SPARC-null mice exhibit increased adiposity without significant differences in overall body weight

Abstract

Secreted protein acidic and rich in cysteine/osteonectin/BM-40 (SPARC) is a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix (ECM). The absence of SPARC in mice gives rise to aberrations in the structure and composition of the ECM that result in generation of cataracts, development of severe osteopenia, and accelerated closure of dermal wounds. In this report we show that SPARC-null mice have greater deposits of s.c. fat and larger epididymal fat pads in comparison with wild-type mice. Similar to earlier studies of SPARC-null dermis, we observed a reduction in collagen I in SPARC-null fat pads in comparison with wild-type. Although elevated levels of serum leptin were observed in SPARC-null mice, their overall body weights were not significantly different from those of wild-type counterparts. The diameters of adipocytes from SPARC-null versus wild-type epididymal fat pads were 252 +/- 61 and 161 +/- 33 microm (means +/- SD), respectively, and there was an increase in adipocyte number within SPARC-null fat pads in comparison with wild-type pads. Thus the absence of SPARC appears to result in an increase in the size of individual adipocytes as well as an increase in the number of adipocytes per fat pad. In fat pads isolated from wild-type mice, SPARC mRNA was associated with both the stromal/vascular and adipocyte fractions. We propose that SPARC limits the accumulation of adipose tissue in mice in part through its demonstrated effects on the regulation of cell shape and production of ECM.

Figure 1

SPARC-null mice display increased accumulation of adipose tissue. Epididymal fat pads removed from 7-month-old wild-type (A) and SPARC-null mice (B) are shown. Although the size of the fat pad was enlarged in the absence of SPARC, the overall body weights of the mice were not significantly different (wild type, 29 g; SPARC null, 30 g). (C) An increased adipose component was also observed in skin. Fragments of skin isolated from 6-month-old SPARC-null (−/−) animals floated in solution, whereas those from wild-type (+/+) mice did not. Skin was removed from three separate animals of each genotype. The skins of SPARC-null animals 5 months old and older floated consistently when wild-type counterparts did not in >10 separate experiments. Sections from 5-month-old wild-type (D) and SPARC-null (E) mice stained with Masson's trichrome reagent are shown also. (Bar, 100 μm.)

Figure 2

SPARC-null fat pads contain less collagen I than equivalent amounts of wild-type fat pads. Equal amounts of wild-type and SPARC-null fat tissue isolated from 6-month-old animals were homogenized and extracted with acetic acid (see Materials and Methods). Equal volumes of wild-type (WT) and SPARC-null extracts were treated with pepsin, and the products were separated by SDS/PAGE. Wild-type extracts (lane 1) contained higher amounts of collagen α1(I) and α2(I) (arrows) in comparison with SPARC-null extracts (lanes 2 and 3) (shown are two separate extractions from SPARC-null pads). The experiment shown is representative of four separate extractions. Equivalent amounts of total protein were present in wild-type and SPARC-null homogenates as determined by Coomassie blue stain of control samples (without pepsin) and immunoblot analysis of tubulin (not shown).

Figure 3

SPARC-null and wild-type mice do not exhibit significant differences in overall body weight but do show increased levels of serum leptin with advancing age. (A) SPARC-null (●) and wild-type (□) mice housed under identical conditions and fed a 4% fat chow diet ad libitum did not show differences in overall weight gain over time. Ten mice or more were used to generate each time point. Error bars represent the standard deviation of the mean. (B) SPARC-null mice show increased levels of serum leptin with advancing age. Wild-type (open bars) and SPARC-null (black bars) mice were subjected to cardiac puncture 4–6 h after food deprivation. Whereas insulin, glucose, cholesterol, high-density lipoprotein, and triglyceride levels showed no significant differences between genotypes, leptin levels at older ages were increased substantially over those of age-matched, wild-type controls. Leptin levels (ng/ml) were normalized to individual body weights and are expressed as percentages of total body weight. The average weights of the mice at 10 weeks were 22.4 g (±1) (wild type) and 22.8 g (±1.4) (SPARC-null) and at >6 months 22.9 g (±3) (wild type) and 24.7 g (±3.4) (SPARC-null). Four mice for each genotype at 10 weeks and at least seven mice at >6 months contributed to the results. Error bars represent the standard error of the mean. *, P = 0.003 for >6 months.

Figure 4

SPARC-null epididymal fat pads contain significantly more large adipocytes than wild-type fat pads. Fat pads were treated with collagenase, and adipocytes were fractionated by centrifugation. Isolated adipocytes were photographed on a hemocytometer slide (  , wild-type adipocytes; ■, SPARC-null adipocytes), and captured images were imported into nih IMAGE software for quantification. (Scale bars, 200 μm.) Wild-type (423 cells, gray bars) and SPARC-null (717 cells, black bars) adipocytes contributed to the distribution. The histogram was generated from the data reported in Tables 2 and 3.

Figure 5

SPARC mRNA was expressed predominantly by stromal/vascular cells in comparison with adipocytes. Fat pads were fractionated into stromal/vascular and adipocyte compartments, and RNA was isolated for RT-PCR analysis (see Materials and Methods). The purity of the stromal/vascular and adipocyte fractions was confirmed by analysis of known tissue-specific genes. The fat fraction contained mRNA encoding peroxisome proliferator-activated receptor γ (PPARγ) and leptin, whereas the stromal/vascular component contained mRNA encoding vascular endothelial (VE)-cadherin (endothelial cells) and smooth muscle (SM) α-actin (smooth muscle cells). That the stromal/vascular component was devoid of detectable fat-specific genes at the number of cycles used indicated very little, if any, fat contamination in this fraction. However, the fat fraction did contain low levels of stromal/vascular-specific mRNAs. The mRNA encoding cyclophilin A, a constitutively expressed peptidyl-prolyl isomerase (25), was used as a control to ensure equal starting amounts of mRNA from each fraction. WT, wild type.