Oncostatin M promotes lipolysis in white adipocytes

Abstract

Oncostatin M (OSM) is a member of the glycoprotein 130 cytokine family that is involved in chronic inflammation and increased in adipose tissue under obesity and insulin resistance. OSM was shown to inhibit adipogenesis, suppress browning, and contribute to insulin resistance in cultured white adipocytes. In contrast, OSM may have a metabolically favourable role on adipocytes in mouse models of obesity and insulin resistance. However, a putative role of OSM in modulating lipolysis has not been investigated in detail to date. To address this, cultured white adipocytes of mouse or human origin were exposed to 10 or 100 ng/ml of OSM for various time periods. In murine 3T3-L1 cells, OSM stimulation directly activated hormone-sensitive lipase (HSL) and other players of the lipolytic machinery, and dose-dependently increased free fatty acid and glycerol release. In parallel, OSM attenuated insulin-mediated suppression of lipolysis and induced phosphorylation of serine-residues on the insulin receptor substrate-1 (IRS1) protein. Key experiments were verified in a second murine and a human adipocyte cell line. Inhibiton of extracellular signal-regulated kinase (ERK)-1/2 activation, abolished OSM-mediated HSL phosphorylation and lipolysis. In conclusion, OSM signalling directly promotes lipolysis in white adipocytes in an ERK1/2-dependent manner.

Keywords: Adipocyte; cytokine; glycoprotein 130; insulin resistance; lipolysis; oncostatin M.

Figure 1.

Prolonged OSM stimulation induces free fatty acid release (FFA) from 3T3-L1 adipocytes. (a) Relative FFA release in 3T3-L1 adipocytes stimulated with OSM and/or insulin. (b) Magnitude of the effect of insulin on FFA release expressed as a percentage of the corresponding OSM concentration without insulin. (c) Relative glycerol release in 3T3-L1 adipocytes stimulated with OSM and/or insulin. Cells were stimulated with OSM for 24 hours. FFA and glycerol were collected during the last 4 in the presence/absence of OSM and/or insulin (n = 7–8 from four independent experiments). Panel A, two-way ANOVA; panel B, one-way ANOVA, both with Tukey’s post-hoc test.

Figure 2.

Phosphorylation kinetics of lipolytic signalling mediators in adipocytes in response to OSM. (a) Representative Western blots of 3T3-L1 adipocytes after treatment with OSM for 15 minutes or 1, 3 or 6 hours. (b-g) Relative expression of indicated phosphorlylated proteins and G0/G1 switch gene 2 (G0S2) at indicated time points after OSM treatment of 3T3-L1 adipocytes (n = 4 from three biological replicates; two-way ANOVA with Tukey’s post-hoc test). Representative Western blots of a subcutaneous murine cell line (h-i) and human SGBS adipocytes (j-k) stimulated for 15 minutes with OSM and the corresponding quantification of HSL phosphorylation relative to control (n = 6 and n = 3, respectively, both from three biological replicates; one-way ANOVA with Tukey’s post-hoc test). White, light grey and dark grey bars represent vehicle, 10 ng/ml OSM and 100 ng/ml OSM, respectively.

Figure 3.

OSM promotes lipolysis in murine and human white adipocytes. (a-c) Relative free fatty acid (FFA) and/or glycerol release upon OSM stimulation of murine 3T3-L1 or subcutaneous adipocytes, respectively. FFA (n = 6) and glycerol (n = 4) were collected during the last 2 hours of a total of 3 hour treatment with the indicated concentrations of OSM and are presented as fold-change over control after correction for total protein content. (d) Glycerol release in OSM-stimulated human adipocytes collected during 4 or 24 hours (n = 3). Panel A-C, one-way ANOVA; panel D, two-way ANOVA, both with Tukey’s post-hoc test.

Figure 4.

ERK1/2 inhibition prevents OSM-mediated HSL phosphorylation and lipolysis. (a) Western blots displaying ERK1/2, STAT3 and HSL phosphorylation in 3T3-L1 adipocytes stimulated for 15 minutes with OSM and pre-treated with DMSO (vehicle), U0126 or Stattic for 1 hour at the indicated concentrations. (b-c) Release of FFA (n = 3–4 from two biological replicates) and glycerol (n = 6 from three biological replicates) from 3T3-L1 adipocytes relative to controls. Cells were pre-treated with vehicle or 5 µM U0126 for one hour followed by three hours of OSM treatment. Lipolytic products were collected during the last 2 hours and are presented relative to controls after correction for total protein content. Panel B-C, two-way ANOVA with Šídák correction.