Preadipocyte transplantation: an in vivo study of direct leptin signaling on adipocyte morphogenesis and cell size

Abstract

Leptin has profound effects on adipose tissue metabolism. However, it remains unclear whether direct leptin signaling in adipocytes is involved. We addressed this question by transplanting inguinal adipose tissue stromal vascular cells (SVCs) from 4- to 5-wk-old wild-type (WT) and leptin receptor-deficient [Lepr(db/db) (db)] mice to inguinal and sternal subcutaneous sites in Ncr nude mice. Both WT and db SVCs gave rise to mature adipocytes with normal morphologies 3 mo after the transplantation. The average adipocyte size (microm(2)/cell) was not significantly different between WT and db transplants at either the inguinal (1,630 +/- 103 vs. 1,491 +/- 74) or the sternal site (1,788 +/- 107 vs. 1,596 +/- 92). Expression levels of beta(3)-adrenergic receptor, a major mediator of lipid mobilization, were indistinguishable between WT and db transplants and similar to those of the hosts. Additionally, adipocyte sizes of inguinal transplants and endogenous inguinal adipose tissues were closely correlated (beta = 0.76, P < 0.001), suggesting that the metabolic milieu of host mice has significant effects on adipocyte size of the transplants. Contrary to the indifference to donor's Lepr genotype, adipocyte size of the transplants was significantly affected by the donor's sex in a leptin receptor-dependent manner. In WT transplants, female SVCs gave rise to smaller adipocytes than male SVCs (1,358 +/- 127 vs. 2,133 +/- 171, P < 0.05). However, this sex difference was not significant in db transplants (1,537 +/- 121 vs. 1,655 +/- 140, P = 0.22). These data suggest that: 1) long-form receptor-mediated direct leptin signaling has no significant cell-autonomous effect on adipocyte differentiation and metabolism in adult mice, 2) sex may affect adipocyte metabolism via genetic and/or epigenetic programming, and 3) leptin may potentiate sexual dimorphism in adipocyte metabolism.

Fig. 1.

Adipose tissue morphogenesis at the sternal (sTx) and inguinal (iTx) transplantation sites. Gross morphology of a sTx (A) and an iTx with the endogenous inguinal adipose tissue (iAT) (B). The transplants were derived from iAT SVCs of the wild-type (WT) male C57BL/6J donor mice. The arrow in A points to blood vessels in the sTx. The arrowhead in B points to the iTx, which is distinct from the paler endogenous iAT by virtue of its redder hue. C–E: tissue morphology of the transplants. Hematoxylin and eosin (HE) staining of a sTx (C), green fluorescent protein (GFP)-immunostaining of an iTx and the surrounding endogenous iAT (D), and HE staining of the endogenous iAT of a host mouse (E). Adipocytes of the iTx are identified by their GFP-staining, as opposed to the GFP-negative adipocytes of the host (D).

Fig. 2.

Quantitative analysis of adipocyte size in the WT and db inguinal transplants. GFP-immunostaining of representative WT (A) and leptin receptor-deficient [Lepr^db/db(db); B] iTx. Donor SVCs were isolated from inguinal adipose tissues of 4- to 5-wk-old GFP-transgenic WT and Lepr^db/db littermates. The bar represents 0.1 mm in two 0.05-mm units. C: quantitative measurements of adipocyte size (the average cross-sectional area of each adipocyte, μm²/cell) in the WT inguinal transplants (iTx-WT, n = 8) and db inguinal transplants (iTx-db, n = 8), and the endogenous iAT of the hosts (iAT-host) (means ± SE). D: correlation in adipocyte size between the iTx and the endogenous iAT tissue (r = 0.76, P < 0.001).

Fig. 3.

Comparison of β₃-adrenergic receptor (β₃-AR) expression in iAT-WT and iAT-db. β₃-AR immunostaining of the representative pairs of iTx-WT (A) and iTx-db (B), the endogenous iAT-host (C), the native iAT of male 8-wk-old WT mice (iAT-WT) (D), and Lepr^db/db mice (iAT-db) (E). The bar represents 0.1 mm. F: quantification of average β₃-AR expression levels on adipocyte plasma membrane of the iTx-WT and iTx-db (n = 8 per genotype), and of the native iAT of the WT and db mice (n = 5 per genotype). The densities of β₃-AR immunostaining in the db transplants and Lepr^db/db iAT are expressed relative to those of the WT transplants and the iAT-WT, respectively. G: correlation of β₃-AR expression levels (in arbitrary units) between the inguinal transplants (iTx), and the hosts' iAT tissues (r = 0.82, P < 0.05 for WT; and r = 0.73, P < 0.05. for db).

Fig. 4.

Effects of sex and Lepr-genotype of donor SVCs on adipocyte size of sTxs. Hematoxylin and eosin-stained tissue sections of representative the WT female (WT-F) (A), WT male (WT-M) (B), db female (db-F) (C), and db male (db-M) (D) sTxs. The bar represents 0.1 mm in two 0.05-mm units. E: quantitative measurements of adipocyte size in sTxs grouped by sex and Lepr-genotype of SVC donors (n = 18). Data are expressed as unweighted means ± SE. A statistically significant difference (P < 0.05) between WT-M and WT-F transplants is indicated by post hoc Tukey tests.