Effect of energy restriction and physical exercise intervention on phenotypic flexibility as examined by transcriptomics analyses of mRNA from adipose tissue and whole body magnetic resonance imaging

Sindre Lee, Frode Norheim, Torgrim M Langleite, Hans J Noreng, Trygve H Storås, Lydia A Afman, Gary Frost, Jimmy D Bell, E Louise Thomas, Kristoffer J Kolnes, Daniel S Tangen, Hans K Stadheim, Gregor D Gilfillan, Hanne L Gulseth, Kåre I Birkeland, Jørgen Jensen, Christian A Drevon, Torgeir Holen, NutriTech Consortium, Sindre Lee, Frode Norheim, Torgrim M Langleite, Hans J Noreng, Trygve H Storås, Lydia A Afman, Gary Frost, Jimmy D Bell, E Louise Thomas, Kristoffer J Kolnes, Daniel S Tangen, Hans K Stadheim, Gregor D Gilfillan, Hanne L Gulseth, Kåre I Birkeland, Jørgen Jensen, Christian A Drevon, Torgeir Holen, NutriTech Consortium

Abstract

Overweight and obesity lead to changes in adipose tissue such as inflammation and reduced insulin sensitivity. The aim of this study was to assess how altered energy balance by reduced food intake or enhanced physical activity affect these processes. We studied sedentary subjects with overweight/obesity in two intervention studies, each lasting 12 weeks affecting energy balance either by energy restriction (~20% reduced intake of energy from food) in one group, or by enhanced energy expenditure due to physical exercise (combined endurance- and strength-training) in the other group. We monitored mRNA expression by microarray and mRNA sequencing from adipose tissue biopsies. We also measured several plasma parameters as well as fat distribution with magnetic resonance imaging and spectroscopy. Comparison of microarray and mRNA sequencing showed strong correlations, which were also confirmed using RT-PCR In the energy restricted subjects (body weight reduced by 5% during a 12 weeks intervention), there were clear signs of enhanced lipolysis as monitored by mRNA in adipose tissue as well as plasma concentration of free-fatty acids. This increase was strongly related to increased expression of markers for M1-like macrophages in adipose tissue. In the exercising subjects (glucose infusion rate increased by 29% during a 12-week intervention), there was a marked reduction in the expression of markers of M2-like macrophages and T cells, suggesting that physical exercise was especially important for reducing inflammation in adipose tissue with insignificant reduction in total body weight. Our data indicate that energy restriction and physical exercise affect energy-related pathways as well as inflammatory processes in different ways, probably related to macrophages in adipose tissue.

Trial registration: ClinicalTrials.gov NCT01803568 NCT01684917.

Keywords: Adipose tissue; energy restriction; exercise; immunometabolism; macrophages; obesity.

© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Figures

Figure 1
Figure 1
Correlations between mRNA sequencing, microarray and RT‐PCR. (A) Correlation between the change in expression of 13 genes (see “Methods”) analyzed by RT‐PCR and microarray. (B) Correlation between the change in expression of 13 genes analyzed by RT‐PCR and mRNA sequencing. (C) Correlation between the change in expression of 13 genes analyzed by microarray and mRNA sequencing. (D) Correlation between the change in expression of 37 immune cell markers (see Table 2) analyzed by microarray and mRNA sequencing. The change (post baseline) was calculated from the average expression from five subjects from the exercise intervention. Spearman's rho correlations were performed.
Figure 2
Figure 2
Markers of immune cell subtypes. Expression of the 37 markers of cell subtypes (see “Table 2”) in human adipose tissue. (A) The median expression of 18 markers of adipose tissue macrophages compared across the median of macrophage‐like cells (n = 12), other immune cells (n = 22), lean adipocytes (n = 20) and obese adipocytes (n = 19). (B) The median expression of 16 markers of M1‐like and M2‐like macrophages compared across the median of activated macrophage‐like cell (n = 6), inactivated macrophage‐like cells (n = 6), other immune cells (n = 22), lean adipocytes (n = 20), and obese adipocytes (n = 19). (C) The median expression of six markers of T cells compared across the median of T cells (n = 10), other immune cells (n = 26), lean adipocytes (n = 20), and obese adipocytes (n = 19). The full expression panel for every marker in every cell type is available in Figure 3. Data are medians + interquartile range.
Figure 3
Figure 3
Expression of mRNA markers of macrophages, M1‐ and M2‐like macrophages and T cells in human adipose tissue. (A) Expression of the 18 markers of macrophages in macrophage‐like cells compared to the expression in other immune cells and adipocytes. (B) Expression of the 11 markers of M1/M2‐like macrophages in stimulated macrophage‐like cells compared to the expression in unstimulated macrophage‐like cells, other immune cells and adipocytes. (C) Expression of six markers of T cells in T cells compared to expression in other immune cells and adipocytes.
Figure 4
Figure 4
The change in expression of immune cell markers in response to exercise and diet. (A) The average expression of markers for adipose tissue macrophage, M2‐like macrophages and T cells were reduced after exercise. (B) The average expression of markers for adipose tissue M1‐like macrophages was increased after energy restriction. (C) No alteration in expression of immune cell markers was observed in lean control subjects after exercise. (D) No alteration in expression of immune cell markers was observed in control subjects. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 baseline versus 12 weeks. RPKM = Reads Per Kilobase of transcript per Million mapped reads; ni = normalized intensities.
Figure 5
Figure 5
Opposite regulation of adipose tissue macrophages after exercise versus energy restriction. (A) Reduced expression (log2[fold‐change] < 0) of 12 of 18 markers of adipose tissue macrophages was observed after exercise. (B) Increased expression (log2[fold‐change] > 0) of seven of 18 markers of adipose tissue macrophages was observed after energy restriction. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 6
Figure 6
Expression of macrophage markers in control subjects. (A) No consistent change in expression of 18 markers of macrophages was observed after exercise in lean control subjects. (B) No change in expression of 18 markers of macrophages was observed in control subjects. (C) No consistent change in expression of 11 markers of M1‐ and M2‐like macrophages was observed after exercise in lean control subjects. (B) No change in expression of 11 markers of M1‐ and M2‐like macrophages was observed in control subjects. Data represent means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 7
Figure 7
Exercise and diet influence adipose tissue macrophage subtypes differently. (A) Reduced expression (log2[fold‐change] < 0) of 4 out of 6 markers of adipose tissue M2‐like macrophages was observed after exercise. (B) Increased expression (log2[fold‐change] > 0) of five of 10 markers of adipose tissue M1‐like macrophages was observed after energy restriction. Data represent means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.
Figure 8
Figure 8
mRNA expression of markers of “metabolically activated” macrophages. No consistent change in two markers of “metabolically activated” macrophages were observed in neither the exercise nor in the diet intervention. Data represent means ± SEM. *P < 0.05.
Figure 9
Figure 9
Macrophage markers correlate with BMI and FFA in both the exercise and diet groups. (A) BMI correlated with adipose tissue macrophage expression at baseline in the exercise group. (B) BMI correlated with adipose tissue macrophage expression at baseline in the diet group. (C) The change in gene expression (12 weeks minus baseline) of adipose tissue macrophages correlated with the change in plasma FFA concentrations in the exercise group. (D) The change in gene expression of adipose tissue macrophages correlated with the change in plasma FFA concentrations in the diet group. RPKM = Reads Per Kilobase of transcript per Million mapped reads, ni = normalized intensities.

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