TNF-? Promotes Palmitic Acid Transport Across Vascular Endothelial Cells And Impairs Cardiac Insulin Sensitivity

Objective:Inflammation and lipid signaling are intertwined modulators of insulin resistance in metabolic syndrome, but the mechanism is unclear. Tumor necrosis factor-a (TNF-a) is an important inflammatory factor in plasma and palmitic acid (PA) is the most abundant saturated long chain fatty acids. In the present study, we investigated whether TNF-a increased the transcytosis of PA across cardiac microvascular endothelial cells (CMECs) and therefore facilitated PA-induced cardiac insulin resistance. And the role of transcription factor nuclear factor-?B (NF-?B) and autophagy signaling pathway were studied.Methods:(1) We established an in-vitro FA transcytosis model to determine FA transcytosis across primary cultured CMECs with a fluorescent labeled FA analogue, C1-BODIPY-C12 (BODIPY-PA). (2) Using this transcytosis model, we detected the effect of TNF-a on FA transcytosis across CMECs with or without PKC, NF-?B, autophagy inhibitors. (3) And then, we detected PKC translocation, NF-?B activity and proteins involved in autophagy with western blot, transcription factor activity assay and GFP-LC3 transfection. (4) After incubated CMECs with TNF-a and inhibitors of NF-?B or autophagy, we added PA into the apical chamber in the CMECs-cardiomyocytes co-culture system. Then, we measured insulin-induced 2-NBDG uptake in cardiomyocytes after PA (transport across CMECs) treatment. (5) To assay the level of heart 18F-FDG uptake, after treated with TNF-a, PA or inhibitors of NF-?B and autophagy, C57BL/6J mice were performed with a whole-body positive emission tomography (PET) scan to obtain heart%ID/cc. Hearts radioactivity was measured using a well gamma-counter and hearts were weighted. And then, western blot and immunohistochemical analysis were performed to detect the expressions of proteins involved in insulin signaling and Fatty acid transport protein 4 (FATP4).Results:(1) We established an in-vitro FA transcytosis model and determinated FA transcytosis across CMECs for 6 hours with a fluorescent FA analogue, BODIPY-PA. BODIPY-PA transport is a time-and concentration-dependent process and could be inhibited by PA treatment. Amount of BODIPY-PA transport through endothelial cell monolayer is calculated by a standard curve. The transcytosis model with 40 ?M BODIPY-PA is used in the following experiment. (2) FATP4 siRNA blocked TNF-a-induced FA transcytosis. Both NF-?B oligodeoxynucleotide (ODN) and autophagy inhibitor prevented the increase of FATP4 expression as well as FA transcytosis enhanced by TNF-a. (3) TNF-a incubation upregated NF-?B activity and autophagy, increased LC3-II expression, Beclinl expression and LC3 puncta. NF-?B ODN suppressed the increased expression of LC3-II, formation of LC3-II puncta and upregulation of Beclinl induced by TNF-a treatment. And 3MA blocked NF-?B activation induced by TNF-a. PKC? translocated into membrane fraction after TNF-a treatment, and PKC? defection by PKC? siRNA disturbed TNF-?-induced FA transcytosis. Also, PKC8 siRNA blocked the up-regulation of NF-?B activity and autophagy flux resulted by TNF-a. (4) PA reduced Akt and GSK3? phosphorylation as well as 2-NBDG uptake in the presence of insulin in a concentration-dependent manner. And the 2-NBDG uptake experiment in co-culture model showed that PA which was transported across CMECs impaired the glucose uptake ability of cardiomyocytes in the presence of insulin, and this effect was facilitated after CMECs were exposed to TNF-a. As expected,3 MA and PDTC blunted the exacerbation of TNF-a. (5) Mice in TNF-a+PA-treated group presented a significant decrease in insulin-induced 18F-FDG uptake compared with mice treated with TNF-a or PA alone. What's more, both 3MA and PDTC attenuated the reduction induced by TNF-a+PA treatment. Result of heart radioactivity measured with a gamma-counter was similar. Compared with TNF-a or PA alone, TNF-a+PA treatment obviously reduced insulin-induced phosphorylation of Akt and GSK3?.And 3MA or PDTC injection enhanced the phosphorylation of Akt and GSK30 stimulated by insulin in mice heart exposed to TNF-a+PA treatment. FATP4 expression in heart microvessel was increased in TNF-a group and TNF-a+PA group compared to control group, and 3MA or PDTC injection decreased the expression of FATP4 compared to TNF-a+PA group.Conculsions:TNF-a increased PA transcytosis across CMECs by activiting NF-?B and autophagy and then exacerbated the PA-induced insulin sensitivity impairment in cardiocmyoctyes.