Pseudostarvation using the AMPK Activator Metformin downregulates Inflammation in Rheumatoid Arthritis Synovial Tissue
Lorna Gallagher, Ursula Fearon, Douglas J. Veale, David Kane, Luke A. O’Neill and Ronan Mullan
Clinical Medicine, Trinity Biosciences Science Institute, Trinity College Dublin
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 through AMPK activation by hypoglycaemic therapy, i.e. Metformin, reverses these effects.
Our aim is to demonstrate AMPK activation in RA synovial tissues (RAST), and investigate the proinflammatory responses in stimulated primary RA synovial fibroblast cells (RASFCs) following pharmacological AMPK activation by Metformin in vitro.
AMPK, activated and inactivated, and ACC, activated and inactivated (P-AMPK, AMPK, P-ACC & ACC, respectively) expression in RAST and RASFCs were analyzed by immunoblotting. RASFCs were stimulated with LPS/TNF?? (10ng/ml) in the presence of Metformin (0–62.5μM) and assessed for wound healing and invasion capabilities. Supernatants were evaluated for IL-6 and IL-8 production by ELISA. RAST, obtained from RA patients during arthroscopy, were stained by immunohistochemistry for P-AMPK, AMPK, P-ACC and ACC.
P-AMPK is present in RAST and in cultured RASFCs. PAMPK expression was upregulated in the presence of Metformin (10μM and 50μM). Stimulated cells with LPS or TNF?? (10ng/ml), in the presence of Metformin (10μM and 50μM) increased P-AMPK expression, greater than that which was observed following LPS/TNF?? stimulation alone. Additionally, LPS stimulated cells in the presence of Metformin (10μM and 50μM) showed P-ACC expression, which was not observed in LPS stimulated cells alone. Immunohistochemistry staining of RAST showed P-AMPK and PACC staining, indicating that AMPK is activated and is activating ACC downstream. Stimulation of RASFCs with LPS or TNF?? (10ng/ml) in the presence of Metformin (15 –62.5μM) decreased IL-6 and IL-8 production in a dose dependent manner. RASFCs stimulated with LPS/TNF?? (10ng/ml) in the presence of Metformin (15–62.5μM) showed a dose dependent decrease in the ability of the cells to heal an induced wound or invade through matrigel.
RAST and RASFCs are capable of responding to pharmacological alterations in cellular metabolic pathways. Metformin both activates AMPK and downregulates proinflammatory effects in RA. AMPK activation occurs in concert with P-ACC . AMPK activation therapy pathways may therefore be a suitable future strategy in the treatment of Rheumatoid Arthritis.