Date: Sunday, November 8, 2015
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.
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 May 11, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/pseudostarvation-using-the-ampk-activator-metformin-downregulates-inflammation-in-rheumatoid-arthritis-synovial-tissue/