PALMITATE INDUCED ENDOTHELIAL DYSFUNCTION: THE ROLE OF CALPAIN, AMPK AND ENOS

Abstract

Obesity is a serious health problem worldwide. Consumption of fat rich food is a common cause of obesity. Some of the food components (i.e. saturated free fatty acids (SFAs)) have been identified as inflammatory inducers (Egger G at al., 2010). After a meal, absorbed free fatty acids (FFAs) will be stored in the liver and adipose tissue. On the luminal surfaces of endothelium in adipose tissue microcirculation, lipoprotein lipase hydrolyses absorbed triglycerides into FFAs. Then, in order to be available for adipocyte storage, FFAs have to cross the capillary endothelium barrier, which connected by tight junctions (Stremmel W et al., 2001). Increased leukocyte infiltration is a featured sign of adipose tissue inflammation found in obesity. Endothelial adhesion molecules up-regulation contributes to leukocyte infiltration during inflammation. Some clinical data suggested an increase of leukocyte-endothelium interaction in healthy volunteers after ingestion of high-fat meals (Shimabukuro M et al., 2007). Other lab results also showed that neutrophil infiltration occurred at a very early stage with high-fat feeding in mice (Talukdar S et al., 2012). However, the detailed mechanism of the above phenomena is still unknown. This thesis provides exciting preliminary data which will guide the further study in this area. First of all, we successfully established a stable protocol that CD31 antibody conjugated microbeads were used to isolate primary microvascular endothelial cells from fresh mice lung tissue. After second sorting, CD31+ cells reach 83.3% by FACS analysis. Previous literatures showed that FFAs activate recruitment of inflammatory cells through up-regulation of endothelial adhesion molecules via reduced eNOS derived eNO production (Rizzo NO et al., 2010; Davenpeck KL et al., 1994; Ahluwalia A et al., 2004). In this thesis, it was found that SFAs palmitate exposure dose dependently reduced endothelial AMPK thr172 and eNOS ser1177 phosphorylation by western blot. Moreover, our study demonstrated that endothelial calpain, a calcium dependent protease associated with endothelial dysfunction, was activated by palmitate, specifically its μ-calpain isoform. Altogether, these data suggested that a new role of calpain as a key mediator of palmitate induced endothelial dysfunction and indicated both AMPK and eNOS1177 phosphorylation contribute to this pathological process. Further investigations are still needed to explore connections among those molecules. This thesis may also lead to a novel way of clinical treatment for the obese related vascular diseases.