Regression analysis of plasma and hepatic markers of inflammation and fibrosis.

<p>Linear regression analysis was used to assess the association between hepatic transcripts involved in hepatic fibrosis, <b>[A]</b>: Col1A1 versus LoxL2; <b>[B]</b>: LoxL2 versus Timp1. This analysis was used to assess the association between plasma TLR2 activators and hepatic transcripts involved in inflammation <b>[C]</b>: <i>Mcp1</i> versus TLR2] and fibrosis <b>[D]</b>: <i>LoxL2</i> versus TLR2]. As indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146942#pone.0146942.t001" target="_blank">Table 1</a>, plasma TLR 2 activators were identified as a highly significant plasma marker of NASH. R² and p-values were as described in Materials and Methods.</p

Time course analysis of hepatic histology of mice fed the LFLC or WD diets.

<p>Liver sections were stained with Sirius Red to reveal fibrosis appearing as red branching fibrosis (black arrows). The stained sections are from mice fed the WD for 24 weeks, the LFLC or WD for 32 weeks and mice fed the WD for 24 weeks and switched to the LFLC diet for 1, 2 or 8 weeks. The stained liver sections are representative of all livers in each group. The magnification of all images is 4X.</p

Time course of effects of diet on plasma markers of MetS.

<p>Plasma levels of glucose <b>[A],</b> triglyceride <b>[B],</b> total cholesterol <b>[C]</b> and free cholesterol <b>[D]</b> were assayed as described in Materials and Methods. Results are represented as plasma values; mean ± SD with 5–6 animals/group. ANOVA: <b>a</b>, p≤0.05 versus the LFLC group; <b>b</b>, p≤0.05 versus the WD-32 group; <b>c</b>, p≤0.05 versus the WD-24 group.</p

Diet effects on the hepatic expression of enzymes involved in desaturation and elongation of saturated, mono- and polyunsaturated fatty acids.

<p><b>[A]</b>: Hepatic transcript abundance of enzymes was quantified as described in Materials and Methods. Results are represented as mRNA-Fold Change; mean ± SD with 5–6 animals/group. <b>[B]</b>: Hepatic PUFA content. Total hepatic fatty acids were quantified as described in Materials and Methods. Hepatic PUFA are represented as Fatty Acid Mole%, mean ± SD with 5–6 animals/group. ANOVA: <b>a</b>, p≤0.05 versus the LFLC group; <b>b</b>, p≤0.05 versus the WD-32 group.</p

Is Western Diet-Induced Nonalcoholic Steatohepatitis <i>in Ldlr^-/-</i> Mice Reversible?

<div><p>Background</p><p>Nonalcoholic fatty liver disease (<b>NAFLD</b>) is a major public health burden in western societies. The progressive form of NAFLD, nonalcoholic steatohepatitis (<b>NASH</b>), is characterized by hepatosteatosis, inflammation, oxidative stress, and hepatic damage that can progress to fibrosis and cirrhosis; risk factors for hepatocellular carcinoma. Given the scope of NASH, validating treatment protocols (i.e., low fat diets and weight loss) is imperative.</p><p>Methods</p><p>We evaluated the efficacy of two diets, a non-purified chow (<b>NP</b>) and purified (low-fat low-cholesterol, <b>LFLC</b>) diet to reverse western diet (<b>WD</b>)-induced NASH and fibrosis in <i>Ldlr</i>^<i>-/-</i> mice.</p><p>Results</p><p>Mice fed WD for 22–24 weeks developed robust hepatosteatosis with mild fibrosis, while mice maintained on the WD an additional 7–8 weeks developed NASH with moderate fibrosis. Returning WD-fed mice to the NP or LFLC diets significantly reduced body weight and plasma markers of metabolic syndrome (dyslipidemia, hyperglycemia) and hepatic gene expression markers of inflammation (<i>Mcp1</i>), oxidative stress (<i>Nox2</i>), fibrosis (<i>Col1A</i>, <i>LoxL2</i>, <i>Timp1</i>) and collagen crosslinking (hydroxyproline). Time course analyses established that plasma triglycerides and hepatic <i>Col1A1</i> mRNA were rapidly reduced following the switch from the WD to the LFLC diet. However, hepatic triglyceride content and fibrosis did not return to normal levels 8 weeks after the change to the LFLC diet. Time course studies further revealed a strong association (r² ≥ 0.52) between plasma markers of inflammation (TLR2 activators) and hepatic fibrosis markers (<i>Col1A</i>, <i>Timp1</i>, <i>LoxL2</i>). Inflammation and fibrosis markers were inversely associated (r² ≥ 0.32) with diet-induced changes in hepatic ω3 and ω6 polyunsaturated fatty acids (<b>PUFA</b>) content.</p><p>Conclusion</p><p>These studies establish a temporal link between plasma markers of inflammation and hepatic PUFA and fibrosis. Low-fat low-cholesterol diets promote reversal of many, but not all, features associated with WD-induced NASH and fibrosis in <i>Ldlr</i>^<i>-/-</i> mice.</p></div

Heat map of the time course of diet effects on reversal of MetS and NASH.

<p>As explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146942#pone.0146942.g002" target="_blank">Fig 2</a>, the heat map is a visualization of changes in entities (rows, e.g., plasma glucose) in mice (columns). This figure represents an analysis of the 6 groups in the time course study: LFLC, WD-24, WD-32, WD-24 to LFLC for 1, 2, or 8 weeks. The number of mice in each group is: 6, 5, 5, 6, 6, 5, respectively. The numbers at the bottom of the heat map represent the mouse identification numbers.</p

Effects of non-purified lab (NP) diet on reversal of WD-induced NASH.

<p>Mice were fed the NP (Lab Chow) or WD diets as described in Materials and Methods. <b>[A]:</b> The left and right panels represent hematoxylin-eosin (H&E) and trichrome (TC) staining of liver sections, respectively. Magnification for the H & E and TC panels was 4X, respectively. Lipid droplets appear as white circles (black arrows), while branching fibrosis (yellow arrows) appears as blue strands in the trichrome stained liver sections. The slides are representative of all livers in each group. <b>[B]:</b> Hepatic triglyceride was quantified as described in Methods and Materials. Results are represented as Triglyceride, μg/mg protein. <b>[C]:</b> Hepatic hydroxyproline content was quantified as described in Methods and Materials. Results are represented as Hydroxyproline μg/mg protein. Statistical analysis used ANOVA plus Tukey’s HSD to establish statistical significance.</p

Time course of effect of diet on plasma markers of hepatic damage, inflammation and leptin.

<p>Plasma levels of alanine aminotransferase (ALT) <b>[A]</b>, activators of toll-like receptor 2 (TRL2), <b>[B]</b> and TLR4 <b>[C]</b>, and leptin <b>[D]</b> were quantified as described in Materials and Methods. Adiponectin was also quantified, but adiponectin was not significantly affected by diet (not shown). Results are represented as plasma values; mean ± SD with 5–6 animals/group. ANOVA: <b>a</b>, p≤0.05 versus the LFLC group; <b>b</b>, p≤0.05 versus the WD-32 group; <b>c</b>, p≤0.05 versus the WD-24 group.</p

Heat maps and volcano plots of the LFLC, WD-32 and WD to LFLC groups.

<p>Bioinformatic analysis of data used the statistical package in MetaboAnalyst 3.0 (<a href="http://www.metaboanalyst.ca/MetaboAnalyst/" target="_blank">http://www.metaboanalyst.ca/MetaboAnalyst/</a>) as described in Materials and Methods. <b>[A]:</b> The heat map is a visualization of the changes in abundance/level of features in rows (e.g., plasma glucose) for each animal (columns). The number at the bottom of the heat map is the animal identification number. The color ranges from deep orange (high abundance or level) to deep blue (low abundance or level); white is no change. <b>[B and C]:</b> All data in the heat map was used to construct volcano plots using the statistics package in MetaboAnalyst 3.0. The volcano plot allows for the visualization of the distribution of p-values versus fold-change for all measured entities, which include body weight, plasma and hepatic parameters, hepatic transcripts and fatty acids.</p

Can a LFLC diet reverse WD-induced NASH? Mice were fed the LFLC or WD diets as described in Materials and Methods.

<p><b>[A]</b>: The left and right panels represent hematoxylin-eosin (H & E) and trichrome (TC) staining of liver sections, respectively. Magnification for the H & E and TC panels was 16X and 10X, respectively. Lipid droplets appear as white circles (black arrows), while fibrosis (branching) appears as blue strands (yellow arrows) in the trichrome stained liver sections. The slides are representative of all livers in each group. <b>[B]</b>: Hepatic hydroxyproline content was quantified as described in Materials and Methods. Results are represented Hydroxyproline μg/mg protein</p