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Abstract Number: 21

Pseudostarvation Using the AMPK Activator Metformin Downregulates Inflammation in Rheumatoid Arthritis Synovial Tissue

Lorna Gallagher1, Ursula Fearon2, Douglas J. Veale3, David Kane4, Luke A. O'Neill5 and Ronan Mullan4, 1Clinical Medicine, School of Medicine, Trinity College Dublin, Dublin, Ireland, 2St. Vincent's University Hospital, Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, Dublin 4, Ireland, 3St Vincent's University Hospital, Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, Dublin 4, Ireland, 4Department of Rheumatology, Tallaght Hospital, TCD, Dublin 24, Ireland, 5Inflammation Research, School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

Meeting: 2015 ACR/ARHP Annual Meeting

Date of first publication: September 29, 2015

Keywords: Fibroblasts, metabolism and rheumatoid arthritis (RA)

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Session Information

Date: Sunday, November 8, 2015

Session Title: Cytokines, Mediators, Cell-cell Adhesion, Cell Trafficking and Angiogenesis Poster I

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose: AMP-activated protein kinase (AMPK) is a highly conserved, regulator of cellular energy status. In inflammation, AMPK inactivation is associated with increased glucose consumption through aerobic glycolysis, and up-regulation of pro-inflammatory effector responses. Pseudostarvation of cells via AMPK activation by hypoglycaemic therapy, i.e. Metformin, reverses these effects.  AMPK activity has a cascade effect on other mediators of cell metabolism, including Acetyl-CoA Carboxylase (ACC). Once activated, phosphorylated AMPK (P-AMPK) in turn phosphorylates ACC (P-ACC). AMPK/ACC interactions play a central role in the regulation of cellular lipid homeostasis. Here we demonstrate AMPK activation in RA synovial tissues (RAST), and inhibition of pro-inflammatory responses in stimulated primary RA synovial fibroblast cells (RASFCs) following pharmacological AMPK activation by Metformin in vitro.

Methods: P-AMPK, AMPK, P-ACC and ACC expression in RAST and RASFCs were analyzed by immunoblotting, normalized against β-actin.  RASFCs were stimulated with LPS or TNFα (10 ng/ml) in the presence of Metformin (0 – 62.5 µM) and assessed for wound healing and invasion capabilities. Culture supernatants were evaluated for IL-6 and IL-8 production by ELISA. RAST, obtained from RA patients during arthroscopy, were stained by immunohistochemistry for Phosphorylated ACC (P-ACC) and ACC, and by Immunofluorescence for P-AMPK and AMPK.

Results: Western blot analysis showed that P-AMPK was present in RAST, and cultured RASFCs. P-AMPK expression was upregulated in the presence of Metformin (10 µM & 50 µM).  Stimulating cells, with either LPS or TNFα (10 ng/ml), in the presence of Metformin (10 µM & 50 µM) increased P-AMPK, expression was greater than what was observed following LPS or TNFα stimulation alone. Additionally, LPS stimulated cells in the presence of Metformin (10 µM & 50 µM) showed P-ACC expression, which was not observed in LPS stimulated cells alone. In vitro immunofluorescence staining of RASFCs showed P-AMPK upregulation both in the presence of Metformin (50 µM) compared to basal and TNFα (10 ng/ml) stimulated RASFCs. Positive immunohistochemistry staining of RAST for P-ACC, indicated that AMPK is activated and in its phosphorylated state, with maximal expression localized around blood vessels. Stimulation of RASFCs with LPS or TNFα (10 ng/ml) in the presence of Metformin (15 – 62.5 µM) decreased IL-6 and IL-8 production in a dose dependent manner. RASFCs stimulated with either LPS or TNFα (10 ng/ml) in the presence of Metformin (15 – 62.5 µM) showed a dose dependent decrease in the ability of the cells to heal an induced wound or invade through matrigel.

Conclusion: Our findings indicate that RAST and RASFCs are capable of responding to pharmacological alterations in cellular metabolic pathways. Metformin both activates AMPK and downregulates pro-inflammatory effects in RA. AMPK activation occurs in concert with P-ACC around blood vessels but not in the lining layer where matrix metalloproteinase mediated joint destruction occurs, in keeping with an anti-inflammatory role for activated AMPK.  AMPK activation therapy pathways may therefore be a suitable future strategy in the treatment of Rheumatoid Arthritis.


Disclosure: L. Gallagher, None; U. Fearon, None; D. J. Veale, None; D. Kane, None; L. A. O'Neill, None; R. Mullan, None.

To cite this abstract in AMA style:

Gallagher L, Fearon U, Veale DJ, Kane D, O'Neill LA, Mullan R. Pseudostarvation Using the AMPK Activator Metformin Downregulates Inflammation in Rheumatoid Arthritis Synovial Tissue [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/pseudostarvation-using-the-ampk-activator-metformin-downregulates-inflammation-in-rheumatoid-arthritis-synovial-tissue/. Accessed April 13, 2021.
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