Adipocyte Gamma-Secretase Inhibition May Alter IL-6 Expression and Trafficking

Abstract

Adipocyte-mediated inflammatory signaling has been proposed to alter adipose physiology in obesity. Activation of adipocytes can increase secretion of inflammatory cytokines such as IL-6. IL-6 secretion is regulated through changes in expression of both IL-6 itself, as well as its specific SNARE trafficking machinery. Recent data suggests that the γ-secretase enzyme complex is involved in both adipose insulin sensitivity and immune system regulation. We therefore hypothesized that adipocyte IL-6 secretion could be altered by γ-secretase inhibition. To explore this hypothesis, 3T3-L1 adipocytes were stimulated with lipopolysaccharide (LPS) in the absence or presence of a γ-secretase inhibitor. Changes in gene expression of inflammatory cytokines and SNARE complex components, including IL-6, MCP1, Stx6, Vti1b, SCAMP5, VAMP3, Stx4, and SNAP23 were examined by qRT-PCR, and immunofluorescence (IF) was used to determine adipocyte IL-6 expression and localization. IL-6 secretion was analyzed by ELISA. Transcription of IL-6 and several of the SNARE complex proteins were not significantly affected by γ-secretase inhibition. However, total cellular IL-6 increased by LPS stimulation by IF, which could be blocked by pre-treatment with a γ-secretase inhibitor. IL-6-associated SNARE component Vti1b mRNA levels were specifically affected by overnight γ-secretase inhibition. LPS stimulation increased Vti1b transcription 3-fold, which was completely blocked by γ-secretase inhibition (p<0.01, n=6 per condition). Increasing amounts of LPS (range 0 to 10 ng/mL) for 6 hours led to an expected increase in IL-6 secretion, which was significantly blocked by overnight pre-treatment with a γ-secretase inhibitor. LPS-stimulated IL-6 secretion could also be decreased by short-term (30 minute) γ-secretase blockade. Taken together, this data suggests that the γ-secretase enzyme complex may be involved in a variety of signaling cascades controlling both the production and secretion of IL-6 in adipocytes. Disclosure D. Sparling: None. N. McCullough: None.