Adipose Tissue Dysfunction Occurs Independently of Obesity in Adipocyte-Specific Oncostatin Receptor Knockout Mice

Abstract

Objective: This study examined the phenotypic effects of adipocyte-specific oncostatin M receptor (OSMR) loss in chow-fed mice.

Methods: Chow-fed adipocyte-specific OSMR knockout (FKO) mice and littermate OSMR^fl/fl controls were studied. Tissue weights, insulin sensitivity, adipokine production, and stromal cell immunophenotypes were assessed in epididymal fat (eWAT); serum adipokine production was also assessed. In vitro, adipocytes were treated with oncostatin M, and adipokine gene expression was assessed.

Results: Body weights, fasting blood glucose levels, and eWAT weights did not differ between genotypes. However, the eWAT of OSMR^FKO mice was modestly less responsive to insulin stimulation than that of OSMR^fl/fl mice. Notably, significant increases in adipokines, including C-reactive protein, lipocalin 2, intercellular adhesion molecule-1, and insulinlike growth factor binding protein 6, were observed in the eWAT of OSMR^FKO mice. In addition, significant increases in fetuin A and intercellular adhesion molecule-1 were detected in OSMR^FKO serum. Flow cytometry revealed a significant increase in leukocyte number and modest, but not statistically significant, increases in B cells and T cells in the eWAT of OSMR^FKO mice.

Conclusions: The chow-fed OSMR^FKO mice exhibited adipose tissue dysfunction and increased proinflammatory adipokine production. These results suggest that intact adipocyte oncostatin M-OSMR signaling is necessary for adipose tissue immune cell homeostasis.

Figure 1. Expression levels of OSMR and gp130 are not altered in non-adipose tissues

Tissue expression of OSMR and gp130 in various adipose depots at the A) gene and B) protein levels in OSMR^fl/fl (CTL) and OSMR^FKO (KO) chow-fed mice. Non-adipose tissue expression of OSMR and gp130 at the C) gene and D) protein levels was also measured. E) Gene expression of OSM itself was also measured in various adipose depots. Total RNA in various tissues was purified and analyzed by real-time PCR. Cyclophilin A (Ppia) was used as an endogenous control. Protein (100 ug for adipose tissues, 50 ug for all other tissues) was subjected to Western blot analysis. Data are shown as mean ± SEM. For PCR, n=8 per genotype (eWAT), n=3–4 per genotype (inguinal WAT (iWAT), brown adipose tissue (BAT), other tissues). ***p < 0.001, *p<0.05 vs. OSMR^fl/fl. AU, arbitrary units.

Figure 2. OSMR^FKO mice are less responsive to an acute OSM challenge

A) Effects of acute OSM or vehicle injection (15 min) on STAT and ERK phosphorylation in eWAT of chow-fed OSMR^fl/fl (n=4) or OSMR^FKO mice (n=4). 75 ug protein was subjected to Western blot analysis. B) Densitometry analyses were conducted using Image Studio software. Data are shown as mean ± SEM and are representative of two independent experiments. AU, arbitrary units.

Figure 3. OSMR^FKO mice exhibit a modest blunting of insulin-induced Akt phosphorylation

A) Body weights, B) 4-hour fasting blood glucose, and C) protein expression levels of Akt, phosphoAkt, and phosphoERK in eWAT from chow-fed OSMR^fl/fl and OSMR^FKO (KO) mice. Body weights and blood glucose levels were not significantly different between genotypes. 100 ug protein was subjected to Western blot analysis. KO mice exhibited a slight diminution in Akt phosphorylation (S473) in eWAT. Densitometry analysis of D) pAkt/Akt ratio was conducted using Image Studio software. For vehicle injections, n=3 per genotype and for insulin injections, n=4 per genotype. Data are shown as mean ± SEM. AU, arbitrary units.

Figure 4. Increased pro-inflammatory adipokine expression in eWAT and serum of OSMR^FKO mice

Protein expression levels of various adipokines in A) eWAT (n=6–7 per genotype) and B) serum (n=3 per genotype) of chow-fed OSMR^fl/fl (grey bars) and OSMR^FKO (black bars) mice. CRP, ICAM-1, IGFBP6, and LCN2 expression levels were significantly increased in eWAT of the OSMR^FKO mice when compared to OSMR^fl/fl mice, and we observed significant increases in FETA and ICAM-1 in the serum of OSMR^FKO mice when compared to OSMR^fl/fl mice. Data are presented as mean ± SEM. *p<0.05 vs. OSMR^fl/fl.

Figure 5. Adipokine gene expression in 3T3-L1 adipocytes in response to OSM treatment and/or ERK inhibition

Fully differentiated 3T3-L1 adipocytes were treated with 1 nM or OSM or vehicle for 6 hours in the presence/absence of the ERK inhibitor U0126. Gene expression levels of A) Lcn2, B) Igfbp6, C) Icam1, Crp, and FetA were assessed, but Crp and FetA were not detected. Total RNA was purified from cells and analyzed by real-time PCR. Ubiquitin b (Ubb) was used as an endogenous control.

Figure 6. Leukocyte accumulation and altered immune cell populations in eWAT of OSMR^FKO mice

A) Epididymal fat pad (eWAT) weight, B) number of SVF cells per gram eWAT and CD45+ leukocytes as determined by flow cytometry in chow-fed OSMR^fl/fl (CTL; grey bars) and OSMR^FKO (KO; black bars) mice (n=4 groups of pooled tissue per genotype; 2–3 mice per pool). Quantification of eWAT stromal vascular cells was performed using flow cytometry and data are presented as % SVCs for C) B cells (CD19+/B220+), D) T cells (CD3+), E) macrophages (CD64+), F) endothelial cells (CD31+), and G) preadipocytes (CD31-/Sca1+). Data presented are mean ± SEM. *p<0.05 and ** p<0.01 vs. OSMR^fl/fl.