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ABSTRACT TITLE�Author Name(s)�Author Affiliation(s)

Abstract

Low-grade chronic inflammation may be a factor in the development of insulin resistance. Studies suggest that chronic inflammation may be due in part to changes in composition and function of gut microflora, which provide an intestinal barrier preventing bacterial lipopolysaccharide (LPS) release. Animal and human studies demonstrate that obesity increases gut permeability leading to elevated plasma LPS levels resulting in inflammation and metabolic dysfunction. We hypothesized that insulin resistance could be produced by inflammatory factors secreted by macrophages when exposed to gut-released LPS. We examined the extracellular signal-regulated kinase (ERK) signaling pathway, which may be responsible for the macrophage inflammatory response. We examined whether inhibition of ERK activity blocked LPS-mediated responses in bone marrow derived macrophages (BMDM). To determine which ERK isoform is involved in the regulation of inflammatory factor production, we used siRNA to knockdown ERK1, ERK2, or both. BMDM were treated with LPS (100 ng/ml, 6hr). LPS induced MCP-1, TNF-α, IFN-β, and RANTES production of 851 ± 73 pg/ml,106371±18250 pg/ml, 454 ± 46 pg/ml, and 11827 ± 1168 pg/ml respectively. Knockdown of ERK1 decreased the release of TNF-α and RANTES to 26534 ± 1471 pg/ml and 5938 ± 806 pg/ml, respectively. Knockdown of ERK2 decreased the release of TNF-α and RANTES to 11600 ± 3610 pg/ml and 7289 ± 265 pg/ml, respectively. Knockdown of either ERK1 or ERK2 did not decrease the release of MCP-1 and IFN-β. However, double knockdown of ERK1 and ERK2 had the greatest inhibition of MCP-1, TNF-α, IFN-β, and RANTES release (549 ± 32 pg/ml, not detected 31 ± 1 pg/ml, and 1540 ± 240 pg/ml, respectively). In summary, knockdown of both ERK isoforms is necessary to completely abrogate the LPS effect in macrophages. We propose that ERK positively regulates LPS-mediated inflammatory responses and inhibition of ERK signaling may protect against development of insulin resistance.

Methods

Hypothesis

Our hypothesis is that insulin resistance could be produced by inflammatory factors secreted by macrophages when exposed to gut-released LPS. ERK1 and ERK2 positively regulate the immune responses mediated downstream of Toll-like Receptor 4 (TLR4).

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• Bone marrow cells were isolated from femurs of 6 – 8 week-old male WT C57Bl/6J mice. Cells were cultured in L929 conditioned medium for differentiation into macrophages. By day 7, cells were 90% macrophages (bone marrow derived macrophages, BMDM) as determined by CD11b+ and F4/80+ via flow cytometry.

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• BMDM were transfected with a control siRNA, mouse ERK1 or ERK2 siRNA or both ERK1 and ERK2 siRNA using a Lipofectamine 3000 kit per manufacture’s instructions. Cells were lysed and analyzed by Western blotting 48 h after transfection.

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• For quantification of MCP-1, TNF-α, RANTES, and IFN-β production, BMDM were treated with LPS (100 ng/ml, 6 h) . Supernatants were analyzed for cytokine and chemokine production by multiplex immunoassay.

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• Statistical analyses of data were performed using one-way ANOVA with multiple comparisons in GraphPad Prism 8 software.

Conclusions

Corresponding author:

email address@urschool.edu

Our findings show that knockdown of ERK1 or ERK2 or both blocked LPS-mediated cytokine and chemokine production in macrophages. ERK1 and ERK2 positively regulate the production of MCP-1, TNF-α, IFN-β, and RANTES. Our results suggest a new role for macrophage ERK1 and ERK2 in regulating LPS-induced immune responses. Our studies contribute new knowledge in understanding the macrophage ERK signaling mechanisms associated with the development and progression of chronic inflammatory diseases such as insulin resistance.

Acknowledgements

This project is supported by…..

Figure 1. ERK1 and ERK2 positive regulation of LPS-mediated immune responses. Activation of TLR4 by LPS leads to the activation of the ERK1/2 module which regulates the production of MCP-1 and TNF-α. TLR4 is internalized to initiate a second signaling cascade leading to the production of IFN-β and RANTES which may also be regulated by the ERK1/2 module. Activation by LPS of both TLR4 pathways may lead to the development of insulin resistance.

Future Studies

• Macrophage specific ERK1 or ERK2 knockout mouse models will be generated.

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• The approach of Cre-LoxP will be used to excise the exons for genes encoding ERK1 (mapk3) or ERK2 (mapk1).

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• The mouse models will undergo a high fat diet to induce insulin resistance and type 2 diabetes.

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• We will assess the role of macrophage ERK signaling deficiency in skeletal muscle inflammation and insulin resistance.

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