficients of variation were 3.9% and 6%, respectively. 2.4. Statistics The study populations comprise 3 different groups who were kept for different time periods at different energy levels under bed rest or controlled physical activity conditions, as previously described.6e9 In the first analysis, the results obtained from the 3 groups of subjects were pooled together. One observation was obtained from each subject of group 1 and 2 while four observations were obtained from each subjects of group 3. The levels of circulating TRAIL were cumulatively considered in relationship with the data relative to change of fat mass over experimental periods of different durations. Pearson’s tests were used to investigate association between variables. To evaluate the effect of bed rest at different energy intake levels, results were analyzed in group 1 by repeated-measures ANOVA with interactions.6 In the parallel group design study performed in group 2,7,8 activity (ambulatory or bed rest) and treatment (higher or lower energy intake) were the within-subject and between-subject factors, respectively. In the crossover design study performed in group 3,9 activity (ambulatory or bed rest) and treatment (adequate or lower energy intake) were the withinsubject factors. Since there was no significant gender or bed rest effects on TRAIL circulating levels, results obtained during the different studies, in ambulatory or bed rest conditions, were pooled together, expressed as means SD and analyzed by paired Student t-test or Mann Withney test where appropriate. P < 0.05 was considered statistically significant. 3. Results 3.1. The circulating levels of TRAIL exhibit a significant correlation with changes of fat mass, but not of insulin sensitivity or lean mass, in healthy subjects kept in experimental physical inactivity In the first analysis, the levels of circulating TRAIL of subjects from group 1, 2 and 3 were cumulatively considered in relation with the data relative to change of fat mass over experimental periods of different durations.6e9 Pre-bed rest values of TRAIL concentrations were not significantly different in male (104 21 pg/ml) and female (94 19 pg/ml) subjects. As shown in Supplemental Fig. 1, a significant (n ¼ 82; R ¼ 0.34; p < 0.001) positive correlation was noticed between levels of circulating TRAIL at the end of the experimental period and energy balance, as determined by individual changes of fat mass (kg) over the experimental period. Insulin sensitivity significantly decreased (groups 1 and 2) after 35 and 60 days of bed rest (HOMA-IR index: from 1.37 0.61 to
1020
G. Biolo et al. / Clinical Nutrition 31 (2012) 1018e1021
Table 1 Effects of energy restriction at different levels of physical activity on soluble TRAIL. Ambulatory Eucaloric Energy intake (kcal/kg/day)b Delta fat mass (kg/14 days) Delta leptin (ng/ml) TRAIL (pg/ml)
45 0.10 0.3 103
3 1.01 1.3 18
Pa
Bed rest (14 days) Hypocaloric 37 1.02 0.9 101
4 0.96 1.3 20
Eucaloric 36 0.28 0.8 102
Hypocaloric
2 0.64 2.2 12
30 1.00 1.2 86
2 0.76 2.5 15
Diet effect
Bed rest effect
Interaction
<0.001 0.01 0.04 0.04
<0.001 0.35 0.37 0.12
0.08 0.24 0.53 0.06
Circulating TRAIL was measured in the post-absorptive state at the end of 14 day periods of bed rest and controlled ambulation in 9 healthy, male young volunteers subjects receiving eucaloric or hypocaloric (80% of total energy expenditure) diets according with a crossover design. Energy intake in bed rest was adapted to decreased requirement. Data are expressed as means SD. a Data were analyzed with the use of repeated-measures ANOVA with activity (ambulatory or bed rest) and diet (eucaloric or hypocaloric) as the 2 factors. b Energy intake is normalized by kg of lean body mass as determined by dual-emission X-ray absorptiometry.
1.80 0.90, plasma insulin: from 6.2 2.8 to 8.1 4.0 U/ml; n ¼ 46, p < 0.001 paired t-test) while body fat did not significantly change (from 13 5 to 13 5 kg). No significant correlation was noticed between bed rest induced changes in insulin sensitivity and levels of circulating TRAIL (HOMA-IR index, R ¼ 0.09; plasma insulin, R ¼ 0.10; n ¼ 46). Lean body mass significantly (p < 0.001) decreased (group 1 and 2) after 35 and 60 days of bed rest (4.7 2.3%). No significant correlation was noticed between bed rest induced changes in lean body mass and levels of circulating TRAIL (R ¼ 0.02; n ¼ 46). 3.2. Physical inactivity combined with overfeeding increases while energy restriction decreases the levels of circulating TRAIL in healthy subjects In order to get insights in the relationship between circulating TRAIL levels and energy intake, we have analyzed the effect of higher or lower energy balance during physical inactivity for 35 days on the levels of circulating TRAIL in a group of young, healthy male volunteers (19 subjects from group 2). Results indicated (Table 1) that there was not significant bed rest effect on TRAIL circulating levels while there was significant bed rest group interaction leading to greater TRAIL changes in the higher energy balance group (þ14%) than in the lower energy balance group (10%) (p ¼ 0.02, Mann Withney test). In parallel, we have analyzed the effects of energy restriction at 80% of requirement in ambulatory conditions and at the end of 14 days of bed rest in another group of young, healthy male volunteers (group 3, n ¼ 9) according to a crossover experimental design.9 As previously shown, fat mass and plasma leptin were significantly (p < 0.05) decreased following energy restriction with respect to the eucaloric periods (Table 2). Of note, energy restriction in bed rest or in ambulatory conditions caused a significant (p < 0.05) decrease in the levels of circulating TRAIL (Table 2). The individual percent changes of energy intake, fat mass and soluble TRAIL from the eucaloric ambulatory period to the other three experimental conditions were calculated in each subject of this group. Of note, there was a significant (R ¼ 0.46; p < 0.001) direct correlation only
between changes in energy intake and changes in circulating TRAIL (Supplemental Fig. 2). 4. Discussion Food intake and energy homeostasis are regulated by a complex physiological network of signals arising from the hypothalamus as well as from peripheral organs, including stomach, intestine, pancreas, liver and adipose tissue. In recent years, great efforts have been made to identify and investigate the pathways involved in the control of energy metabolism. Disturbance of food intake and energy balance can lead to complex dysfunctions, characterized by either obesity or cachexia. While over-nutrition and obesity have been rapidly increasing worldwide and constitute a major social and medical problem, on the other hand, cachexia is marked by loss of weight as a result of hypercatabolic state, which is characteristic for many subacute and chronic diseases, including cancer, congestive heart failure and sepsis. In this study, we have demonstrated for the first time the existence of a positive correlation between levels of circulating TRAIL and changes in fat mass and energy intake in three distinct groups of young healthy participants under bed-rest conditions. These findings were somewhat surprising taking into account that several previous studies have demonstrated that circulating TRAIL levels are inversely related to the risk of mortality in patients affected by cardiovascular disease.2,3 In two distinct groups of healthy volunteers, 35-days of experimental overfeeding increased soluble TRAIL by about 15% while 14-days of energy restriction decreased its circulating levels by about 10%. Moreover, a significant direct correlation was noticed only between changes in energy intake and fat mass with changes in circulating TRAIL, while changes in level of insulin sensitivity did not influence soluble TRAIL. Thus, although we have not addressed the intracellular pathways regulating the production and release of TRAIL in response to metabolic changes, our data has allowed to establish for the first time a link between energy balance and levels of circulating TRAIL. Although the nature of this study does not allow to ascertain the physiopathological significance of the changes in
Table 2 Effects of overfeeding and physical inactivity on soluble TRAIL.
TRAIL (pg/ml) Leptin (ng/ml) Fat mass (kg)
Bed rest at higher energy balance
Bed rest at lower energy balance
Baseline
35 days
Baseline
35 days
108 20 3.4 3.8 12.9 7.2
119 23 6.9 8.2b 15.0 7.02
108 28 2.1 1.8 10.6 3.7
95 18 3.2 3.6 11.0 3.8
Pa activity effect
Pa interaction
0.83 0.01 <0.001
0.03 0.04 <0.001
Circulating TRAIL was measured in the post-absorptive state before and at the end of 35 days of bed rest in nineteen healthy, young male volunteers maintained at higher (n ¼ 10) or lower (n ¼ 9) energy balance. Changes in body fat in the higher or lower energy balance groups averaged 2.6 1.0 and 1.0 1.5 kg, respectively. Data are expressed as means SD. a Data were analyzed by using a 2-factor (group activity) ANOVA with interaction. There was not significant group effect. b Significantly different from the ambulatory adaptation condition, P < 0.025 (Bonferroni’s post hoc analysis).
G. Biolo et al. / Clinical Nutrition 31 (2012) 1018e1021
circulating TRAIL in response to energy intake, we and other groups of investigators have previously shown that TRAIL shows antiatherosclerotic and vasoeprotective activities in animal models.1 Therefore, it is tempting to speculate that the raise of circulating TRAIL levels might represent a compensatory mechanism aimed to protect the vascular system following increase energy intake. Our results may contribute to understand the unexpected protective effect of moderate obesity and overweight on cardiovascular outcome.10 Future studies are required in order to address at the cellular level the mechanisms involved in promoting increased TRAIL release in response to increased energy intake.
Conflict of interest The authors have no conflict of interest.
Acknowledgments Italian Association for Cancer Research (AIRC) to GZ.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clnu.2012.04.016.
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References 1. Di Pietro R, Zauli G. Emerging non-apoptotic functions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L. J Cell Physiol 2004;201:331e40. 2. Secchiero P, Corallini F, Ceconi C, Parrinello G, Volpato S, Ferrari R, et al. Potential prognostic significance of decreased serum levels of TRAIL after acute myocardial infarction. PLoS One 2009;4:e4442. 3. Volpato S, Ferrucci L, Secchiero P, Corallini F, Zuliani G, Fellin R, et al. Association of tumor necrosis factor-related apoptosis-inducing ligand with total and cardiovascular mortality in older adults. Atherosclerosis 2011;215:452e8. 4. Choi JW, Song JS, Pai SH. Associations of serum TRAIL concentrations, anthropometric variables, and serum lipid parameters in healthy adults. Ann Clin Lab Sci 2004;34:400e4. 5. Ashley DT, O’Sullivan EP, Davenport C, Devlin N, Crowley RK, McCaffrey N, et al. Similar to adiponectin, serum levels of osteoprotegerin are associated with obesity in healthy subjects. Metabolism 2011;60:994e1000. 6. Trudel G, Payne M, Mädler B, Ramachandran N, Lecompte M, Wade C, et al. Bone marrow fat accumulation after 60 days of bed rest persisted 1 year after activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study. J Appl Physiol 2009;107:540e8. 7. Mazzucco S, Agostini F, Mangogna A, Cattin L, Biolo G. Prolonged inactivity upregulates cholesteryl ester transfer protein independently of body fat changes in humans. J Clin Endocrinol Metab 2010;95:2508e12. 8. Biolo G, Agostini F, Simunic B, Sturma M, Torelli L, Preiser JC, et al. Positive energy balance is associated with accelerated muscle atrophy and increased erythrocyte glutathione turnover during 5 wk of bed rest. Am J Clin Nutr 2008;88:950e8. 9. Biolo G, Ciocchi B, Stulle M, Bosutti A, Barazzoni R, Zanetti M, et al. Calorie restriction accelerates the catabolism of lean body mass during 2 wk of bed rest. Am J Clin Nutr 2007;86:366e72. 10. Kadakia MB, Fox CS, Scirica BM, Murphy SA, Bonaca MP, Morrow DA. Central obesity and cardiovascular outcomes in patients with acute coronary syndrome: observations from the MERLIN-TIMI 36 trial. Heart 2011;97:1782e7.
Contents lists available at SciVerse ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Short communication
The energy balance positively regulates the levels of circulating TNF-related apoptosis inducing ligand in humans Gianni Biolo a, d, Paola Secchiero b, d, Sara De Giorgi a, Veronica Tisato b, Giorgio Zauli c, * a
Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy Department of Morphology and Embryology and LTTA Centre, University of Ferrara, Ferrara, Italy c Institute for Maternal and Child Health IRCCS “Burlo Garofolo”, Via dell’Istria 65/1, 34137 Trieste, Italy b
a r t i c l e i n f o
s u m m a r y
Article history: Received 3 February 2012 Accepted 27 April 2012
Background & aims: Although decreased levels of circulating TRAIL have been associated to cardiovascular risk and overall mortality, the mechanisms controlling TRAIL levels in physiopathological conditions are currently unknown. The aim of the present study was to investigate whether changes in the energy intake and insulin sensitivity may influence circulating TRAIL, and to analyze potential relationships between circulating TRAIL and changes in fat mass in healthy subjects receiving hypocaloric or hypercaloric diets. Methods: Three distinct groups of participants were studied, at the end of a 14-day (n ¼ 9), 35-day (n ¼ 30) or 60-day (n ¼ 16) period of experimental bed rest to induce insulin resistance and during controlled ambulation, after receiving eucaloric, hypocaloric or hypercaloric diets. Results: After bed rest conditions, energy restriction significantly decreased circulating TRAIL, while overfeeding significantly increased TRAIL levels with respect to eucaloric control subjects. Moreover, a positive correlation was found between levels of circulating TRAIL and energy intake as well as between circulating TRAIL and energy balance, as determined by changes in fat mass in these subjects. Conclusions: Circulating levels of TRAIL exhibit a clear-cut positive correlation with the energy intake and balance in healthy subjects during experimental physical inactivity. Ó 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: TRAIL Energy balance Bed rest
1. Introduction TNF-related apoptosis inducing ligand (TRAIL) is a TNF family member either expressed by several cell types as a type II transmembrane protein or, similarly to other membrane-bound ligands of the TNF superfamily, as a soluble protein, which is detectable in the plasma/serum under physiological conditions.1 Although the best characterized biological activity of TRAIL, also known as Apo2 ligand, is represented by a potent induction of apoptosis in a variety of cancer cell types, accumulating evidence has shown that TRAIL plays multiple non-apoptotic functions in a variety of tissues, including the vascular system.1 It is also noteworthy that while the induction of apoptosis in cancer cells usually requires high concentrations of recombinant soluble
Abbreviations: TRAIL, TNF-related apoptosis inducing ligand. * Corresponding author. Tel.: þ39 0 40 378478. E-mail address: [email protected] (G. Zauli). d The first two authors equally contributed to this work.
TRAIL (>10 ng/ml), TRAIL activates intracellular signal transduction pathways involved in cell survival, migration and proliferation in a variety of normal cells at lower concentrations (10e100 pg/ml),1 comparable to those found physiologically in serum or plasma.2e5 Although it has been shown that circulating TRAIL levels are inversely related to the risk of mortality in patients affected by cardiovascular disease,2,3 only few studies have attempted to correlate circulating TRAIL with body adiposity and serum lipid levels.4,5 In one of these studies,4 increased levels of TRAIL were associated with greater body fat and LDL cholesterol as well as with diminished lean body mass. It is presently unknown, however, whether changes in energy balance, adipose tissue and skeletal muscle mass may affect the levels of circulating TRAIL. On these bases, the aim of the present study was to evaluate whether the levels of soluble TRAIL in humans can be modulated in response to specific interventions that affect the energy balance in healthy volunteers, kept for different time periods under overfeeding or energy restriction conditions combined with experimental bed rest to induce changes in muscle mass.
0261-5614/$ e see front matter Ó 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2012.04.016
G. Biolo et al. / Clinical Nutrition 31 (2012) 1018e1021
2. Materials and methods 2.1. Subjects The study populations comprise three different groups of healthy volunteers who have been previously characterized in studies aimed to investigate the effect of different periods of either calorie restriction or overfeeding coupled to experimental bed rest on the lean and fatty body mass.6e9 All subjects had a normal body mass index and underwent a series of clinical and biochemical evaluations, which have been previously described.6e9 Briefly, group 1 was composed by 16 healthy females aged 31 4 years, body mass index 21 2 kg/m2, who were enrolled at MEDES Clinical Research Facility of the Rangueil University Hospital (Toulouse, France).6 Group 2 was composed by 30 healthy males aged 23 0.4 years, body mass index 24 0.4 kg/m2, who were enrolled for the present study at the Valdoltra Hospital, University of Primorska (Ankaran-Capodistria, Slovenia).7,8 Group 3 was composed by 9 healthy males aged 24 1 years, body mass index 23 1 kg/m2, who were enrolled for the present study at the Clinical Research Center of the German Aerospace Institute (Cologne, Germany).9 All subjects were physically active before the study. Each subject signed an informed consent form upon admission. The study was performed in accordance with the Declaration of Helsinki for human studies and relative amendments. 2.2. Experimental design In order to investigate the potential contribution of body fat, energy balance, muscle mass and insulin sensitivity in regulating the levels of circulating TRAIL, we have used a well-characterized bed rest protocol as a model of experimental muscle atrophy and insulin resistance in humans. In particular, we took advantage of previous studies performed on healthy volunteers, who underwent experimental bed rest for 60 days (group 1, n ¼ 16), 35 days (group 2, n ¼ 30) and 14 days (group 3, n ¼ 9) at different levels of energy intake (calorie restriction, near-neutral energy balance, overfeeding) according to different experimental protocols.6e9 Energy balance was calculated according to changes in fat mass during the experimental periods. Fat mass was determined by dual-emission X-ray absorptiometry (DXA) in group 1 and 3 and by bioimpedance in group 2. Subjects of group 1 were randomized to complete 60 days of strict bed rest (n ¼ 8) or to combine bed rest with daily resistance or aerobic exercise routines in supine position (n ¼ 8), energy balance resulted near-neutral and slightly negative in the two sub-groups, respectively.6 Subjects of group 2 completed 35 days of strict bed rest at different levels of energy intakes.7 Nineteen out of the 30 subjects of group 2 were randomized to combine strict bed rest with overfeeding (n ¼ 10) or with near-adequate energy intake (n ¼ 9) according to a parallel design.8 Subjects of group 3 have been studied 4 times for 14-day periods over 2 years in bed rest or in ambulatory condition in combination with eucaloric or hypocaloric diets using a crossover experimental design. Energy intakes were individually tailored to account for the decrease in requirement during bed rest and then decreased by about 20% during the hypocaloric periods.9 2.3. Analyses Insulin, glucose and leptin concentrations were measured by laboratory analyses as referenced.6e9 Body fat mass was determined by DEXA or bioimpedance, as previously described.6e9 Insulin sensitivity was calculated according to the formula of the
1019
homeostasis model assessment (HOMA-IR index) method: insulin resistance ¼ fasting plasma insulin (mUI/ml) fasting plasma glucose (mmol/l)/22.5. The index is highly correlated with the insulin resistance index assessed by the euglycemichyperinsulinemic clamp, which is the gold standard of insulin resistance measurement and is widely adopted in clinical studies for individuals with various degree of insulin sensitivity. Plasma TRAIL was measured in duplicate by using a specific, commercially available ELISA kit, which detect total TRAIL levels (both free and bound to other proteins, such as osteoprotegerin) in accordance with the manufacturer’s instructions (R&D Systems, Minneapolis, MN) in frozen (80 C) aliquots of plasma, obtained at the time of the original studies.6e9 It is important to point out that the present study was possible thanks to the good stability of circulating TRAIL in plasma samples correctly frozen 80 C at over periods of many years.1 Sensitivity of the assay was 2.86 pg/ml and the intra- and inter-assay coefficients of variation were 3.9% and 6%, respectively. 2.4. Statistics The study populations comprise 3 different groups who were kept for different time periods at different energy levels under bed rest or controlled physical activity conditions, as previously described.6e9 In the first analysis, the results obtained from the 3 groups of subjects were pooled together. One observation was obtained from each subject of group 1 and 2 while four observations were obtained from each subjects of group 3. The levels of circulating TRAIL were cumulatively considered in relationship with the data relative to change of fat mass over experimental periods of different durations. Pearson’s tests were used to investigate association between variables. To evaluate the effect of bed rest at different energy intake levels, results were analyzed in group 1 by repeated-measures ANOVA with interactions.6 In the parallel group design study performed in group 2,7,8 activity (ambulatory or bed rest) and treatment (higher or lower energy intake) were the within-subject and between-subject factors, respectively. In the crossover design study performed in group 3,9 activity (ambulatory or bed rest) and treatment (adequate or lower energy intake) were the withinsubject factors. Since there was no significant gender or bed rest effects on TRAIL circulating levels, results obtained during the different studies, in ambulatory or bed rest conditions, were pooled together, expressed as means SD and analyzed by paired Student t-test or Mann Withney test where appropriate. P < 0.05 was considered statistically significant. 3. Results 3.1. The circulating levels of TRAIL exhibit a significant correlation with changes of fat mass, but not of insulin sensitivity or lean mass, in healthy subjects kept in experimental physical inactivity In the first analysis, the levels of circulating TRAIL of subjects from group 1, 2 and 3 were cumulatively considered in relation with the data relative to change of fat mass over experimental periods of different durations.6e9 Pre-bed rest values of TRAIL concentrations were not significantly different in male (104 21 pg/ml) and female (94 19 pg/ml) subjects. As shown in Supplemental Fig. 1, a significant (n ¼ 82; R ¼ 0.34; p < 0.001) positive correlation was noticed between levels of circulating TRAIL at the end of the experimental period and energy balance, as determined by individual changes of fat mass (kg) over the experimental period. Insulin sensitivity significantly decreased (groups 1 and 2) after 35 and 60 days of bed rest (HOMA-IR index: from 1.37 0.61 to
1020
G. Biolo et al. / Clinical Nutrition 31 (2012) 1018e1021
Table 1 Effects of energy restriction at different levels of physical activity on soluble TRAIL. Ambulatory Eucaloric Energy intake (kcal/kg/day)b Delta fat mass (kg/14 days) Delta leptin (ng/ml) TRAIL (pg/ml)
45 0.10 0.3 103
3 1.01 1.3 18
Pa
Bed rest (14 days) Hypocaloric 37 1.02 0.9 101
4 0.96 1.3 20
Eucaloric 36 0.28 0.8 102
Hypocaloric
2 0.64 2.2 12
30 1.00 1.2 86
2 0.76 2.5 15
Diet effect
Bed rest effect
Interaction
<0.001 0.01 0.04 0.04
<0.001 0.35 0.37 0.12
0.08 0.24 0.53 0.06
Circulating TRAIL was measured in the post-absorptive state at the end of 14 day periods of bed rest and controlled ambulation in 9 healthy, male young volunteers subjects receiving eucaloric or hypocaloric (80% of total energy expenditure) diets according with a crossover design. Energy intake in bed rest was adapted to decreased requirement. Data are expressed as means SD. a Data were analyzed with the use of repeated-measures ANOVA with activity (ambulatory or bed rest) and diet (eucaloric or hypocaloric) as the 2 factors. b Energy intake is normalized by kg of lean body mass as determined by dual-emission X-ray absorptiometry.
1.80 0.90, plasma insulin: from 6.2 2.8 to 8.1 4.0 U/ml; n ¼ 46, p < 0.001 paired t-test) while body fat did not significantly change (from 13 5 to 13 5 kg). No significant correlation was noticed between bed rest induced changes in insulin sensitivity and levels of circulating TRAIL (HOMA-IR index, R ¼ 0.09; plasma insulin, R ¼ 0.10; n ¼ 46). Lean body mass significantly (p < 0.001) decreased (group 1 and 2) after 35 and 60 days of bed rest (4.7 2.3%). No significant correlation was noticed between bed rest induced changes in lean body mass and levels of circulating TRAIL (R ¼ 0.02; n ¼ 46). 3.2. Physical inactivity combined with overfeeding increases while energy restriction decreases the levels of circulating TRAIL in healthy subjects In order to get insights in the relationship between circulating TRAIL levels and energy intake, we have analyzed the effect of higher or lower energy balance during physical inactivity for 35 days on the levels of circulating TRAIL in a group of young, healthy male volunteers (19 subjects from group 2). Results indicated (Table 1) that there was not significant bed rest effect on TRAIL circulating levels while there was significant bed rest group interaction leading to greater TRAIL changes in the higher energy balance group (þ14%) than in the lower energy balance group (10%) (p ¼ 0.02, Mann Withney test). In parallel, we have analyzed the effects of energy restriction at 80% of requirement in ambulatory conditions and at the end of 14 days of bed rest in another group of young, healthy male volunteers (group 3, n ¼ 9) according to a crossover experimental design.9 As previously shown, fat mass and plasma leptin were significantly (p < 0.05) decreased following energy restriction with respect to the eucaloric periods (Table 2). Of note, energy restriction in bed rest or in ambulatory conditions caused a significant (p < 0.05) decrease in the levels of circulating TRAIL (Table 2). The individual percent changes of energy intake, fat mass and soluble TRAIL from the eucaloric ambulatory period to the other three experimental conditions were calculated in each subject of this group. Of note, there was a significant (R ¼ 0.46; p < 0.001) direct correlation only
between changes in energy intake and changes in circulating TRAIL (Supplemental Fig. 2). 4. Discussion Food intake and energy homeostasis are regulated by a complex physiological network of signals arising from the hypothalamus as well as from peripheral organs, including stomach, intestine, pancreas, liver and adipose tissue. In recent years, great efforts have been made to identify and investigate the pathways involved in the control of energy metabolism. Disturbance of food intake and energy balance can lead to complex dysfunctions, characterized by either obesity or cachexia. While over-nutrition and obesity have been rapidly increasing worldwide and constitute a major social and medical problem, on the other hand, cachexia is marked by loss of weight as a result of hypercatabolic state, which is characteristic for many subacute and chronic diseases, including cancer, congestive heart failure and sepsis. In this study, we have demonstrated for the first time the existence of a positive correlation between levels of circulating TRAIL and changes in fat mass and energy intake in three distinct groups of young healthy participants under bed-rest conditions. These findings were somewhat surprising taking into account that several previous studies have demonstrated that circulating TRAIL levels are inversely related to the risk of mortality in patients affected by cardiovascular disease.2,3 In two distinct groups of healthy volunteers, 35-days of experimental overfeeding increased soluble TRAIL by about 15% while 14-days of energy restriction decreased its circulating levels by about 10%. Moreover, a significant direct correlation was noticed only between changes in energy intake and fat mass with changes in circulating TRAIL, while changes in level of insulin sensitivity did not influence soluble TRAIL. Thus, although we have not addressed the intracellular pathways regulating the production and release of TRAIL in response to metabolic changes, our data has allowed to establish for the first time a link between energy balance and levels of circulating TRAIL. Although the nature of this study does not allow to ascertain the physiopathological significance of the changes in
Table 2 Effects of overfeeding and physical inactivity on soluble TRAIL.
TRAIL (pg/ml) Leptin (ng/ml) Fat mass (kg)
Bed rest at higher energy balance
Bed rest at lower energy balance
Baseline
35 days
Baseline
35 days
108 20 3.4 3.8 12.9 7.2
119 23 6.9 8.2b 15.0 7.02
108 28 2.1 1.8 10.6 3.7
95 18 3.2 3.6 11.0 3.8
Pa activity effect
Pa interaction
0.83 0.01 <0.001
0.03 0.04 <0.001
Circulating TRAIL was measured in the post-absorptive state before and at the end of 35 days of bed rest in nineteen healthy, young male volunteers maintained at higher (n ¼ 10) or lower (n ¼ 9) energy balance. Changes in body fat in the higher or lower energy balance groups averaged 2.6 1.0 and 1.0 1.5 kg, respectively. Data are expressed as means SD. a Data were analyzed by using a 2-factor (group activity) ANOVA with interaction. There was not significant group effect. b Significantly different from the ambulatory adaptation condition, P < 0.025 (Bonferroni’s post hoc analysis).
G. Biolo et al. / Clinical Nutrition 31 (2012) 1018e1021
circulating TRAIL in response to energy intake, we and other groups of investigators have previously shown that TRAIL shows antiatherosclerotic and vasoeprotective activities in animal models.1 Therefore, it is tempting to speculate that the raise of circulating TRAIL levels might represent a compensatory mechanism aimed to protect the vascular system following increase energy intake. Our results may contribute to understand the unexpected protective effect of moderate obesity and overweight on cardiovascular outcome.10 Future studies are required in order to address at the cellular level the mechanisms involved in promoting increased TRAIL release in response to increased energy intake.
Conflict of interest The authors have no conflict of interest.
Acknowledgments Italian Association for Cancer Research (AIRC) to GZ.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clnu.2012.04.016.
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