benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Non-alcoholic-Fatty-Liver-Disease

The pathogenesis of non-alcoholic steatohepatitis (NASH) is not fully understood. In the present study, both in vitro and in vivo vimentin expression and secretion in NASH were investigated. The exposure of palmitate and lipopolysaccharide (LPS) to HepG2 cells enhanced caspase-3 activity and vimentin expression, respectively. The combined effects of both treatments on vimentin expression and caspase-3 activation appeared to be synergic. In contrast, blockade of caspase-3 activity by zVADfmk resulted in a significant reduction of cleaved vimentin and secreted vimentin into the culture supernatant. Similarly, lipid accumulation and inflammation occurred in mice fed a methionine-choline-deficient diet; thus, vimentin expression and serum cleaved vimentin levels were increased. However, vimentin was not significantly upregulated, and no cleavage occurred in mice fed a high-fat diet. It was conclusively determined that lipid accumulation in hepatocytes induces apoptosis through a caspase-3 dependent pathway; whereas, LPS stimulates vimentin expression, leading to its cleavage and secretion. Increased vimentin fragment levels indicated the existence of substantial hepatocellular death via an apoptotic mechanism.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Diet; Disease Models, Animal; Disease Progression; Hep G2 Cells; Humans; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Palmitates; Up-Regulation; Vimentin

Ballooned hepatocytes in non-alcoholic steatohepatitis (NASH) generate sonic hedgehog (SHH). This observation is consistent with a cellular phenotype in which the cell death program has been initiated but cannot be executed. Our aim was to determine whether ballooned hepatocytes have potentially disabled the cell death execution machinery, and if so, can their functional biology be modeled in vitro.. Immunohistochemistry was performed on human NASH specimens. In vitro studies were performed using HuH-7 cells with shRNA targeted knockdown of caspase 9 (shC9 cells) or primary hepatocytes from caspase 3(-/-) mice.. Ballooned hepatocytes in NASH display diminished expression of caspase 9. This phenotype was modeled using shC9 cells; these cells were resistant to lipoapoptosis by palmitate (PA) or lysophosphatidylcholine (LPC) despite lipid droplet formation. During lipid loading by either PA or LPC, shC9 cells activate JNK which induces SHH expression via AP-1. An autocrine hedgehog survival signaling pathway was further delineated in both shC9 and caspase 3(-/-) cells during lipotoxic stress.. Ballooned hepatocytes in NASH downregulate caspase 9, a pivotal caspase executing the mitochondrial pathway of apoptosis. Hepatocytes engineered to reduce caspase 9 expression are resistant to lipoapoptosis, in part, due to a hedgehog autocrine survival signaling pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 1; Caspase 9; Cell Line, Tumor; Down-Regulation; Fatty Liver; Gene Knockdown Techniques; Hedgehog Proteins; Hepatocytes; Humans; JNK Mitogen-Activated Protein Kinases; Lysophosphatidylcholines; Minute Virus of Mice; Non-alcoholic Fatty Liver Disease; Palmitates; Phosphorylation; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transcription Factor AP-1