Lipid Metabolism, Oxidative Stress and Cell Death Are Regulated by PKC Delta in a Dietary Model of Nonalcoholic Steatohepatitis

<div><p>Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKCδ) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKCδ in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKCδ^−/− mice and wildtype littermates a control or MCD diet. PKCδ^−/− primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKCδ^−/− mice fed the MCD diet. The hepatic expression of key regulators of β-oxidation and plasma triglyceride metabolism was significantly reduced in PKCδ^−/− mice and changes in serum triglyceride were blocked in PKCδ^−/− mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKCδ^−/− mice. MCD diet-induced NADPH oxidase activity and p47^phox membrane translocation were blunted and blocked, respectively, in PKCδ^−/− mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKCδ^−/− mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKCδ^−/− mice. Our results suggest that PKCδ plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.</p></div

The effect of MCD diet on PKC isoform gene expression and activation in PKCδ^+/+ and PKCδ^−/− mice.

<p>(A) PKCα, PKCβII, PKCε, and PKCθ expression was determined in four week fed PKCδ^+/+ (WT) and PKCδ^−/− (KO) mice by quantitative real time PCR and normalized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085848#s2" target="_blank">Materials and Methods</a>. Fold change is shown as the means +/− SE relative to control fed WT mice. (B) An equivalent amount of cytosolic and membrane protein was analyzed by Western blotting for PKCα, PKCβII, PKCε, and PKCθ expression. Quantitation of the immunoreactive bands minus background is shown as the means +/− SE. (*, p<0.05 versus control diet).</p

Markers of apoptosis and apoptosis related gene expression.

<p>(A) and (B) TUNEL staining of liver sections from four (A) or eight (B) week fed PKCδ^+/+ (WT) and PKCδ^−/− (KO) mice (means +/− SE). (C). Assessment of Caspase cleavage. Total cell lysate (70 µg of protein) from liver tissue was analyzed by immunoblotting for Caspase 9 (mouse specific), Caspase 3, cleaved Caspase 3 (Asp175), and αTubulin expression. Arrows indicate the cleaved 19 and 37 kDa Caspase 3 and 9 fragments, respectively. (D) and (E) Relative expression of hepatic cell cycle (D) and apoptosis (E) genes from four (<i>left panel</i>) or eight (<i>right panel</i>) weeks fed mice. Normalized gene expression and fold change (means +/− SE) relative to Con fed WT mice. (*, p<0.05 versus Con diet fed WT mice).</p

Hepatic Masson's trichrome and Sirius Red staining and fibrosis gene expression.

<p>(A) Scoring of Masson's trichrome stained liver sections (means +/− SE). (B) Scoring of Sirius Red stained liver sections (means +/− SE). (C) Relative expression of hepatic fibrosis related genes from Con or MCD diet fed mice for four weeks. Normalized gene expression and fold change (means +/− SE) relative to Con fed WT mice. (*, p<0.05 versus Con diet fed WT mice).</p

Markers of oxidative stress and oxidative stress gene expression.

<p>(A) 4-HNE stained liver sections from four week fed PKCδ^+/+ (WT) and PKCδ^−/− (KO) mice (<i>left panel</i>) and quantitation (means +/− SE) (<i>right panel</i>). (B) Quantitation of hepatic TBARS (means +/− SE). (C). Relative expression of hepatic NADPH oxidase subunit genes from Con or MCD diet fed mice for four (<i>upper panel</i>) or eight (<i>lower panel</i>) weeks or hepatocytes treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085848#pone-0085848-g001" target="_blank">Fig. 1</a>. Normalized gene expression and fold change (means +/− SE) relative to Con fed WT mice. (*, p<0.05 versus Con diet fed WT mice). (D) Hepatic NADPH oxidase subunit protein expression. Total cell lysate (70 µg of protein) from liver tissue was analyzed by immunoblotting for p22^phox, p47^phox, p67^phox, and Nox2 (p91^phox) and αTubulin expression. Representative immunoblots are shown (<i>upper panel</i>) and quantitation of the immunoreactive bands minus background is shown as the means +/− SE (<i>lower panel</i>). (*, p<0.05 versus control diet). (E) Hepatic NADPH oxidase activity. Membrane protein (50 µg) was assayed for NADPH oxidase activity as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085848#s2" target="_blank">Materials and methods</a>. (*, p<0.05 versus WT). (F). Hepatic NADPH oxidase subunit membrane and cytosolic protein expression. An equivalent amount of membrane and cytosolic protein was analyzed by Western blotting for p22^phox, p47^phox, calnexin or αTubulin expression (<i>upper panels</i>). Quantitation of the immunoreactive bands minus background of the membrane protein divided by the cytosolic protein (translocation) is shown as the means +/− SE (<i>lower panels</i>). (*, p<0.05 versus control diet).</p

Hepatic inflammation and inflammation gene expression.

<p>(A) Scoring of hematoxylin and eosin stained liver sections (means +/− SE) from 6–8 mice per group. (B) Relative expression of hepatic pro and anti-inflammatory genes from Con or MCD diet fed mice (n = 6) for four weeks (means +/− SE). (*, p<0.05 versus Con diet fed WT mice).</p

Weights and serum metabolic parameters in male PKCδ^+/+ and PKCδ^−/− mice.

<p>Con, control diet; MCD, methonine and choline deficient diet; NEFA, non-esterified fatty acids.</p><p>Values represent the means ± SEM for n = 5–8.</p><p>compared to Con,</p><p>p, >0.05.</p

Weights and serum metabolic parameters in female PKCδ^+/+ and PKCδ^−/− mice.

<p>Con, control diet; MCD, methonine and choline deficient diet; NEFA, non-esterified fatty acids.</p><p>Values represent the means ± SEM for n = 5–8.</p><p>compared to Con,</p><p>p, >0.05.</p