Targeting AMP Kinase in Myeloid Cells to Reduce Atherosclerosis

Subjects with diabetes have a 1.8 times greater risk of having a heart attack than subjects without diabetes. Although cardiovascular mortality has decreased by 30.8% in the last decade, it is still responsible for 1 in 3 deaths in the U.S. (1). In spite of targeting the common risk factors, there is still a residual burden for atherosclerotic cardiovascular disease (ASCVD) (2). As inflammatory processes are involved in every stage of atherosclerosis, targeting inflammation has become an attractive strategy for further reducing the incidence of ASCVD (3).

AMP activated kinase (AMPK) is an enzyme that increases cellular ATP generation and diminishes ATP use for less critical processes (4). It regulates the transport of glucose, the synthesis of lipids and proteins, and the rate of fuel metabolism. It is also involved in regulating inflammation and oxidative and ER stress (5). AMPK may thus provide the link between nutrient, metabolic, and inflammatory stimuli. Dysregulation of AMPK may therefore play a role in the pathogenesis of diabetes, hypertension, and ASCVD. On the basis of the information cited above, activation of AMPK could prevent or treat these conditions.

Although there is evidence that AMPK activation is atheroprotective, the specific mechanism responsible for this effect is unclear (6). To explore the hypothesis that the atheroprotective effect of AMPK is due to the suppression of macrophage inflammation, Cao et al. (7) have investigated whether myeloid deletion of α1AMPK exacerbates atherosclerosis and whether this is associated with an increase in macrophage inflammation and chemotaxis and an alteration in cholesterol and lipid metabolism. To investigate this hypothesis they created a myeloid α1AMPK knockout (MAKO) in the LDL receptor knockout (LDLRKO) mice (MAKO/LDLRKO). Control floxed/LDLRKO and MAKO/LDLRKO mice were fed an atherogenic diet for 16 weeks, and various analyses were done to quantify atherosclerosis, macrophage inflammation, chemotaxis, lipid and cholesterol metabolism, and liver inflammation. α1AMPK mRNA and protein were decreased specifically in bone marrow–derived macrophages (BMDMs) and peritoneal macrophages in the MAKO/LDLRKO mice. The MAKO/LDLRKO mice were similar in weight and insulin sensitivity but had higher total and LDL cholesterol than controls. Atherosclerotic lesions were increased by 38 and 63% in the aortic roots and whole aorta, respectively, in the MAKO/LDLRKO mice. Deletion of α1AMPK increased basal and lipopolysaccharide-stimulated proinflammatory genes and macrophage adhesion to endothelial cells in BMDMs isolated from MAKO/LDLRKO mice. The macrophage content in atherosclerotic plaque was increased in the MAKO/LDLRKO mice along with an increase in chemotactic chemokines. Inflammatory genes were also upregulated in macrophages isolated from the atherosclerotic plaques of MAKO/LDLRKO mice. The higher cholesterol levels in MAKO/LDLRKO mice were associated with an increase in apolipoprotein B (apoB100 and apoB48) in plasma and in apoB mRNA and protein and expression of proinflammatory genes in the liver. The increase in cholesterol was not associated with an increase in peritoneal macrophage cholesterol content and had no effect on cholesterol efflux. On the basis of these observations, the authors concluded that α1AMPK is atheroprotective and may serve as a therapeutic target for prevention and treatment of atherosclerosis.

An interesting observation in the study by Cao et al. (7) was the absence of an effect on insulin sensitivity in the MAKO/LDLRKO mice fed an atherogenic diet compared with the development of insulin resistance in MAKO mice with intact LDL receptors fed a high-fat diet (HFD). This suggests that macrophage inflammation by itself may not lead to insulin resistance. While the MAKO mice with an intact LDL receptor were more obese than the MAKO/LDLRKO mice, the investigators have also raised the possibility of insulin resistance being affected by the composition of the meal. There is evidence to show that a single 900-calorie high-fat, high-carbohydrate (HFHC) meal increases inflammatory mediators and the generation of reactive oxygen species and induces endotoxemia and the expression of Toll-like receptor 4, the receptor for endotoxin (8,9). It also increases the expression of the suppressor of cytokine signaling 3, which interferes with insulin and leptin signal transduction (9). In contrast, an equicaloric meal rich in fruit and fiber does not induce any of these changes (10). An HFHC meal also increases postprandial glucagon and decreases postprandial levels of incretins compared with a fruit-and-fiber meal (10). Free fatty acid infusions acutely induce oxidative and inflammatory stress over 4 h, whereas over 48 h they induce insulin resistance and hypertension (11,12). Therefore, macronutrients have been shown to induce both inflammation and insulin resistance, and the HFD could have been responsible for the reduction in insulin sensitivity in the MAKO mice. This confusion could have been resolved by conducting additional investigations on MAKO/LDLRKO mice with and without the administration of an HFD.

Another observation was the increase in LDL cholesterol without any increase in the cellular lipid content of the macrophages expressing higher basal and stimulated proinflammatory genes. Therefore, inflammation by itself was sufficient to increase the risk of atherosclerosis in MAKO/LDLRKO mice. These findings clearly suggest that a multifaceted approach targeting the common risk factors along with strategies to reduce inflammation may be needed to further lower the risk of ASCVD.

The observations of Cao et al. (7) support a role for AMPK in atherogenesis through the modulation of macrophage inflammation and provide the rationale for the pursuit of AMPK activators for preventing and treating ASCVD. In this regard, exercise and calorie restriction are known to activate AMPK and are also associated with a reduction in cardiovascular disease (CVD) (13,14). Similarly, metformin, thiazolidinediones, and glucagon-like peptide 1 receptor agonists, which are used for the treatment of diabetes, are also known to be AMPK activators and have been shown to have beneficial effects on CVD and diabetes prevention (15–18) (Fig. 1). Although generalized AMPK activation may be a reasonable strategy for atheroprotection, this study suggests that upregulation of AMPK specifically in macrophages may also prevent CVD. Such an approach would alleviate the possible concerns of the deleterious effects of generalized AMPK activation on the conduction system of the heart and of its detrimental effect secondary to increased fatty acid oxidation during myocardial ischemia and reperfusion (19,20). However, such an approach may not provide the comprehensive benefits of AMPK activation on both insulin resistance and CVD as is seen with the activation of AMPK in multiple tissues.