The Impact of Long-term Physical Inactivity on Adipose Tissue Immunometabolism

Abstract

Context: Adipose tissue and physical inactivity both influence metabolic health and systemic inflammation, but how adipose tissue responds to chronic physical inactivity is unknown.

Objective: This work aimed to characterize the impact of chronic physical inactivity on adipose tissue in healthy, young males.

Methods: We collected subcutaneous adipose tissue from 20 healthy, young men before and after 60 days of complete bed rest with energy intake reduced to maintain energy balance and fat mass. We used RNA sequencing, flow cytometry, ex vivo tissue culture, and targeted protein analyses to examine adipose tissue phenotype.

Results: Our results indicate that the adipose tissue transcriptome, stromal cellular compartment, and insulin signaling protein abundance are largely unaffected by bed rest when fat mass is kept stable. However, there was an increase in the circulating concentration of several adipokines, including plasma leptin, which was associated with inactivity-induced increases in plasma insulin and absent from adipose tissue cultured ex vivo under standardized culture conditions.

Conclusion: Physical inactivity-induced disturbances to adipokine concentrations such as leptin, without changes to fat mass, could have profound metabolic implications outside a clinical facility when energy intake is not tightly controlled.

Trial registration: ClinicalTrials.gov NCT03594799.

Keywords: Adipose tissue; immunometabolism; physical inactivity.

© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.

Figures

Figure 1.

Overview of adipose tissue transcriptional responses to long-term bed rest. (A) Volcano plot of all transcripts by Log2FC against DESeq2-produced p-values. Horizontal dotted lines indicate significance threshold (P < .01). Vertical dotted lines represent 0.5 Log2 fold change thresholds. Red dots = significantly downregulated; blue dots = significantly upregulated; grey dots = non-differentially expressed genes. (B) Quantification of differentially expressed genes identified in (A). (C) Top-10 significantly down/upregulated transcripts, by magnitude of Log2FC (presented as mean ± SEM). (D) Top 25 most significantly DEGs ranked by significance, from left to right (presented as mean ± SEM). (E) Significantly enriched (P < .05; EASE score) pathways associated with DESeq2 DEGs from (A) using DAVID bioinformatics resources 6.8. P values are presented as –Log10 values. Pathways and terms are ranked in ascending order by significance (EASE score). The specific number of differentially expressed genes within a given pathway related to differentially expressed genes put into the analyses are presented within each respective bar. A minimum threshold of 2 genes within a given pathway/term was set for consideration. BP, biological process; CC, cellular compartment; DAVID, database for annotation, visualization, and integrated discovery; DEG, differentially expressed gene; EASE, expression analysis systematic explorer; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; MF, molecular function.

Figure 2.

Plasma glucose and insulin, and metabolic gene and protein expression in adipose tissue in response to bed rest. (A) Fasting plasma insulin concentrations before and after bed rest. Data represent group means at each time point with individual responses overlaid. (B) Fasting plasma glucose concentrations before and after bed rest. Data represent group means at each time point with individual responses overlaid. (C) Relative gene expression of several genes associated with adipose tissue metabolism at post bed rest relative to pre bed rest. Ratios represent the fold change at post bed rest compared with pre bed rest expression levels. Data were normalized to peptidylprolyl isomerase A, pre bed rest, and internal calibrator using the ∆∆Ct method. Dashed line represents no change relative to pre bed rest. Data are presented as mean ± SEM, with the sample size indicated at the bottom of each bar. (D, E) Changes in protein expression of key insulin signaling proteins in (D) adipose tissue and (E) paired adipocytes as a ratio of post bed rest compared with pre bed rest levels, with representative blots (right of respective figure). Solid lines represent mean ± SEM, with individual responses overlaid. Dashed lines in Panel C represent no change relative to pre bed rest. Sample sizes are indicated underneath each variable in A, B, D, and E, and within bars in C.

Figure 3.

Relative change in plasma and adipose tissue supernatant concentrations of inflammatory and metabolic proteins in response to bed rest. (A) Peripheral blood plasma protein concentration expressed as the average change, calculated from each individual’s change from pre to post bed rest. Data are presented as mean ± SEM. (B) Three-hour ex vivo culture of adipose tissue explant protein secretion expressed as the average change, calculated from each individual’s change from pre to post bed rest. Data are presented as mean ± SEM. Sample sizes are indicated at the base of each respective bar. Where concentrations fell below detectable limits for the assay, n.d. is written. *P < .05. CCL, chemokine (C-C motif) ligand; CXCL, chemokine (C-X-C motif) ligand; FGF, fibroblast growth factor; GM-CSF, granulocyte-macrophage colony stimulating factor; ICAM, intracellular adhesion molecule; IFN, interferon; IL, interleukin; IP, interferon gamma inducible protein; MCP, monocyte chemotactic protein; MIP, monocyte inflammatory protein; RANTES, regulated on activation, normal T-cell expressed and secreted; SAA, serum amyloid-A; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factory.

Figure 4.

Adipose tissue and peripheral blood leukocyte responses to bed rest. (A) Adipose tissue CD4+ and CD8+ T-cells expressed as number of cells per gram of tissue. (B) Adipose tissue CD206+ macrophages and NK cells expressed as number of cells per gram of tissue. (C) Adipose tissue CD206+ macrophage HLA-DR surface expression (MFI). (D) Adipose tissue CD4+ T-cell subpopulations expressed as number of cells per gram of tissue. (E) Adipose tissue CD8+ T-cell subpopulations expressed as number of cells per gram of tissue. (F) Adipose tissue endothelial cells expressed as number of cells per gram of tissue. (G) Adipose tissue progenitor cells expressed as number of cells per gram of tissue. (H) Adipose tissue MSCA-1+ progenitor cells expressed as number of cells per gram of tissue. (I) Adipose tissue MSCA-1+ progenitor cell MSCA-1+ surface expression (MFI). (J) Peripheral blood CD4+ and CD8+ T-cells expressed as cells per μL. (K) Peripheral blood monocytes subpopulations expressed as cells per μL. (L) Peripheral blood CD4+ T-cell subpopulations expressed as cells per μL. (M) Peripheral blood CD8+ T-cell subpopulations expressed as cells per μL. All data are presented as group means within individual responses overlaid. T-cell subsets presented in (D), (E), (J), and (K) represent CD45RA+CD27+ (NA) cells; CD45RA−CD27+ (CM) cells; CD45RA−CD27− (EM) cells; CD45RA+CD27− (EMRA) cells. Adipose tissue progenitor cells represent CD45−CD31−CD34+ cells. Adipose tissue endothelial cells represent CD45−CD31+CD34+ cells. Sample sizes are indicated underneath each variable. CD, cluster of differentiation; CM, central memory; EM, effector memory; EMRA, effector memory re-expressing CD45RA; NA, naïve; PBMC, peripheral blood mononuclear cell; MFI, median fluorescence intensity; MSCA–1, mesenchymal stem cell antigen-1.

Figure 5.

CD4+ T–Cell, CD8+ T–cell, and monocyte subpopulation HLA-DR expression in peripheral blood in response to bed rest. (A-D) CD4+ T-cell subpopulation HLA-DR surface expression for (A) naïve; (B) central memory; (C) effector memory; and (D) terminally differentiated effector memory cells. (E–H) CD8+ T-cell subpopulation HLA-DR surface expression for (E) naïve); (F) central memory; (G) effector memory; and (H) terminally differentiated effector memory cells. (I–K) Monocyte subpopulation HLA-DR surface expression for (I) nonclassical; (J) classical; and (K) intermediate monocytes. T-cell subsets presented in (A-H) represent CD45RA+CD27+ (NA) cells; CD45RA−CD27+ (CM) cells; CD45RA−CD27− (EM) cells; CD45RA+CD27− (EMRA) cells. Data represent HLA-DR cell surface expression on a per-cell basis. Data are presented as group means with individual responses overlaid. Sample sizes are indicated underneath each variable. CD, cluster of differentiation; CM, central memory; EM, effector memory; EMRA, effector memory re-expressing CD45RA; HLA–DR, human leukocyte antigen–DR isotype; MFI, median fluorescence intensity; NA, naïve.