Evidence for the regulatory role of lipocalin 2 in high-fat diet-induced adipose tissue remodeling in male mice

Abstract

Lipocalin 2 (Lcn2) has previously been characterized as an adipokine/cytokine playing a role in glucose and lipid homeostasis. In this study, we investigate the role of Lcn2 in adipose tissue remodeling during high-fat diet (HFD)-induced obesity. We find that Lcn2 protein is highly abundant selectively in inguinal adipose tissue. During 16 weeks of HFD feeding, the inguinal fat depot expanded continuously, whereas the expansion of the epididymal fat depot was reduced in both wild-type (WT) and Lcn2(-/-) mice. Interestingly, the depot-specific effect of HFD on fat mass was exacerbated and appeared more pronounced and faster in Lcn2(-/-) mice than in WT mice. In Lcn2(-/-) mice, adipocyte hypertrophy in both inguinal and epididymal adipose tissue was more profoundly induced by age and HFD when compared with WT mice. The expression of peroxisome proliferator-activated receptor-γ protein was significantly down-regulated, whereas the gene expression of extracellular matrix proteins was up-regulated selectively in epididymal adipocytes of Lcn2(-/-) mice. Consistent with these observations, collagen deposition was selectively higher in the epididymal, but not in the inguinal adipose depot of Lcn2(-/-) mice. Administration of the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (Rosi) restored adipogenic gene expression. However, Lcn2 deficiency did not alter the responsiveness of adipose tissue to Rosi effects on the extracellular matrix expression. Rosi treatment led to the further enlargement of adipocytes with improved metabolic activity in Lcn2(-/-) mice, which may be associated with a more pronounced effect of Rosi treatment in reducing TGF-β in Lcn2(-/-) adipose tissue. Consistent with these in vivo observations, Lcn2 deficiency reduces the adipocyte differentiation capacity of stromal-vascular cells isolated from HFD-fed mice in these cells. Herein Rosi treatment was again able to stimulate adipocyte differentiation to a similar extent in WT and Lcn2(-/-) inguinal and epididymal stromal-vascular cells. Thus, combined, our data indicate that Lcn2 has a depot-specific role in HFD-induced adipose tissue remodeling.

Figure 1.

A, Lcn2 protein is more abundant in inguinal adipose tissue. B–D, Lcn2 protein expression in Epi-WAT (B), Ing-WAT (C), and serum Lcn2 (D) in normal mice under RCD and HFD conditions. E–J, Time course of RCD or HFD effect on body weight gain (E and F) and inguinal (G and I), and epididymal (H and J) fat mass increase in WT and Lcn2^−/− mice. Results of E–J represent mean ± SE of 7 to 10 animals. *, P < .05; **, P < .01; WT vs Lcn2^−/−.

Figure 2.

A–D, Age effect on fat cell size distribution of epididymal (A and C) and inguinal (B and D) adipose tissue of WT and Lcn2^−/− mice fed RCD. E–H, HFD effect on fat cell size distribution of epididymal (E and G) and inguinal (F and H) fat depots of WT and Lcn2^−/− mice. Results represent mean of 3 animals.

Figure 3.

A and B, The mRNA expression of adipogenic genes in primary inguinal (A) and epididymal (B) adipocytes isolated from WT and Lcn2^−/− mice fed an HFD for 16 weeks. C and D, PPARγ protein expression in Epi-WAT (C) and Ing-WAT (D) of HFD-fed WT and Lcn2^−/− mice. E–H, The mRNA expression of ECM molecules in epididymal adipocytes (E) and SV cells (F) and inguinal adipocytes (G) and SV cells (H) isolated from WT and Lcn2^−/− mice fed an HFD. I, Picrosirius staining of epididymal and inguinal adipose tissue from HFD-fed WT and Lcn2^−/− mice; collagen fibers are stained with red. Results (A and B and E–H) represent mean ± SE of 4 to 6 animals. *, P < .05; **, P < .01; WT vs Lcn2^−/−; #, P < .05; ##, P < .01, H₂O vs Rosi.

Figure 4.

The mRNA expression of ECM molecules in primary epididymal and inguinal adipocytes and SV cells isolated from control and TZD-treated WT and Lcn2^−/− mice. A–D, Primary inguinal SV cells (A), primary inguinal adipocytes (B), primary epididymal SV cells (C), and primary epididymal adipocytes (D) from HFD WT and Lcn2^−/− mice with or without TZD treatment. E and F, TGF-β protein expression in Epi-WAT (E) and Ing-WAT (F) of HFD WT and Lcn2^−/− with or without TZD treatment. Results (A–D) represent mean ± SE of 4 to 6 animals. *, P < .05; **, P < .01; WT vs Lcn2^−/− (KO); #, P < .05; ##, P < .01, H₂O vs Rosi.

Figure 5.

A, Oil-red O staining of inguinal SV cell cultures with or without Rosi treatment during the 9-day differentiation process. B, The mRNA expression of adipogenic genes in differentiated inguinal adipocytes with or without TZD treatment. C, The morphology of epididymal SV cell cultures with or without TZD treatment. D, The mRNA expression of adipogenic genes in differentiated epididymal adipocytes with or without TZD treatment. The morphological results of adipocytes (A and C) represents 4 independent experiments. Results of gene expression represent mean ± SE of 2 independent SV cell cultures from 4 animals per experiment. *, P < .05; **, P < .01, WT vs Lcn2^−/−; #, P < .05; ##, P < .01, H₂O vs Rosi. Abbreviation: Ctrl, control.

Figure 6.

A–D, TZD effect on fat cell size distribution of epididymal (A and B) and inguinal (C and D) adipose tissue of WT and Lcn2^−/− mice. Results represent mean of 3 animals. D–H, Comparison of mRNA expression of adipogenic genes and genes involved in lipid metabolism in epididymal and inguinal adipocytes between HFD-fed WT and Lcn2^−/− mice (E and F) and between HFD-fed Lcn2^−/− mice with and without TZD treatment (G and H). Results represent mean ± SE of 4 to 6 animals. *, P < .05; **, P < .01, WT vs Lcn2^−/−; #, P < .05; ##, P < .01, H₂O vs Rosi.