ER stress inhibition enhances formation of triacylglcerols and protects endothelial cells from lipotoxicity.

Elevated concentrations of palmitate in serum of obese individuals can impair endothelial function, contributing to development of cardiovascular disease. Although several molecular mechanisms of palmitate-induced endothelial dysfunction have been proposed, there is no consensus on what signaling event is the initial trigger of detrimental palmitate effects. Here we report that inhibitors of ER stress or ceramid synthesis can rescue palmitate-induced autophagy impairment in macro- and microvascular endothelial cells. Furthermore, palmitate-induced cholesterol synthesis was reverted using these inhibitors. Similar to cell culture data, autophagy markers were increased in serum of obese individuals. Subsequent lipidomic analysis revealed that palmitate changed the composition of membrane phospholipids in endothelial cells and that these effects were not reverted upon application of above-mentioned inhibitors. However, ER stress inhibition in palmitate-treated cells enhanced the synthesis of trilglycerides and restored ceramide levels to control condition. Our results suggest that palmitate induces ER-stress presumably by shift in membrane architecture, leading to impaired synthesis of triglycerides and enhanced production of ceramides and cholesterol, which altogether enhances lipotoxicity of palmitate in endothelial cells.

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts.

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.

Lysophospholipids secreted by splenic macrophages induce chemotherapy resistance via interference with the DNA damage response.

Host responses to systemic anti-cancer treatment play important roles in the development of anti-cancer drug resistance. Here we show that F4/80(+)/CD11b(low) splenocytes mediate the resistance to DNA-damaging chemotherapeutics induced by two platinum-induced fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) in xenograft mouse models. Splenectomy or depletion of splenic macrophages by liposomal clodronate protects against PIFA-induced chemoresistance. In addition, we find that 12-S-HHT, but not 16:4(n-3), functions via leukotriene B4 receptor 2 (BLT2). Genetic loss or chemical inhibition of BLT2 prevents 12-S-HHT-mediated resistance. Mass spectrometry analysis of conditioned medium derived from PIFA-stimulated splenic macrophages identifies several lysophosphatidylcholines as the resistance-inducing molecules. When comparing cisplatin and PIFA-treated tumours with cisplatin alone treated tumours we found overall less γH2AX, a measure for DNA damage. Taken together, we have identified an intricate network of lysophospholipid signalling by splenic macrophages that induces systemic chemoresistance in vivo via an altered DNA damage response.