Using discovery replication strategy and large-scale proteomics data, we identified consistent positive associations between leptin and A-FABP in two independent cohorts of patients with type 2 diabetes, an association which appeared stronger in men than in women. This association was similar after additional multivariable adjustment for cardiovascular risk factors. In longitudinal analyses, the association between leptin and MACE was slightly attenuated after adding A-FABP to the multivariate model which may indicate that A-FABP mediate some, but not all, of the link between leptin and CVD.
This is, to our knowledge, the first study to report associations between circulating levels of leptin and a multiplex proteomics assay. We are aware of only one previous study that have reported the association between circulating leptin and A-FABP. In that study, Reinehr et al studied obese children before and after weight loss, showing a significant correlation between A-FABP and leptin and percentage of bodyfat, but not with other markers of the metabolic syndrome (20).
Potential mechanisms
Adipose tissue has been studied extensively regarding its role in metabolic regulation through lipid signaling (21). A-FABP, also known as adipocyte protein 2, aP2 or FABP4, is a fatty acid binding protein which can be found in white adipocytes and macrophages. In some studies, A-FABP has been identified as a circulating biomarker for metabolic syndrome, diabetes mellitus type 2, and cardiovascular events (22, 23). A-FABP is thought to be a central mediator of obesity-related CVD, and the production of A-FABP in adipocytes is thought to lead to insulin resistance and the expression of proinflammatory genes (24). Tuncman et al provided genetic support for the involvement of A-FABP in atherosclerosis in humans, where a reduction in A-FABP activity generated a metabolically favorable phenotype (25) while Furuhashi et al showed that inhibiting A-FABP is effective against severe atherosclerosis and type 2 diabetes in mice (26). Human studies showed an association between A-FABP and coronary heart disease (27) and has also been shown to be associated with peripheral arterial disease (28). According to one study, A-FABP reduces the expression of leptin in mice adipocytes (29), while another study in leptin deficient mice showed an impaired gene expression for lipid utilization for, amongst other proteins, A-FABP (30). One study showed that A-FABP has a greater impact on atherosclerosis in women than in men, possibly related to the higher fat percentage in women (31). Another showed that A-FABP serum concentration correlates with gender and serum leptin as well as BMI (32).
High serum leptin levels have been observed in patients with CHD (33). There is a strong correlation between both circulating and adipose tissue levels of leptin and serum c-reactive protein (CRP) in obese women (34). There is a known difference between leptin levels in men and women, where women have been shown to have about two times higher serum leptin levels than men at each level of BMI, a difference shown in both obese and non-obese (35, 36). In clinical studies, correlations between leptin levels and established vascular risk factors, markers of impaired fibrinolysis, vascular dysfunction and inflammation have been shown (37). A-FABP and leptin both act as interfaces between metabolic and inflammatory pathways, both expressed by adipocytes as well as being inflammatory mediators, where leptin can promote monocyte-macrophage-activation and A-FABP is produced by macrophages. Circulating levels of classical monocytes have been shown to be independently associated with cardiovascular events in both a cohort consisting of nearly a thousand coronary patients (38), and in a randomly selected cohort of 700 subjects (39). Monocytes in the plaque can give rise to foam cells, and those may in turn play an important role in plaque instability (40).
Our study suggests that a small portion of the association between leptin and cardiovascular disease could be mediated via A-FABP, especially in men. However, our study cannot clarify whether leptin affects A-FABP or the other way around, and we can also not exclude the possibility that A-FABP is simply a confounder and not at all casually involved in the development of CVD. Leptin and A-FABP are both connected to macrophages in different stages, possibly connecting white adipose tissue with the inflammatory response.
Clinical implications
Diabetes and CVD pose major disease burdens on the world’s population and understanding the mechanisms driving these diseases are of utmost importance. Proteomics analyses could be an important part in finding new associations between proteins and new drug targets. Whether A-FABP could be used as a target for intervention to influence leptin levels cannot be established in the present study.
Strengths and limitations
Strengths of our study include the discovery/replication design, the longitudinal data and outcomes, and the consistent findings between different cohorts despite the small size of the cohorts. Strengths also include the use of registry data with high quality for mortality and morbidity. Limitations include the moderate sample size with limited power to detect weak associations, particularly in the replication cohort. The Olink assay does not give absolute concentrations of the proteins which may limit the generalizability and applicability of our results to other cohorts. Generalizability is limited to middle-aged to elderly Caucasian adults (55–70 years of age) with type 2 diabetes. BMI is not an ideal measurement of body fat, but associations were similar when adding waist circumference of sagittal abdominal diameter to the model.