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Z. Zhang et al.
Results
Adipocyte-derived stem cell factor is not essential for brown fat function in vivo
To determine whether adipocyte-derived SCF cell- autonomously regulates BAT function in vivo, we generat- ed fat-specific SCF KO mice by crossing Adiponectin (Adipoq)-Cre mice with Kitlfl/fl mice. The adipose stromal vascular faction (SVF) cells derived from Kitlfl/fl controls and Adipoq-Cre+;Kitlfl/fl KO mice were isolated and differentiat- ed into adipocytes in culture. The Kitl gene was specifical- ly deleted (Online Supplementary Figure S1A) and the Kitl mRNA reduced its level (Online Supplementary Figure S1B) in KO cells when Adipoq-Cre started to be expressed after adipogenic induction (Online Supplementary Figure S1B). Control and KO SVF cells showed similar capacity in adi- pogenic differentiation, determined by Oil Red O staining (Figure 1A). Consistent with the previous finding that SCF is required for UCP1 expression,37 we found that differen- tiated brown adipocytes from Adipoq-Cre+;Kitlfl/fl mice had much lower levels of UCP1 protein compared to Kitlfl/fl cells (Figure 1B and Online Supplementary Figure S1C). Expression of PGC-1a and PERILIPIN was unaffected (Figure 1B).
We then sought to determine gene expression in ther- mogenic fat tissues in vivo. The levels of UCP1, PGC-1a, COX4, and PPARγ proteins in BAT and inguinal WAT (iWAT) were essentially the same between Kitlfl/fl and Adipoq-Cre+;Kitlfl/fl mice (Figure 1C and D and Online Supplementary Figure S1D and E). Kitlfl/fl and Adipoq- Cre+;Kitlfl/fl mice had similar body weight at 7, 14, and 28 weeks of age (Online Supplementary Figure S1F-H). No changes in the mass of BAT, iWAT, and gonadal WAT (gWAT) (Online Supplementary Figure S1F), composition of lean and fat mass (Online Supplementary Figure S1G), or fasting body weight (Online Supplementary Figure S1H) were observed. In addition, systemic glucose metabolism shown by the glucose tolerance test was also comparable between the two genotypes (Online Supplementary Figure S1I).
To assess sympathetic nerve-activated adaptive thermo- genesis, we treated mice with a β3-adrenoceptor agonist, CL 316,243 for seven days. qRT-PCR and western blotting showed no difference in levels of Ucp1 mRNA (Figure 1E) or UCP1 protein (Figure 1F and Online Supplementary Figure S1J and K) in BAT or iWAT between Kitlfl/fl and Adipoq- Cre+;Kitlfl/fl mice. Together, these data demonstrate that SCF secreted by adipocytes is essential for UCP1 expres- sion in vitro but is not essential for energy metabolism in vivo. It is possible that SCF from non-adipose cells or tis- sues may compensate for the loss of SCF in adipocytes, as no change in serum levels of SCF was observed in Adipoq- Cre+;Kitlfl/fl mice (Online Supplementary Figure S1L).
Adipocyte-derived stem cell factor controls steady-state hematopoiesis
Marrow adipose tissue is an endocrine organ important for hematopoiesis and systemic metabolism.27-30 MAT pro- motes the regeneration of hematopoietic stem cells after irradiation by secreting SCF.32 We first confirmed that most BM adipocytes (approx. 77% in cMAT and approx. 80% in rMAT) expressed SCF by performing perilipin immuno- fluorescent staining on bone sections of KitlEGFP knockin mice (Figure 2A and Online Supplementary Figure S2A). Compared to BAT and iWAT, the flushed BM expressed
similar levels of the long isoform of Kitl that can be tran- scribed and cleaved into the soluble form of SCF, but much less of the short Kitl transcript that encodes the membrane-bound SCF (Online Supplementary Figure S2B).34 Note that there are significant numbers of non-adipocytes in the adipose tissues and BM analyzed. In BM, Adipoq- Cre was recently shown to only label mature adipocytes, but not bone stroma, adipogenic progenitors, hematopoi- etic cells, bone lining cells, or osteoblast cells.41 In Adipoq- Cre+;Kitlfl/fl mice, the Kitl gene was specifically knocked down in marrow adipocytes (Online Supplementary Figure S2C). We could observe a significant loss of Kitl transcript in the flushed BM (Figure 2B) and SCF protein in the BM supernatant (Figure 2C), suggesting that MAT is a major source of SCF in the BM.
We then quantified hematopoietic stem and progenitor cells (HSPC) in the BM of Adipoq-Cre+;Kitlfl/fl mice by flow cytometry (Online Supplementary Figure S3).12,39 We first characterized the Adipoq-Cre+ line and could not observe any potential Cre-specific defects in BM cellularity or HSPC numbers when compared to wild-type mice (Online Supplementary Figure S4). Thus, Kitlfl/fl mice were used as controls for comparison in the following experiments. Loss of SCF specifically in adipocytes reduced marrow cel- lularity in male mice (Figure 2D). The frequency and also absolute number of lineage–Sca–1+c-Kit+ (LSK) HSPC, phe- notypic long-term (LT)-HSC (e.g. CD150+CD48– LSK cells), and myeloid progenitors (MP) were all down-regu- lated in Adipoq-Cre+; Kitlfl/fl male mice (Figure 2E and F). Within MP, common myeloid progenitors (CMP), megakaryocyte-erythrocyte progenitors (MEP), and gran- ulocyte-monocyte progenitors (GMP) all showed decreased frequency and number (Figure 2G and H). Interestingly, there was a reduction in the ratio of MEP to GMP (Figure 2I), suggesting that the extent of dependence on SCF varies between different myeloid progenitors. On the other hand, common lymphoid progenitors (CLP) maintained their number (Figure 2J) and the ratio of CMP/CLP was reduced (Figure 2K) in Adipoq-Cre+;Kitlfl/fl mice. Colony formation assay showed that BM HSPC, though reducing their numbers in the SCF-deficient envi- ronment, did not show functional decline when assessed in a complete medium in vitro (Figure 2L). It suggests that loss of adipose SCF results in defects in the niche environ- ment but not intrinsically in HSPC. Whether BM adipocyte-derived SCF supports the long-term prolifera- tion and self-renewal of HSPC requires future investiga- tion. Hematopoietic stresses can mobilize HSPC outside the BM to sites like the spleen to expand hematopoiesis.42 We found that spleen in Adipoq-Cre+;Kitlfl/fl mice was slight- ly heavier (Figure 2M) and splenic cells from Adipoq- Cre+;Kitlfl/fl mice formed significantly more colony-forming units (CFU) in vitro (Figure 2N), indicating a compensatory induction of splenic hematopoiesis when BM hematopoiesis was defective.
Similar phenotypes were observed in female mice. There was a trending decline in BM cellularity in Adipoq- Cre+;Kitlfl/fl female mice, compared to their control counter- parts (Online Supplementary Figure S5A). Both the absolute number and frequency of LSK cells, MP, CMP, MEP, and GMP were declined when SCF was absent (Online Supplementary Figure S5B and C). CLP remained unchanged (Online Supplementary Figure S5B and C). Adipoq-Cre+;Kitlfl/fl female mice also had reduced ratio of MEP to GMP (Online Supplementary Figure S5D). These data demon-
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haematologica | 2019; 104(9)