Differences in Hematopoietic Stem Cells Contribute to Sexually Dimorphic Inflammatory Responses to High Fat Diet-induced Obesity

Abstract

Women of reproductive age are protected from metabolic disease relative to postmenopausal women and men. Most preclinical rodent studies are skewed toward the use of male mice to study obesity-induced metabolic dysfunction because of a similar protection observed in female mice. How sex differences in obesity-induced inflammatory responses contribute to these observations is unknown. We have compared and contrasted the effects of high fat diet-induced obesity on glucose metabolism and leukocyte activation in multiple depots in male and female C57Bl/6 mice. With both short term and long term high fat diet, male mice demonstrated increased weight gain and CD11c(+) adipose tissue macrophage content compared with female mice despite similar degrees of adipocyte hypertrophy. Competitive bone marrow transplant studies demonstrated that obesity induced a preferential contribution of male hematopoietic cells to circulating leukocytes and adipose tissue macrophages compared with female cells independent of the sex of the recipient. Sex differences in macrophage and hematopoietic cell in vitro activation in response to obesogenic cues were observed to explain these results. In summary, this report demonstrates that male and female leukocytes and hematopoietic stem cells have cell-autonomous differences in their response to obesity that contribute to an amplified response in males compared with females.

Keywords: adipose tissue; adipose tissue inflammation; diet-induced obesity; hematopoiesis; inflammation; metabolism; metainflammation; obesity; sexually dimorphic.

FIGURE 1.

Sexually dimorphic body composition responses to high fat diet feeding in mice. Male and female C57Bl6 mice were fed ND or 60% HFD diets starting at 6 weeks of age for up to 16 weeks. A, animals were weighed weekly. Shown are weight (B) and GWAT (C) after 6 weeks of diet. D, body composition performed based on Minispec NMR. Shown are food intake (E) and energy expenditure (F) in light and dark hours and in HFD mice over the 48-h period. G, respiratory exchange ratio (RER), fat and glucose oxidation, oxygen consumption (VO2), and activity. Shown are change in weight after 16 weeks (H) and final weight after 16 weeks of diet (I). Shown are GWAT fat pad weight (J), GWAT weight as a percentage of body weight (K), and inguinal fat in grams (L) and as a percentage of total body weight (M). N, adipocyte cross-sectional area at 6 weeks and distributions of adipocyte cross-sectional area. O, adipocyte cross-sectional area at 16 weeks and distributions of adipocyte cross-sectional area. n = 4–8/group; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ***, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.

FIGURE 2.

Sexually dimorphic metabolic responses to high fat diet feeding in mice. Shown are fasting glucose (A) and insulin levels (B) assessed at 4 weeks of diet challenge and 10 weeks of diet challenge (C). Shown is glucose tolerance testing in male and female mice after 12 weeks of diet feeding in ND (D) and HFD feeding (E). 16-week-fed glucose (F) and insulin (G) are shown. Shown are H&E images of livers after 6 weeks (H) and 16 weeks (I) of HFD. n = 4; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ***, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.

FIGURE 3.

Adipose tissue inflammatory gene expression in GWAT and IWAT. A, H&E images of GWAT in male and female mice after 16 weeks of HFD. B and C, GWAT inflammatory gene expression; D and E, IWAT gene expression. A.U., arbitrary units. n = 4; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.

FIGURE 4.

Compared with females, male mice have increased numbers of CD11c⁺ ATMs and myeloid progenitors with high fat diet. A, flow cytometry plots for ATM subtypes and adipose tissue dendritic cells based on CD64 and CD11c staining. Shown are quantitations of CD45⁺ cells in SVF (B), total ATMs (C), ATM/g of GWAT (D), and CD11c⁺ ATMs (E) in gonadal adipose tissue in male and female mice after 16 weeks of diet challenge. F, representative flow cytometry histogram of CD11c expression in CD64⁺ ATM for males and female mice (ND (black) and HFD (gray)). G, CD11c⁻ ATMs; H, ratio of CD11c⁺/CD11c⁻ ATMs; I, adipose tissue dendritic cell populations. Shown are quantitations of total circulating CD115⁺ monocytes (J) and Ly6c^hi classical monocytes (K) by flow cytometry after 12 weeks of diet challenge. L, quantitation of HSC precursors after 16 weeks of diet. n = 4; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.

FIGURE 5.

Male mice accumulate CD11c⁺ ATMs more rapidly with short term high fat compared with females. Male and female C57Bl6 mice were fed either ND or 60% HFD chow for 6 weeks. A, gonadal gene expression in arbitrary units (A.U.). Shown are flow cytometry quantitations of total and CD11c⁺ ATMs in GWAT (B), CD115⁺ monocytes and Ly6c^hi blood monocytes (C), and bone marrow myeloid progenitors and stem cells (D). n = 8; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.

FIGURE 6.

In a competitive BMT model, HSCs from male mice preferentially contribute to myeloid cells and CD11c⁺ ATMs after high fat diet. A, experimental scheme. Lethally irradiated female and male recipients (CD45.1/CD45.2 heterozygotes) were injected with male (CD45.2) and female (CD45.1) BM mixed in a 1:1 ratio. Animals were weighed weekly. B, representative flow cytometry analysis of mice after 2 weeks of diet (8 weeks after BMT). Shown is a quantitation of the ratio of male to female (CD45.2/CD45.1) cells in total leukocytes (C), Ly6c^hi monocytes (D), and CD3⁺ cells (E) prior to HFD exposure and after 2 weeks of HFD feeding. F, representative flow cytometry plots of male and female marrow contributions to total GWAT ATMs after 16 weeks of diet challenge. G, quantitation of total ATMs, CD11c subsets, and adipose tissue dendritic cells (DC) in male and female recipients. H, ratio of male to female (CD45.2/CD45.1) cells in GWAT ATMs and dendritic cells. I, quantitation of total BM HSCs and myeloid progenitors in male and female recipient mice. J, donor contribution to HSC and myeloid progenitor subsets in the bone marrow. n = 7 males, and n = 9 females. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.

FIGURE 7.

Males have increased bone marrow inflammatory responses to palmitic acid, high fat diet, and LPS compared with females. A, male and female BM was isolated and plated in methocult media for granulocyte and macrophage colony-forming units. 10,000 cells were isolated and stimulated with FA free BSA or 10 μm palmitic acid with BSA. B, similarly, male and female marrow after in vivo diet challenge were plated for cfu. Bone marrow mononuclear phagocytes (C) and bone marrow-derived dendritic cells (D) of male mice have increased proinflammatory profiles after stimulation with LPS compared with females. A.U., arbitrary units. n = 8; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001. Comparisons between sexes are shown as follows: #, p < 0.05; ##, p < 0.01; ###, p < 0.005; ####, p < 0.001. Error bars, S.E.