Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus.

Chronic ethanol consumption disrupts several metabolic pathways including β-oxidation and lipid biosynthesis, facilitating the development of alcoholic fatty liver disease. Many of these same metabolic pathways are directly regulated by cell autonomous circadian clocks, and recent studies suggest that disruption of daily rhythms in metabolism contributes to multiple common cardiometabolic diseases (including non-alcoholic fatty liver disease). However, it is not known whether ethanol disrupts the core molecular clock in the liver, nor whether this, in turn, alters rhythms in lipid metabolism. Herein, we tested the hypothesis that chronic ethanol consumption disrupts the molecular circadian clock in the liver and potentially changes the diurnal expression patterns of lipid metabolism genes. Consistent with previous studies, male C57BL/6J mice fed an ethanol-containing diet exhibited higher levels of liver triglycerides compared to control mice, indicating hepatic steatosis. Further, the diurnal oscillations of core clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) and clock-controlled genes (Dbp, Hlf, Nocturnin, Npas2, Rev-erbα, and Tef) were altered in livers from ethanol-fed mice. In contrast, ethanol had only minor effects on the expression of core clock genes in the suprachiasmatic nucleus (SCN). These results were confirmed in Per2(Luciferase) knock-in mice, in which ethanol induced a phase advance in PER2::LUC bioluminescence oscillations in liver, but not SCN. Further, there was greater variability in the phase of PER2::LUC oscillations in livers from ethanol-fed mice. Ethanol consumption also affected the diurnal oscillations of metabolic genes, including Adh1, Cpt1a, Cyp2e1, Pck1, Pdk4, Ppargc1a, Ppargc1b and Srebp1c, in the livers of C57BL/6J mice. In summary, chronic ethanol consumption alters the function of the circadian clock in liver. Importantly, these results suggest that chronic ethanol consumption, at levels sufficient to cause steatosis, disrupts the core hepatic clock as well as the diurnal rhythms of key lipid metabolism genes

Ozone inhalation modifies the rat liver proteome

Ozone (O3) is a serious public health concern. Recent findings indicate that the damaging health effects of O3 extend to multiple systemic organ systems. Herein, we hypothesize that O3 inhalation will cause downstream alterations to the liver. To test this, male Sprague-Dawley rats were exposed to 0.5 ppm O3 for 8 h/day for 5 days. Plasma liver enzyme measurements showed that 5 day O3 exposure did not cause liver cell death. Proteomic and mass spectrometry analysis identified 10 proteins in the liver that were significantly altered in abundance following short-term O3 exposure and these included several stress responsive proteins. Glucose-regulated protein 78 and protein disulfide isomerase increased, whereas glutathione S-transferase M1 was significantly decreased by O3 inhalation. In contrast, no significant changes were detected for the stress response protein heme oxygenase-1 or cytochrome P450 2E1 and 2B in liver of O3 exposed rats compared to controls. In summary, these results show that an environmentally-relevant exposure to inhaled O3 can alter the expression of select proteins in the liver. We propose that O3 inhalation may represent an important unrecognized factor that can modulate hepatic metabolic functions

Cosinor analysis of clock genes in the SCN.

<p>Cosinor analysis of clock genes in the SCN.</p

The methyl donor S-adenosylmethionine prevents liver hypoxia and dysregulation of mitochondrial bioenergetic function in a rat model of alcohol-induced fatty liver disease

Background: Mitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. Methods: For this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. Results: Chronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. Conclusion: Collectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the attenuation of hypoxic stress

Biochemical measurements in C57BL/6J mice.

<p>A) Blood alcohol concentration (BAC) in ethanol-fed C57BL/6J mice. One-way ANOVA analysis revealed significant changes in BAC over a 24 hour period (p<0.001). Data are presented as mean ± SEM for 3–5 ethanol-fed mice at each ZT time. B) Triglyceride content in livers from control (▪) or ethanol-fed (grey square) mice. Two-way ANOVA was performed to determine significant effects of time and/or treatment on triglyceride content. There was no main effect of time (ZT) on liver triglyceride levels (<i>F</i>(5,52) = 1.595, p = 0.178) and no significant interaction between ZT time and diet (<i>F</i>(5,52) = 1.995, <i>p = </i>0.095). However, there was a significant main effect of diet (<i>F</i>(1,52) = 91.125, p<0.0001). Significant differences in mean triglyceride content across all ZT times were determined using a t-test. Data are presented as mean±SEM for 4–6 control and ethanol-fed mice at each time point with the mean triglyceride content across all times shown in the last set of bars (n = 28 control and 28 ethanol-fed mice). ***p<0.0001, compared to control. C) Plasma ALT levels in control (▪) and ethanol-fed (grey square) mice. ALT levels were significantly increased in ethanol-fed mice as determined by Mann-Whitney Rank Sum Test (*p = 0.019). Data are presented as mean ± SEM for 21 control mice and 16 ethanol-fed mice.</p

Expression of oscillating metabolic genes in the liver of wild-type C57BL/6J mice.

<p>Gene expression of A) <i>Acaca</i>, B) <i>Adh1</i>, C) <i>Cpt1a</i>, D) <i>Cyp2e1</i>, E) <i>Dgat2</i>, F) <i>Nampt</i>, G) <i>Pck1</i>, H) <i>Pdk4</i>, I) <i>Ppargc1a</i>, J) <i>Ppargc1a</i>, and K) <i>Srebp1c</i> in livers from mice fed control (♦) or ethanol-containing (grey square) liquid diets was determined using real-time quantitative PCR. Expression levels were normalized to the <i>Gapdh</i> housekeeping gene and are displayed as fold-change from a single control mouse. Cosinor analysis for curve fitting was performed using the nonlinear regression module in SPSS. Cosinor analysis results are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071684#pone-0071684-t003" target="_blank">Table 3</a>. Data are presented as mean ± SEM for n = 4–6 control and ethanol-fed mice at each time point.</p

Expression of clock genes in the suprachiasmatic nucleus (SCN) of C57BL/6J mice.

<p>Gene expression of a) <i>Bmal1</i>, b) <i>Cry1</i>, c) <i>Per1</i>, d) <i>Per2</i>, and e) <i>Rev-erbα</i> in SCN from mice fed control (♦) or ethanol-containing (grey square) liquid diets was determined using real-time quantitative PCR. Expression levels were normalized to the <i>Gapdh</i> housekeeping gene and are displayed as fold-change from a single control mouse. Cosinor analysis for curve fitting was performed using the nonlinear regression module in SPSS. Cosinor analysis results are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071684#pone-0071684-t002" target="_blank">Table 2</a>. There were no statistically significant differences between the two feeding groups in mesor or amplitude in any genes examined in the SCN. Data are presented as mean ± SEM for n = 4 control and ethanol-fed mice at each time point.</p

Cosinor analysis of clock genes in the liver.

<p>Cosinor analysis of clock genes in the liver.</p

Expression of non-oscillating metabolism genes in the liver of C57BL/6J mice.

<p>Gene expression of A) <i>Adipor1</i>, B) <i>Aldh2</i>, C) <i>Fabp1</i>, D) <i>Ppara</i>, E) <i>Pparg</i>, F) <i>Rora</i>, and G) <i>Sirt1</i> was determined in liver from mice fed control (▪) or ethanol-containing (grey square) diets. Expression was normalized to the <i>Gapdh</i> housekeeping gene and is represented as fold-change compared to a single control mouse. Two-way ANOVA was used to determine significant effects of time and/or treatment on gene expression. Data are presented as mean ± SEM for n = 4–6 control and ethanol-fed mice at each time point.</p

PER2::LUC expression in SCN and liver tissue explants.

<p>Representative traces of PER2::LUC bioluminescence in liver and SCN are shown for a pair-fed A) control and B) ethanol-fed mouse. Peak phases of PER2::LUC expression in C–D) SCN and E–F) liver tissue cultures from individual control (•) and ethanol-fed (▴) mice are shown on the first full cycle following culture. Corresponding colors between ethanol-fed and control mice indicate those that were pair-fed. Further, correspondingly colored shapes between SCN and liver indicate that those tissues were obtained from the same mouse. The black arrow indicates the mean phase vector of the samples, where length is inversely proportional to the phase variance, and the direction indicates timing relative to the previous light cycle in LD. The plots were generated using Oriana 3 software. Data represent results from n = 9 control and 9 ethanol-fed mice.</p