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. 2020 Nov;32(11):e12854.
doi: 10.1111/jne.12854. Epub 2020 Apr 29.

GHR-/- Mice are protected from obesity-related white adipose tissue inflammation

Jonathan A Young  1   2 , Brooke E Henry  1   3   4 , Fabian Benencia  2 , Stephen Bell  4 , Edward O List  1 , John J Kopchick  1   2 , Darlene E Berryman  1   2   4
Affiliations

GHR-/- Mice are protected from obesity-related white adipose tissue inflammation

Jonathan A Young et al. J Neuroendocrinol. 2020 Nov.

Abstract

Growth hormone (GH) excess in bovine (b)GH transgenic mice has been shown to alter white adipose tissue (WAT) immune cell populations. The present study aimed to evaluate the effects of GH resistance on WAT immune cell populations using GH receptor knockout (GHR-/- ) mice. Eight- and 24-month-old, male GHR-/- and wild-type mice were used. Body composition and tissue weights were determined, and systemic inflammation was assessed by measuring serum cytokine levels. The stromal vascular fraction (SVF) was isolated from three distinct WAT depots, and immune cell populations were quantified using flow cytometry. GHR-/- mice at both ages had decreased body weight but were obese. Although no significant changes were observed in serum levels of the measured cytokines, SVF cell alterations were seen and differed from depot to depot. Total SVF cells were decreased in epidydimal (Epi) depots, whereas SVF cells per gram adipose tissue weight were increased in mesenteric (Mes) depots of GHR-/- mice relative to controls. T cells and T helper cells were increased in Mes at 8 months old, whereas cytotoxic T cells were decreased in subcutaneous (SubQ) at 24 months old. Other cells were unchanged at both ages measured. The present study demonstrates that removal of GH action results in modest and depot-specific changes to several immune cell populations in WAT of intra-abdominal depots (Epi and Mes), which are somewhat surprising results because the SubQ has the largest change in size, whereas the Mes has no size change. Taken together with previous results from bovine GH transgenic mice, these data suggest that GH induces changes in the immune cell population of WAT in a depot-specific manner. Notably, GHR-/- mice appear to be protected from age-related WAT inflammation and immune cell infiltration despite obesity.

Keywords: ATM; GHR−/− mice; SVF; T cells; WAT; adipose tissue macrophages; leukocyte; stromal vascular fraction; white adipose tissue.

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Figures

Figure 1.
Figure 1.
Characteristics of 8-month-old WT and GHR−/− mice. A. Body weight, p<0.0001, t=22.17, df=13. B. Body composition as percent of body weight: Fat p<0.0001, t=10.47, df=13; Lean p=0.0004, t=4.773, df=13; Fluid p=0.1218, t=1.655, df=13. C. Adipose tissue depot weights expressed as percent of total body weight: SubQ p<0.0001, t=9.126, df=13; Epi p=0.0018, Welch-corrected t=4.326, df=9.289; Mes p=0.0446, t=2.223, df=13. D. Serum cytokine levels: IL-6 p=0.3969, U=20; MCP-1 p=0.2654, t=1.937, df=13; TNFα p=0.075, t=1.937, df=13. Data are expressed as mean ± SEM. WT n = 8 and GHR−/− n = 8. * indicates a significant difference; p < 0.05.
Figure 2.
Figure 2.
Quantification of SVF, CD45+ leukocytes, and MHC-II antigen presenting cells in 8 month old WT and GHR−/− mice. A. Estimated total SVF cells normalized to gram of WAT: SubQ p=0.6903, t=0.4075, df=13; Epi p=0.2552, t=1.2, df=11; Mes p=0.014, U=7. B. Number of CD45+ cells per gram of tissue: SubQ p=0.6943, U=24; Epi p=0.1226, Welch-corrected t=1.705, df=8.955; Mes p=0.2345, U=20. C. Number of activated (MHC-II+) CD45+ cells normalized to gram of WAT: SubQ p=0.4634, U=21; Epi p=0.065, U=14; Mes p=0.6454, U=27. Data are expressed as mean ± SEM. WT n = 8 and GHR−/− n = 8. * indicates a significant difference; p < 0.05.
Figure 3.
Figure 3.
Quantification of adipose tissue T cells in WT and GHR−/− WAT depots at 8 months of age. A. Dot plot distribution of T cells (CD3+ CD45+) in WT and GHR−/− male mice. The y-axis and x-axis of the dot plots represent fluorescent intensity. B. Number of CD3+CD45+ T cells normalized to depot weight in WT and GHR−/− male mice: SubQ p=0.4634, U=21; Epi p=0.1304, U=17; Mes p=0.0281, U=11. Data are expressed as mean ± SEM. WT n = 8 and GHR−/− n = 8. * indicates a significant difference; p < 0.05
Figure 4.
Figure 4.
Quantification of WAT T cell subsets and MHC-II+ activated adipose tissue macrophages (ATMs) at 8 months of age. A. Number of CD3+CD4+ T helper cells: SubQ p=0.9551, U=27; Epi p=0.4347, t=0.8042, df=14; Mes 118% increase; p=0.0148, U=9. B. Number of CD3+CD4- cytotoxic T cells: SubQ p=0.9551, U=27; Epi p=0.2786, U=21; Mes p=0.0662, t=1.993, df=14. C. Number of MHCII+ activated ATMs: SubQ p=0.4634, U=21; Epi p=0.4418, U=24; Mes p=0.1679, t=1.454, df=14. Data are reported as the mean of the number of cells per gram of tissue ± SEM. WT n = 8 and GHR−/− n = 8. * indicates a significant difference; p < 0.05.
Figure 5.
Figure 5.
Comparisons of M2 and M1 populations in WT and GHR−/− mice at 8 months of age. A and C. Distribution and quantification of M2 macrophages (A) and M1 macrophages (C) in WT and GHR−/− male mice. The y-axis represents relative number of events normalized to mode and the x-axis represents the intensity of the fluorescent signal. Isotype control, WT and GHR−/− are represented by gray, blue and red peaks respectively. B and D. Distribution and quantification of M2 macrophages (B: SubQ p=0.28, t=1.127, df=13; Epi p=0.3823, U=23; Mes p=0.1605, U=18) and M1 macrophages (D: SubQ p=0.2319, U=17; Epi p=0.1944, U=19; Mes p=0.3823, U=23) in WT and GHR−/− male mice. M2 and M1 populations were derived from F480+CD11b+CD45+ ATM parent population. It should be noted that because cells over a certain fluorescence threshold were counted, the variation in intensity of the fluorescence shown in A and C is not reflected in the enumeration of cells reported in B and D. Data are reported as the mean of the number of cells per gram of tissue ± SEM.
Figure 6.
Figure 6.
Notable results from the 24 month cohort of GHR−/− mice. A. Body weight p<0.0001, t=12.95, df=12. B. Body composition expressed as percent of body weight: Fat p<0.0001, t=5.815, df=12; lean p=0.0006, U=0; fluid p=0.0024 t=3.826, df=12. C. Serum cytokine levels: IL-6 p=0.6402, U=27; MCP-1 p=0.2092, Welch-corrected t=1.356, df=8.704; TNF-α p=0.4462, U=24.5. D. Number of CD3+CD8+ cytotoxic T cells, reported as the mean of the number of cells per gram of tissue ± SEM: SubQ p=0.0105, U=5.5; Epi p=0.7523, U=18.5. WT n = 8 and GHR−/− n = 8. * indicates a significant difference; p < 0.05.
Figure 7.
Figure 7.
Proportion of immune cells and other SVF cells in WAT depots in WT and GHR−/− male mice at 8 months of age. Number of T cytotoxic cells, T helper cells, M1 macrophages, M2 macrophages, and other CD45+ leukocytes in WT and GHR−/− male mice normalized to gram of adipose tissue. WT n = 8 and GHR−/− n = 8.
Figure 8.
Figure 8.
Summary of flow cytometry results. The flow cytometry results of three different cohorts of GH-altered mice (bGH(16), GHR−/− 8 months, and GHR−/− 24 months) are summarized in this figure. A significant increase is indicated by a blue cell, significant decrease an orange cell, no significant change is indicated by a grey cell and no measurement is a signified by a white cell.
See this image and copyright information in PMC

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