Pleiotropic Benefits of Metformin: Macrophage Targeting Its Anti-inflammatory Mechanisms

Metformin is a biguanide family member used in the treatment of type 2 diabetes and one of the most widely prescribed antidiabetes drugs. This drug increases the peripheral uptake of glucose, decreases hepatic glucose production, and reduces insulin resistance in liver and skeletal muscle. The exact molecular mechanisms responsible for its effect on glucose homeostasis are still not completely understood but may include the triggering of the AMPK pathway (1), although it has been indicated that the acute inhibitory effects of high doses of metformin on hepatic gluconeogenesis are independent of AMPK activation (2). Interestingly, experimental and clinical studies have shed new light on an array of potential benefits of metformin, not only in the treatment of diabetes. Notably, accumulating evidence suggests that the cardiovascular protective role of metformin is largely beyond its hypoglycemic action and is ascribed to pleiotropic effects (3–5).

Previous studies have described the anti-inflammatory effects of metformin on different types of cells, including human vascular endothelial cells and smooth muscle cells (6,7). Recent reports have also demonstrated that metformin can attenuate lipopolysaccharide (LPS)-induced or oxidized LDL–induced proinflammatory responses in monocytes and macrophages (8,9). Although the crucial mechanisms underlying the anti-inflammatory effects of metformin remain to be fully elucidated, the current concept of atherosclerosis as an inflammatory disorder may imply that such anti-inflammatory properties could contribute, at least in part, to the anti-atherosclerotic effects of metformin beyond glucose lowering.

In this issue of Diabetes, Vasamsetti et al. (10) demonstrate that metformin inhibits monocyte-to-macrophage differentiation in THP-1 cells, a human monocytic leukemia cell line, stimulated with phorbol myristate acetate (PMA). THP-1 cells are one of the most widely used models for investigating monocytic differentiation and subsequent biological functions of differentiated cells (11), although immortalized THP-1 cells may not always be of sufficient relevance to human macrophages. PMA treatment, which activates protein kinase C, can induce a greater degree of differentiation in THP-1 cells, as reflected by changes in morphology, adherence, phagocytosis, expression of surface markers, or the release of prostaglandin E and tumor necrosis factor-α (TNF-α) associated with macrophage differentiation (11,12). The differentiation of monocytes into macrophages is a pivotal step in the early immune response: In response to injury or inflammatory stimuli, migration of circulating monocytes into the inflamed tissues is accelerated, and subsequent differentiation into macrophages occurs rapidly. Upon differentiation, this maturation process allows the cells to actively participate in the inflammatory and immune responses. Thus, the ability of metformin to inhibit PMA-induced monocyte-to-macrophage differentiation in THP-1 cells discovered by Vasamsetti et al. (10) may serve as a novel mechanism underlying its anti-inflammatory actions. It must be kept in mind, however, that the inhibition of PMA-induced monocyte-to-macrophage differentiation was observed at a dose range of 0.5–2 mmol/L, which is much higher than the peak serum concentrations under clinically relevant dosing conditions (13).

The study by Vasamsetti et al. (10) confirms that the inhibition by metformin of monocyte-to-macrophage differentiation was AMPK dependent. Furthermore, AMPK-mediated signaling events in THP-1 cells are found to be downregulated in the presence of external stimuli, such as PMA and LPS (10). These observations suggest that AMPK may play a regulatory role in the differentiation of monocytes into macrophages in response to stimulus inducers. Caution is required, however, in the AMPK activator/inhibitor used in this study because compound C can generate many off-target effects through inhibition of different protein kinases (14) and AICAR is a nonspecific activator of AMPK and has the potential to activate other AMP-sensitive enzymes (15). In this regard, it would have been better to use A-769662, a potent and reversible activator of AMPK that mimics the function of AMP on AMPKβ-1 by allosteric activation and the inhibition of dephosphorylation of AMPK (16).

Vasamsetti et al. (10) propose that metformin inhibits monocyte-to-macrophage differentiation by reducing STAT3 activity due to increased AMPK activation (Fig. 1). Indeed, metformin inhibited PMA-induced STAT3 phosphorylation in THP-1 cells in a concentration-dependent manner. Moreover, the STAT3 inhibitor stattic not only inhibited monocyte-to-macrophage differentiation but also reduced production of proinflammatory cytokines, such as TNF-α, in PMA-stimulated THP-1 cells. It should be noted, however, that the role of STAT3 in cytokine production by macrophages does not achieve a general agreement. Previous reports have shown that mice lacking STAT3-deficient macrophages are characterized by excessive cytokine release (17,18).

Macrophages exhibit marked phenotype heterogeneity. Phenotypically polarized macrophages are now generally termed proinflammatory M1 and anti-inflammatory M2. On the other hand, based on expression levels of Ly6C (Gr-1), an inflammatory monocyte marker, mouse monocytes subsets are grouped as Ly6C⁺ monocytes and Ly6C⁻ monocytes (19). In general terms, both human classical and intermediate monocytes exhibit inflammatory properties reminiscent of murine Ly6C⁺ monocytes, and human nonclassical monocytes show patrolling properties similar to those of murine Ly6C⁻ monocytes. Both human and mouse inflammatory monocytes express high levels of the chemokine receptor CCR2 and low levels of the chemokine receptor CX3CR1, whereas patrolling monocytes display a reverse pattern (20). The Ly6C⁺ monocyte–derived cells have been compared with M1 macrophages, while, in vascular inflammation, Ly6C⁻ monocytes are recruited to tissues and are more likely to differentiate into M2 macrophages, which secrete anti-inflammatory cytokines and contribute to tissue repair (21). Accordingly, how metformin can modulate the differentiation of Ly6C⁻ monocytes into M2 macrophages remains the subject of ongoing interesting studies.

Previous work using murine bone marrow–derived macrophages and human monocyte-derived macrophages has revealed that activation of the AMPK signaling pathway suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype (22). The study by Vasamsetti et al. (10), together with the above report (22), suggests that metformin displays anti-inflammatory potentials at least in part by modulating macrophage differentiation and polarization. Macrophages are now emerging as an important player in the pathogenesis of insulin resistance. Thus, insulin resistance can result from a combination of altered functions of insulin-targeted tissues, adipocytes, skeletal muscles, and liver and the accumulation of inflammatory macrophages that are major sources of proinflammatory cytokines, such as TNF-α (23). Furthermore, several lines of evidence indicate that the presence of macrophages in tumors are more likely to contribute to cancer progression (24), and metformin may have a potential use in the treatment of different cancers in several clinical trials under way. Conclusively, the anti-inflammatory benefits of metformin targeting macrophage differentiation/polarization may help explain its usable value for prevention and/or treatment of vascular injury, atherosclerosis, certain cancers, and insulin resistance (Fig. 1).