The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis

金沢大学医薬保健研究域医学系Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH. © 2009 Elsevier Inc. All rights reserved

Feeding Cues and Injected Nutrients Induce Acute Expression of Multiple Clock Genes in the Mouse Liver

The circadian clock is closely associated with energy metabolism. The liver clock can rapidly adapt to a new feeding cycle within a few days, whereas the lung clock is gradually entrained over one week. However, the mechanism underlying tissue-specific clock resetting is not fully understood. To characterize the rapid response to feeding cues in the liver clock, we examined the effects of a single time-delayed feeding on circadian rhythms in the liver and lungs of Per2::Luc reporter knockin mice. After adapting to a night-time restricted feeding schedule, the mice were fed according to a 4, 8, or 13 h delayed schedule on the last day. The phase of the liver clock was delayed in all groups with delayed feeding, whereas the lung clock remained unaffected. We then examined the acute response of clock and metabolism-related genes in the liver using focused DNA-microarrays. Clock mutant mice were bred under constant light to attenuate the endogenous circadian rhythm, and gene expression profiles were determined during 24 h of fasting followed by 8 h of feeding. Per2 and Dec1 were significantly increased within 1 h of feeding. Real-time RT-PCR analysis revealed a similarly acute response in hepatic clock gene expression caused by feeding wild type mice after an overnight fast. In addition to Per2 and Dec1, the expression of Per1 increased, and that of Rev-erbα decreased in the liver within 1 h of feeding after fasting, whereas none of these clock genes were affected in the lung. Moreover, an intraperitoneal injection of glucose combined with amino acids, but not either alone, reproduced a similar hepatic response. Our findings show that multiple clock genes respond to nutritional cues within 1 h in the liver but not in the lung

Reduction of Type IV Collagen by Upregulated <em>miR-29</em> in Normal Elderly Mouse and <em>klotho</em>-Deficient, Senescence-Model Mouse

<div><p>MicroRNA (miRNA), a small non-coding RNA that functions as a mediator in gene silencing, plays important roles in gene regulation in various vital functions and activities. Here we show that the <em>miR-29</em> members are upregulated in <em>klotho</em>-deficient [<em>klotho(−/−)</em>] mice, a senescence-model animal, and also in normal elderly ICR mice relative to wild-type littermates and young ICR mice. In addition, levels of type IV collagen, a major component of basement membranes and a putative target of <em>miR-29</em>, were lower in <em>klotho(−/−)</em> and elderly ICR mice than in wild-type littermates and young ICR mice. RNA degradation mediated by <em>miR-29</em> may participate in the suppression of type IV collagen, both <em>in vivo</em> and <em>in vitro</em>. Taken together, our current findings suggest that the <em>miR-29</em> upregulated in aging may be involved in the downregulation of type IV collagen, leading to a possible weakening of the basal membrane in senescent tissues, and <em>miR-29</em> may be a useful molecular marker of senescence.</p> </div

Assessment of knockdown potency using reporter genes.

<p>(<b>A</b>) Schematic drawing of constructed reporter genes. The psiCheck2-backbone reporter plasmid encoding the <i>Renilla luciferase</i> gene carrying the target sequence of <i>miR-29a</i> (psiCheck2-miR29a) or <i>miR-29b</i> (psiCheck2-miR29b) was constructed. The SV40 promoter, <i>Renilla luciferase</i> coding region (CDR) and 3′ untranslated region (3′UTR) of the reporter genes are indicated. The target sequences of <i>miR-29a</i> and <i>miR-29b</i> are indicated in the 3′UTRs. (<b>B, C</b>) Suppression of reporter genes by synthetic <i>miR-29</i> mimics. The reporter plasmids indicated were transfected together with synthetic miRNA mimics of <i>miR-29a</i> (mimic_29a), <i>miR-29b</i> (mimic_29b) or a negative control miRNA (negative_miR) into N2a cells. 24 hr after transfection, dual-luciferase assay was carried out. The target (<i>Renilla</i>) luciferase activity was normalized to control (<i>Photinus</i>) luciferase activity and further normalized to the data obtained from N2a cells transfected with only the reporter plasmid (non-treatment). Data are averages of four independent experiments. Error bars represent standard deviations. The normalized data were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett's test. Significant differences are indicated by * (<i>P</i><0.05).</p

<i>miR-29a</i> and <i>miR-29b</i> expression in young and elderly ICR mice.

<p>Total RNAs were extracted from indicated tissues of young (1-month-old: 1 m) and elderly (14- or 18-month-old: 14 m or 18 m) ICR mice, and subjected to RT-qPCR analysis. The <i>miR-29a</i>, <i>miR-29b</i> and <i>snoRNA202</i> (as a control) expressions were examined and analyzed by the delta-delta Ct method. Five individual mice in each group were investigated to account for individual variability, and three measurements by qPCR per sample were carried out. Error bars represent standard deviations. The normalized <i>miR-29a</i> and <i>miR-29b</i> expression levels in the young (gray bars) and elderly (red bars) groups in each tissue were further examined by Student's <i>t</i>-test, and significant differences between the two groups in all the tissues examined were detected (* <i>P</i><0.05, **<i>P</i><0.01).</p

<i>In vivo</i> knockdown potency mediated by endogenous <i>miR-29a</i> or <i>miR-29b</i>.

<p>The constructed reporter plasmid carrying the <i>miR-29a</i> or <i>miR-29b</i> target sequence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048974#pone-0048974-g002" target="_blank">Fig. 2A</a>) and the <i>β-galactosidase</i> expression plasmid as a control were systemically administrated to young (5-week-old: 5w) and elderly (8∼18-month-old: ≈1 y) ICR mice by a hydrodynamic delivery method. Three days after administration, kidney cell extracts were prepared from the treated mice, and then the luciferase and β-galactosidase activities were examined. The expression levels of luciferase were normalized to those of β-galactosidase, and further normalized to the data obtained with a control empty vector (Empty). Data are averages of three different individual's data. Error bars represent standard deviations. The normalized data in the young (5w) and elderly (≈1 y) groups were analyzed by Student's <i>t</i>-test (one-tailed), and significant differences from the data of each empty vector are indicated by * (<i>P</i><0.05).</p

Gene expression profiles in <i>miR-29</i>-treated N2a cells and young ICR mice.

<p>(<b>A</b>) Expression profiles in <i>miR-29</i>-treated N2a cells. MISSION microRNA Mimic (Sigma-Aldrich) <i>has-miR-29a</i> (mimic_29a), <i>has-miR-29b</i> (mimic_29b) or negative control miRNA (negative_miR) was transfected into N2a cells. Three days after transfection, total RNAs were extracted and examined by RT-qPCR followed by analysis using the delta-delta Ct method using the expression levels of <i>Gapdh</i> as a control. The data were further normalized to the data obtained with the negative_miR. Error bars represent standard deviations. The genes examined are indicated: <i>Col4α1</i>, <i>Col4α2</i>, <i>Dicer1</i>, <i>Dnmt1</i>, <i>Lamc1</i> and <i>Cabin1</i>. The <i>Col4α3–α6</i> genes were examined as well, but their expression was not detected in the cells. (<b>B</b>, <b>C</b>) Gene expression profiles in <i>miR-29b</i>-treated ICR mice. Normal ICR mice (5-week-old) were subjected to four systemic administrations (Day 1, 4, 8, and 12) of <i>miR-29b</i> duplex or siControl (as a negative control). Two days after the last administration, the treated mice were examined. RT-qPCR analyses were carried out as in A. The examined tissues and genes are indicated: (<b>B</b>) <i>type IV collagen</i> gene family and (<b>C</b>) other genes. Statistically significant decrease in the expression of genes in the <i>miR-29b</i>-treated mice is indicated by * (<i>p</i><0.05).</p

Cell viability of N2a cells treated with <i>miR-29</i> mimics.

<p>The <i>miR-29</i> mimics (mimic_29a and mimic_29b) and a negative control miRNA (negative_miR) were transfected into N2a cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048974#pone-0048974-g006" target="_blank">Fig. 6A</a>. Three days after transfection, cell viability assay was performed. Data are averages of four measurements. Error bars represent standard deviations. Difference between non-treated cells and each of the treated cells was statistically analyzed by ANOVA, followed by the Dunnett's test (* <i>P</i><0.05).</p

Plasma creatinine, HEL, and 8OHdG levels in elderly and young mice and <i>miRNA</i>-treated mice.

<p>Plasma samples were prepared from young (5-week-old; 5w) and elderly (8∼18-month-old: ≈1 y) ICR mice and also from miR-29b- and siControl (siCont)-treated ICR mice (5-week-old), and then subjected to plasma biochemical tests. The level of creatinine was measured as an index of renal function, and HEL and 8OHdG were each examined as a stress marker. Data are averages of three different individual's measurements. Error bars represent standard deviations. Differences were statistically analyzed by Student's <i>t</i>-test. n.s., no statistical significance.</p