ApoA1 Deficiency Reshapes the Phenotypic and Molecular Characteristics of Bone Marrow Adipocytes in Mice

Abstract

In the present study, we studied the effect of apolipoprotein A-1 (APOA1) on the spatial and molecular characteristics of bone marrow adipocytes, using well-characterized ApoA1 knockout mice. APOA1 is a central regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, and thus HDL; our recent work showed that deficiency of APOA1 increases bone marrow adiposity in mice. We found that ApoA1 deficient mice have greatly elevated adipocytes within their bone marrow compared to wild type counterparts. Morphologically, the increased adipocytes were similar to white adipocytes, and displayed proximal tibial-end localization. Marrow adipocytes from wild type mice were significantly fewer and did not display a bone-end distribution pattern. The mRNA levels of the brown/beige adipocyte-specific markers Ucp1, Dio2, Pat2, and Pgc1a; and the expression of leptin were greatly reduced in the ApoA1 knock-out in comparison to the wild-type mice. In the knock-out mice, adiponectin was remarkably elevated. In keeping with the close ties of hematopoietic stem cells and marrow adipocytes, using flow cytometry we found that the elevated adiposity in the ApoA1 knockout mice is associated with a significant reduction in the compartments of hematopoietic stem cells and common myeloid, but not of the common lymphoid, progenitors. Moreover, the 'beiging'-related marker osteopontin and the angiogenic factor VEGF were also reduced in the ApoA1 knock-out mice, further supporting the notion that APOA1-and most probably HDL-C-regulate bone marrow microenvironment, favoring beige/brown adipocyte characteristics.

Keywords: apolipoprotein A-1; beige (hybrid) adipose tissue; brown adipose tissue; high-density lipoprotein; white adipose tissue.

Figure 1

Hematoxylin and eosin sections of ApoA1 deficient and wild type (WT) mouse tibiae; (A,B) The bone marrow of ApoA1 (knockout) KO mice display significant adiposity. The adipocytes are spherical and unilocular, with a single, large lipid vacuole that pushes the nucleus towards the cell periphery; these features are typical of WAT. In addition, these adipocytes are located at the proximal end of tibiae, within red bone marrow; (C,D) On the contrary, bone marrow of WT mice has very low adiposity. The few adipocytes are similar to those of WAT. Unlike bone marrow fat cells of the ApoA1 KO mice, their occurrence is random, without any overt tendency for proximal end distribution. Not the reduced number of bone spicules of KO mice tibiae, in line with previous studies [9]. Scale Bar (100 μm) applies to all figures.

Figure 2

The secondary effects of ApoA1 deficiency on adipocyte molecular phenotype. All graphs indicate mean ± SD. In all studies, n = 5 for both wild-type and ApoA1 mRNA isolates. Expression of the brown/beige adipose tissue markers Ucp1, Dio2, Pat2, and Pgc1a were strongly reduced in the ApoA1 KO mice in comparison to the wild-type counterparts, suggesting a role of APOA1 and probably HDL in the process of bone marrow adipocyte browning. The mRNA of the two major adipokines, leptin and adiponectin, are also shown. Our findings that the mRNA expression of leptin markers is significantly reduced, whereas the mRNA expression of adiponectin (Adipoq) is greatly elevated in the ApoA1 deficient mice, compared to the controls, is consistent with the hypothesis the APOA1 plays a role in the ‘white-to-brown’ switch in bone marrow adipose tissue. (*: p ≤ 0.05, **: p ≤ 0.01, ****: p ≤ 0.0001).

Figure 3

Flow cytometric analysis of whole bone marrow cells. Four animals of each group were tested; (A) Gating strategy for hematopoietic stem cells (HSC). The live Lin-population indicated by the bar is further analyzed for c-kit and Sca-1 expression; (B) The HSC (Lin-c-kit + Sca1+) and the common myeloid progenitors (CMP; Lin-c-kit + Sca1−) compartment, were significantly reduced in the ApoA1 KO compared to their wild-type (WT) littermates. On the contrary, the common lymphoid progenitors compartment (CLP; Lin-c-kit low Sca1 low) was unaffected. Data from one representative experiment is shown; (C) Reduction in Alcam1 (activated leukocyte cell adhesion molecule 1) expression in the ApoA1 KO compared to the wild type mice (****: p ≤ 0.0001).

Figure 4

Graphical summaries of mRNA expression of osteopontin (Opn) and VEGFA (Vegfa) in mice. The mRNA of osteopontin (Opn) revealed a strong trend for reduction, without reaching the level of statistical significance. VEGFa expression was greatly reduced in the ApoA1 deficient mice, consistent with a role of this growth factor in bone marrow adipose tissue ‘beiging’. (**: p ≤ 0.01).