P-selectin glycoprotein ligand-1 deficiency is protective against obesity-related insulin resistance

Abstract

Objective: An inflammatory process is involved in the mechanism of obesity-related insulin resistance. Recent studies indicate that monocyte chemoattractant protein-1 (MCP-1) is a major chemokine that promotes monocyte infiltration into adipose tissues; however, the adhesion pathway in adipose tissues remains unclear. We aimed to clarify the adhesion molecules that mediate monocyte infiltration into adipose tissue.

Research design and methods: We used a DNA microarray to compare the gene expression profiles in epididymal white adipose tissues (eWAT) between db/db mice and C57/BL6 mice each fed a high-fat diet (HFD) or a low-fat diet (LFD). We investigated the change of insulin resistance and inflammation in eWAT in P-selectin glycoprotein ligand-1 (PSGL-1) homozygous knockout (PSGL-1⁻(/)⁻) mice compared with wild-type (WT) mice fed HFD.

Results: DNA microarray analysis revealed that PSGL-1, a major ligand for selectins, is upregulated in eWAT from both db/db mice and WT mice fed HFD. Quantitative real-time RT-PCR and immunohistochemistry showed that PSGL-1 is expressed on both endothelial cells and macrophages in eWAT of obese mice. PSGL-1⁻(/)⁻ mice fed HFD showed a remarkable reduction of macrophage accumulation and expression of proinflammatory genes, including MCP-1 in eWAT. Moreover, adipocyte hypertrophy, insulin resistance, lipid metabolism, and hepatic fatty change were improved in PSGL-1⁻(/) ⁻mice compared with WT mice fed HFD.

Conclusions: These results indicate that PSGL-1 is a crucial adhesion molecule for the recruitment of monocytes into adipose tissues in obese mice, making it a candidate for a novel therapeutic target for the prevention of obesity-related insulin resistance.

FIG. 1.

A: Metabolic characteristics of db/db and WT mice. Metabolic parameters of 8-week-old WT mice (□) and db/db mice (■) are shown. B: Gene expression in epididymal fat from 8-week-old WT mice (□) and db/db mice (■) analyzed by quantitative real-time RT-PCR. Data are means ± SE. *P < 0.05, **P < 0.005 vs. WT. n = 10 for each group.

FIG. 2.

A: Immunohistochemical localization of PSGL-1, macrophages, and endothelial cells in adipose tissue. Epididymal fat pads from 8-week-old db/db mice and WT mice were stained with anti–MAC-3 (left-hand panels) and anti–PSGL-1 antibodies (right-hand panels). Macrophages and PSGL-1 expressed around the small vessels in the interstitium of adipose tissue in db/db mice are shown. The scale bars represent 50 μm. B: Double immunofluorescence staining of adipose tissue from db/db mice with the antibodies against PSGL-1 (green) and leukocyte (CD45, red). PSGL-1 and CD45 were stained in the interstitium of adipose tissue and are colocalized in the merged picture. C: Double immunofluorescence staining of adipose tissue from db/db mice with the antibodies against PSGL-1 (green) and endothelial cell (CD31, red). PSGL-1 and CD31 were stained along small vessels of adipose tissue and are colocalized in the merged picture. D–F: The expression of PSGL-1 on cells in WT mice and db/db mice was analyzed using flow cytometry. D: The expression of PSGL-1 in PBMCs. E: The expression of PSGL-1 in F4/80⁺ macrophages in the SVF from adipose tissue. F: The expression of PSGL-1 in CD31⁺ endothelial cells in the SVF from adipose tissue. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

A: Metabolic characteristics of C57/BL6 mice fed LFD or HFD from 7 to 19 weeks old. Metabolic parameters of mice fed LFD diet (○, □) or HFD (●, ■) are shown (n = 9 [LF]; n = 11 [HF]). B: Blood glucose level (left-hand panel) and plasma insulin levels (right-hand panel) during the glucose tolerance test (1.2 g/kg body mass) (n = 9 [LF], ○; n = 11 [HF], ●). C: Blood glucose level during the insulin tolerance test (0.7 units/kg body mass) (n = 9 [LF], ○; n = 11 [HF], ●). D: Gene expression in epididymal fat from C57/BL6 mice fed a LFD (□) or HFD (■) from 7 to 19 weeks old analyzed by quantitative real-time RT-PCR (n = 9 [LF], n = 10 [HF]). Data are means ± SE. *P < 0.05 vs. LFD, **P < 0.005 vs. LFD.

FIG. 4.

A: Metabolic characteristics of WT mice and PSGL-1^−/− (KO) mice fed HFD from 7 to 17 weeks old. Body composition and food intake in WT mice (□) and PSGL-1^−/− mice (■) fed HFD (n = 7 [WT-HF]; n = 8 [KO-HF]) is shown. B: Metabolic parameters of WT mice (□) and PSGL-1^−/− mice (■) fed HFD (n = 7 [WT-HF]; n = 8 [KO-HF]). C: Blood glucose level (upper panel) and plasma insulin levels (lower panel) during the glucose tolerance test (1.2 g/kg body mass) (n = 9 [WT-HF], ○; n = 8 [KO-HF], ●). D: Blood glucose level during the insulin tolerance test (0.7 units/kg body mass) (n = 9 [WT-HF], ○; n = 8 [KO-HF], ●). Data are means ± SE. *P < 0.05 vs. WT-HFD, **P < 0.005 vs. WT-HFD. E: Equal amounts of protein in total lysates of liver and muscle were immunoblotted with anti–phospho-Akt (pAkt) and anti-Akt antibodies. The relative ratio of Akt phosphorylation was calculated after normalization with the Akt signal (n = 5 [WT-HF], □; n = 5 [KO-HF], ■). Data are means ± SE. *P < 0.05 vs. WT-HFD. FFA, free fatty acid.

FIG. 5.

A: Periodical acid Schiff staining of epididymal fat sections from WT mice (left-hand panel) and PSGL-1^−/− (KO) mice (right-hand panel) fed HFD for 10 weeks. The scale bars represent 100 μm. B: Distribution of adipocyte size in epididymal fat tissues from WT mice (□) and PSGL-1^−/− mice (■). Data are the mean from analysis of six high-power fields from each of five mice. C: Immunohistochemical detection of Mac-3 in epididymal fat tissue from WT mice (upper panels) and PSGL-1^−/− mice (lower panels) fed HFD. Macrophage infiltration into epididymal fat tissue decreased in PSGL-1^−/− mice. Scale bars, 50 μm. D: Gene expression of F4/80, CD11c, IL-10, MCP-1, IL-6, iNOS, leptin, and LPL in epididymal fat tissues from WT mice (□) and PSGL-1^−/− mice (■) fed HFD analyzed by quantitative real-time RT-PCR (n = 7 [WT-HF]; n = 7 [KO-HF]). Data are means ± SE. *P < 0.05, **P < 0.01, ***P < 0.0001 vs. WT-HFD. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

A: Liver weight (left) and hepatic triglyceride (right) in WT mice (□) and PSGL-1^−/− (KO) mice (■) fed HFD for 10 weeks (n = 5 [WT-HF]; n = 8 [KO-HF]). Data are means ± SE. *P < 0.05 vs. WT-HFD. B: Gene expression of CD68 in liver from WT mice (□) and PSGL-1^−/− mice (■) fed HFD diet analyzed by quantitative real-time RT-PCR (n = 5 [WT-HF]; n = 8 [KO-HF]). Data are means ± SE. C: Hematoxylin and eosin stain. Hepatic steatosis is prominent in the liver of WT mice fed HFD. The scale bars represent 100 μm. (A high-quality digital representation of this figure is available in the online issue.)