Osteopontin is required for the early onset of high fat diet-induced insulin resistance in mice

Abstract

Background: Insulin resistance is manifested in muscle, adipose tissue, and liver and is associated with adipose tissue inflammation. The cellular components and mechanisms that regulate the onset of diet-induced insulin resistance are not clearly defined.

Methodology and principal findings: We initially observed osteopontin (OPN) mRNA over-expression in adipose tissue of obese, insulin resistant humans and rats which was normalized by thiazolidinedione (TZD) treatment in both species. OPN regulates inflammation and is implicated in pathogenic maladies resulting from chronic obesity. Thus, we tested the hypothesis that OPN is involved in the early development of insulin resistance using a 2-4 week high fat diet (HFD) model. OPN KO mice fed HFD for 2 weeks were completely protected from the severe skeletal muscle, liver and adipose tissue insulin resistance that developed in wild type (WT) controls, as determined by hyperinsulinemic euglycemic clamp and acute insulin-stimulation studies. Although two-week HFD did not alter body weight or plasma free fatty acids and cytokines in either strain, HFD-induced hyperleptinemia, increased adipose tissue inflammation (macrophages and cytokines), and adipocyte hypertrophy were significant in WT mice and blunted or absent in OPN KO mice. Adipose tissue OPN protein isoform expression was significantly altered in 2- and 4-week HFD-fed WT mice but total OPN protein was unchanged. OPN KO bone marrow stromal cells were more osteogenic and less adipogenic than WT cells in vitro. Interestingly, the two differentiation pathways were inversely affected by HFD in WT cells in vitro.

Conclusions: The OPN KO phenotypes we report reflect protection from insulin resistance that is associated with changes in adipocyte biology and adipose tissue inflammatory status. OPN is a key component in the development of HFD-induced insulin resistance.

Figure 1. Adipose tissue OPN RNA levels are elevated in obese rats and humans and are suppressed by pioglitazone treatment.

(A) Epididymal white adipose tissue OPN RNA levels from Zucker lean, obese, and pioglitazone-treated obese rats. 6 rats per group. (B) Subcutaneous white adipose tissue OPN RNA levels in lean subjects with normal insulin sensitivity and obese subjects with insulin resistance, before (black bars) and after (grey bars) pioglitazone treatment. 4–7 subjects per group. Values are averages ± standard error. * p<0.05 vs lean; # p<0.05 vs obese, before treatment. (C) Correlation between baseline adipose tissue OPN RNA levels and Rd in lean (filled symbols) and obese subjects (open symbols).

Figure 2. OPN KO mice are protected from HFD-induced insulin resistance.

Euglycemic hyperinsulinemic clamp study results are shown. (A) Ginf, (B) GDR, and (C) HGO rates in WT (black bars) and OPN KO (grey bars) mice fed normal chow or HFD. Values are averages ± standard error. 7–9 mice per group. * p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched, normal chow.

Figure 3. Insulin-stimulated Akt phosphorylation is enhanced in HFD-fed OPN KO mice.

Phosphorylation of Ser⁴⁷³-Akt (pS-Akt) after 15 min in vivo insulin stimulation was measured in tissue lysates by ELISA and western blotting. Ratios of pS-Akt∶total Akt protein are normalized to basal levels in WT controls. WT fed HFD (black bars), OPN KO fed HFD (grey bars). A representative western blot from each tissue is shown directly above the matching bar graph. Muscle – gastrocnemius, scWAT – subcutaneous white adipose tissue, eWAT – epididymal white adipose tissue. Values are averages ± standard error. 8–10 mice per group. * p<0.05 vs WT.

Figure 4. Adipocyte hypertrophy is blunted in OPN KO mice.

(A) Representative histological images of eWAT from mouse groups. (B) Quantitation of subcutaneous WAT (scWAT) and epididymal WAT (eWAT) adipocyte size. WT scWAT (black bars), WT eWAT (dark grey bars), OPN KO scWAT (light grey bars), OPN KO eWAT (white bars). 7 mice per group. Values are averages ± standard error. * p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched, normal chow. (C) Correlation of eWAT fat pad weight and adipocyte size in HFD-fed WT and OPN KO mice. WT (filled symbols), OPN KO (open symbols). 7 mice per group. (D) GDR normalized to eWAT cell size in HFD-fed WT (black bars) and OPN KO (grey bars) mice. 5–6 mice per group. Values are averages ± standard error. * p<0.05 vs WT.

Figure 5. HFD-induced elevation of plasma leptin levels is blunted in OPN KO mice.

(A) Leptin levels were measured by ELISA. WT (black bars) and OPN KO (grey bars) mice fed normal chow or HFD. Values are averages ± standard error. 8–10 mice per group. * p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched, normal chow. (B) Correlation of plasma leptin levels with eWAT adipocyte size in WT and OPN KO mice fed HFD. WT (filled symbols), OPN KO (open symbols). 7 mice per group.

Figure 6. Inflammatory macrophage content and OPN expression in adipose tissue SVCs are blunted in HFD-fed OPN KO mice.

(A) FACS detection of M1-type, F4/80+CD11b+CD11c+ pro-inflammatory macrophages in adipose tissue SVCs. Data are presented as fold change in the percentage of live adipose tissue SVCs expressing all three markers (F4/80, CD11b and CD11c) in the HFD vs normal chow (NC) groups. (B) FACS detection of percent of live adipose tissue SVC F4/80+CD11b+ macrophages that are CD11c+, M1-type. Data are presented as fold change vs strain-matched normal chow (NC) groups. (C) OPN expression increases in adipose tissue SVCs after HFD feeding in WT mice. SVCs were isolated during sample preparation for the FACS studies. NC (white bars), 2-week HFD (grey bars), 4-week HFD (black bars). 2–4 mice per group. *p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched NC, § p<0.05 vs strain-matched 2-week HFD.

Figure 7. HFD-induced elevation of adipose tissue cytokine levels is blunted in OPN KO mice.

Cytokine protein levels were measured in eWAT lysates from WT (black bars) and OPN KO (grey bars) mice fed normal chow or HFD. Values are averages ± standard error. 8–10 mice per group. * p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched, normal chow.

Figure 8. HFD alters adipose tissue OPN isoform expression.

(A) Representative western blot of OPN isoforms with apparent molecular weights of approximately 40 and 55kD. (B) Paired ratios of 40kD∶55kD isoforms in all WT samples. (C) Sum of 40 and 55kD isoforms in all WT samples. Values are averages ± standard error. 4–10 mice per group. ^# p<0.05 vs normal chow (NC).

Figure 9. OPN KO and HFD effects on osteogenic and adipogenic differentiation of bone marrow stromal cells.

BMSCs from the bone marrow of WT and OPN KO mice fed NC or HFD were cultured for 14 days and then subjected to osteogenic or adipogenic differentiation cocktails for the number of days shown. (A) Osteogenic differentiation was gauged by Akp2 and OSX RNA expression. Insert bar graphs are data from WT BMSCs shown separately. (B) Adipogenic differentiation was gauged by PPARγ RNA expression. Experiments were conducted in triplicate using BMSCs isolated from 4 mice per group. Gene expression data for all genes was normalized to GAPDH RNA expression. WT mice fed NC (black bars), WT mice fed HFD (white bars), OPN KO mice fed NC (dark grey bars), OPN KO mice fed HFD (light grey bars). Values are averages ± standard error. * p<0.05 vs diet-matched WT, # p<0.05 vs strain-matched, normal chow.