Page 1 of 37
Diabetes
Metformin inhibits monocyte-to-macrophage differentiation via AMPK mediated inhibition of STAT3 activation: Potential role in atherosclerosis Sathish Babu Vasamsetti1, Santosh Karnewar1, Anantha Koteswararao Kanugula1, Avinash T. Raj2, Jerald M. Kumar2* and Srigiridhar Kotamraju1*
1
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad-500007, India. 2 CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India. *Address for Correspondence: Dr. Srigiridhar Kotamraju1, Senior Scientist, Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad-500007, India. +91-40-27191867 Email:
[email protected] Dr. Jerald M. Kumar2, Senior Scientist, CSIR- Centre for Cellular and Molecular Biology, Hyderabad-500007, India. +91-40-27192872 Email:
[email protected]
Diabetes Publish Ahead of Print, published online December 31, 2014
Diabetes
Page 2 of 37
Abstract Monocyte-to-macrophage differentiation is a critical event that accentuates atherosclerosis by promoting an inflammatory environment within the vessel wall. In this study, we investigated the molecular mechanisms responsible for monocyte-to-macrophage differentiation and subsequently, the effect of metformin in regressing Angiotensin-II-mediated atheromatous plaque formation in ApoE-/- mice. AMPK activity was dose- and time-dependently downregulated
during
PMA-induced
monocyte-to-macrophage
differentiation
which
was
accompanied by an up-regulation of pro-inflammatory cytokines production. Interestingly, AMPK activators metformin and AICAR significantly attenuated PMA-induced monocyte-tomacrophage differentiation and pro-inflammatory cytokines production. However, inhibition of AMPK activity alone by compound C was ineffective in promoting monocyte-to-macrophage differentiation in the absence of PMA. On the other hand, inhibition of JNK activity inhibited PMA-induced inflammation but not differentiation. Thereby suggesting that, inflammation and differentiation are independent events. In contrast, inhibition of STAT3 activity inhibited both inflammation and monocyte-to-macrophage differentiation. Metformin and AICAR by decreasing STAT3 phosphorylation via increased AMPK activation caused inhibition of monocyte-to-macrophage differentiation. Metformin attenuated Ang-II-induced atheromatous plaque formation and aortic aneurysm in ApoE-/- mice partly by reducing monocyte infiltration. We conclude that AMPK-STAT3 axis plays a pivotal role in regulating monocyte-tomacrophage differentiation and that AMPK activators by decreasing STAT3 phosphorylation through increased AMPK activity inhibit monocyte-tomacrophage differentiation.
Page 3 of 37
Diabetes
Introduction: Substantial evidence implicates that macrophages are abundantly present at all stages of atherosclerotic disease process (1). Macrophages fuel an inflammatory environment in atherosclerotic neointima by exuding a diverse repertoire of inflammatory mediators (2). Continuous production of pro-inflammatory cytokines and chemokines augment the influx and retention of other inflammatory cells migration of vascular smooth muscle cells from media to intima (2, 3). All these events lead to the proximal exacerbation of arterial damage. Restrain of monocytes /macrophage recruitment into the aortic wall may attenuate the risk of atherosclerosis and hence, strategies to prevent monocyte infiltration and differentiation constitute an attractive approach for the treatment of atherosclerosis and other related vascular disorders.
Metformin, a widely used anti-diabetic drug has beneficial effects in cardiovascular complications besides glycemic control (4). The UKPDS has demonstrated that usage of metformin was associated with a significant decrease in the incidence of myocardial infarction (5).
Another clinical study has reported that metformin administration in diabetic patients
significantly attenuated the progression of carotid artery intima-media thickness, a known index of atherosclerotic progression (6). Earlier studies have shown that the pleiotropic effects of metformin are in part mediated by the activation of adenosine monophosphate-activated protein kinase (AMPK) (7,8). AMPK functions as a fuel gauge, senses the changes in the energy status of the cell and plays a critical role in regulating the systemic energy balance (9, 10). AMPK is activated allosterically by an increase in the intracellular AMP/ATP ratio (10). AMPK is also sensitive to the ‘lipid status’ of a cell and its activation is influenced by the availability of
intracellular fat deposit (11). AMPK-mediated signaling events were down-regulated by LPS,
Diabetes
free fatty acids, high fat diets and lipid infusion in macrophages and endothelial cells (12, 13).
Activation of AMPK signaling pathway suppressed pro-inflammatory responses and promoted macrophage polarization to an anti-inflammatory functional phenotype in macrophages (14). In addition, it was shown that decreased AMPK activity with increased STAT3 in smooth muscle cells promoted RAGE signaling-induced neointima formation in response arterial injury (15). Monocyte-to-macrophage differentiation is characterized by the activation of various metabolic and inflammatory signaling networks. Despite the potential role of AMPK in mediating antiinflammatory effects against various stimulations is known, specifically its role in regulating monocyte-to-macrophage differentiation still remains elusive. In this study, we investigated the effects of metformin and AICAR on monocyte- to-macrophage differentiation process using human monocytic leukemia (THP-1) cell line. AMPK activators attenuated the monocyte-tomacrophage differentiation and also the pro-inflammatory signaling events associated during the differentiation process by a novel mechanism involving AMPK-1α-mediated STAT3 regulation. Furthermore, metformin significantly attenuated Ang-II-induced plaque formation and aortic aneurysm (AA) in ApoE-/- mice, possibly by impairing monocyte recruitment and its differentiation into macrophage in the arterial vessel wall.
Research Design and Methods Cell culture and differentiation: Cell culture procedures are described in the Online Data Supplement. For differentiation of monocytes to macrophages, THP-1 cells were seeded at a density of 2 x 105/ ml and stimulated with Phorbol Myristate-Acetate (PMA).
Page 4 of 37
Page 5 of 37
Diabetes
Animal experiments. Experiments were conducted in two months old male C57 BALB/c apolipoprotein E knockout (ApoE-/-) mice according to the guidelines formulated for care and use of animals in scientific research (ICMR, India) at a CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) registered animal facility. The experimental protocols were approved by the Institutional Animal Ethical Committee at CSIRIICT (IICT/CB/SK/20/12/2013/10). Animals were randomly divided into 3 groups each n=12; 1) control 2) Ang-II treatment and 3) metformin+Ang-II treatment. Ang-II and metformin treatment groups received Ang-II (sigma) at a dose of 1.44 mg/kg/day as described previously (16-18) for 6 weeks through sub-cutaneous route where as control group received normal saline. Metformin treatment group received the drug at a dose of 100 mg/kg/day in normal drinking water. All the animals were fed on normal chow throughout the study. After 6 weeks, animals were sacrificed as per the standard protocols by euthanasia. Please see supplementary file for other methodologies adopted in this study. Statistical analysis. Data are expressed as Mean ± SD. The significance of differences between groups was examined using either Student’s t test or ONE-WAY ANOVA as appropriate. P values
