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O0040 FREE FATTY ACIDS PROMOTE HEPATIC LIPOTOXICITY BY STIMULATING TNF-ALPHA EXPRESSION VIA A LYSOSOMAL PATHWAY

Feldstein, A. E.1; Canbay, A.2; Guicciardi, M. E.2; Gores, G. J.2

Journal of Pediatric Gastroenterology and Nutrition: June 2004 - Volume 39 - Issue - p S22-S23
ABSTRACTS: Oral Presentation Abstracts
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1Gastroenterology,2Gastroenterology, Mayo Clinic, Rochester, MN, United States

Submitted by: feldstein.ariel@mayo.edu

Introduction: Nonalcoholic fatty liver disease (NAFLD) is a serious public health problem and represents a form of lipotoxicity. However the molecular basis of lipotoxicity remains poorly understood. TNF-alpha has been implicated in NAFLD, and this cytokine can signal lysosomal permeabilization. Thus, our AIMS were to determine if free fatty acids (FFA)-mediate hepatic lipotoxicity via lysosomal destabilization.

Methods: Cells were transfected with cathespsin B (ctsb)-GFP, a lyososomal protease, and its cellular compartmentation determined by confocal microscopy. TNF-[alpha] expression was assessed by Real Time PCR. In selective experiments, HepG2 cells were infected with the adenovirus Ad5I[kappa]B superre-spressor to block NF-[kappa]B activation or empty virus Ad5ƒ’E1. NF-[kappa]B cellular localization was examined by immunofluorescence and confocal microscopy. I[kappa]B expression was assessed by immunoblot analysis. Ctsb compartmentation was also examined by immunfluorescence in liver biopsies from 35 patients with NAFLD and 15 controls. Finally, ctsb wildtype and knockout mice were fed either a high sucrose diet or a standard diet for 16 wk. A fasting metabolic profile was measure at various time points. Liver injury was assessed by histopathology and serum ALT values.

Results: FFA (2:1 oleate:palmitate) treatment of HepG2 cells resulted in lysosomal destabilization with translocation of ctsb-GFP from lysosomes into the cytosol. Interestingly, the lysosomal permeabilization was ctsb-dependent as lysosomal permeabilization, which occurred in wild type hepatocytes with FFA treatment, was not observed in hepatocytes from ctsb deficient animals. Importantly, ctsb release into the cytoplasm was also observed in human liver specimens from patients with NAFLD, but not normal livers, and correlated with severity of steatohepatitis. FFA-induced lysosomal destabilization was associated with NF-[kappa]B activation and markedly enhanced TNF-[alpha] expression in Hep G2 cells. In a dietary murine model of NAFLD, genetic inactivation of ctsb protected against development of hepatic steatosis and liver injury, and significantly improved insulin resistance and hyperleptinemia.

Conclusion: These data support a lipotoxic model of FFA-mediated ctsb-dependent lysosomal permeabilization causing TNF-[alpha] expression, which in turn promotes hepatic steatosis and steatohepatitis. Ctsb is a new potential therapeutic target for lipotoxicity-mediated processes like NAFLD.

© 2004 Lippincott Williams & Wilkins, Inc.