Impact of Hepatitis C Infection on Very Low Density Lipoprotein Secretion by the LiverMahra Nourbakhsh, Donna Douglas, Norman M Kneteman

Department of Surgery, University of Alberta, Edmonton, Canada

Hepatitis C virus (HCV) infection is a major health concern as 170 million people are chronically infected with HCV world-wide. Acute HCV infection progresses to chronic hepatitis in 70%-80% of infected patients, characterized by steatosis, progressive liver fibrosis, cirrhosis and hepatocellular carcinoma. Most of the fatal complications of HCV are related to the expansion of fibrosis and the factors that accelerate the rate of fibrosis worsen the prognosis of chronic hepatitis C (CHC) infection. Over the last decade, it has become apparent that HCV infection is significantly associated with liver steatosis. HCV-associated steatosis is clinically relevant because it correlates with more rapid disease progression and reduced response to antiviral therapy. The mechanism of HCV-associated steatosis is not completely understood, although, hepatic steatosis will develop whenever hepatic production/sequestration of triglyceride (TG) exceeds TG secretion or metabolism. In CHC, patients have abnormalities in serum lipids. Specifically they exhibit hypobetalipoproteinemia characterized by low serum level of Very Low Density Lipoproteins (VLDL) and their lipoprotein derivatives. VLDL is exclusively secreted by liver and its assembly and secretion is the major pathway for the export of lipids (mostly TG) by the liver. As the liver is the main repository for HCV infection, it is tempting to speculate a direct role of HCV on the regulation of lipid export from the thus contributing to steatosis. Hypothesis: HCV infection can result in impaired VLDL secretion, leading to hepatic intracellular accumulation of triglycerides and hence steatosis.

VLDL Secretion by the Liver: In the liver, VLDLs are formed in the luminal space within the endoplasmic reticulum (ER), whereas the lipids (mostly TGs) that are required for their formation are synthesized on the cytosolic side of the ER membrane (Fig-1). Apolipoprotein B100 (ApoB) is the major structural protein associated with VLDL. In the presence of sufficient lipid, mainly TG, ApoB is efficiently assembled into secretion-competent VLDL particles. There is substantial evidence that the bulk of TG secreted with VLDL arises from the lipolysis of intracellular cytosolic TG stores, followed by re-esterification and finally lipid transfer to nascent ApoB in a process that requires microsomal transfer protein (MTP), the cytosolic TG stores are mobilized for the lipidation of nascent ApoB. MTP is also thought to play a role in the formation of luminal TG droplet which maybe the direct TG source for the ApoB lipidation. Poorly lipidated ApoB is directed to the proteosome for degradation. Therefore, lipolysis and re-esterification of stored TG, MTP activity, ApoB synthesis and lipidation represent key steps in the hepatic export of VLDL-bound TG.

Objective: To determine if impaired secretion of VLDL is a possible mechanism contributing to HCV associated steatosisAims: To determine if HCV infection alters ApoB100 synthesis, secretion or degradation by modulation of MTP and/or cellular lipolytic activity required for TG mobilization.

Introduction and background

Fig-1: JFH-1 (Japanese Fulminant Hepatitis)-1 HCV genotype 2a strain replicates efficiently in Human Hepatocellular Carcinoma (Huh7.5).

Fig-2: JFH-1 Infection specifically impairs mass secretion of ApoB-100 (not Albumin).

Fig-3: JFH-1 Infection impairs secretion of newly synthesizedApoB-100.

Fig-4: * Cellular lysates from JFH-1 infected culture have significantly reduced MTP activity in vitro(P<0.05, t-test).

Fig-5: * Cellular lysates from JFH-1 infected culture have significantly reduced cellular lipase activity (P<0.05, t-test).

Fig-6: Relative to human hepatocytes Huh7.5 cells have impaired secretion of TG stores despite having appreciable amounts of intracellular TG stores present even 8h after withdrawal of exogenously supplied OA.

Methods and Results

Conclusions:

Taken from Wang et. al., JBC 2007 282(45): p.33218-26.

Significantly decreased MTP and cellular lipolytic activities may contribute to observed decreased secretion of preformed and newly synthesized ApoB-100. Impaired ApoB-100 secretion by HCV infection may be a mechanism contributing to HCV-associated steatosis.

Future Directions:1- Determine if observed decrease in the secretion of ApoB-100 is a significantly result with JFH-1 infected cultures.2-generate Huh7.5 cells having stable expression of Triacylglycerol Hydrolase (TGH). Note: TGH has shown to be absent from all Hepatoma derived cell line and its expression has been shown to increase the mobilization of TG stores for VLDL assembly/secretion. We will evaluate TGH as a potential target for disruption by HCV.

3- It is known that HCV-associated steatosis is more prevalent with HCV genotype 3 infected CHC patients. A specific role for the HCV core protein has been established. I have generated a chimeric JFH-1 virus in which the core coding region has been replaced with the core coding region obtained from a HCV 3a patient. This chimeric virus has similar infection characteristics as the origin JFH-1 strain (data not shown). We will determine if HCV core protein from HCV-3 has a distinct role in the modulation of VLDL synthesis and secretion.