Fasting-induced changes in SIRT3 and PGC-1α mRNA and protein expression of lean and obese dam offspring.

<p>(<b>A</b>) SIRT3 mRNA expression, (<b>B</b>) SIRT3 mitochondrial protein content, and (<b>C</b>) PGC-1α mRNA expression in liver from fed and fasted offspring of lean and obese dams at PND 21 (N = 8–15 per group). Representative blot is also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024068#pone-0024068-g005" target="_blank">FIG 5B</a> for SIRT3 protein content (N = 4 per group). Values are expressed as means±SE, different letter superscripts indicate statistical significance (p<0.05).</p

Electron transport chain complexes from mitochondrial fractions of livers from offspring of lean and obese dams at PND 21.

<p>(A) Representative blots and (B) densitometric quantitation (N = 3–4 pools representing a total of 6–8 animals/group). Values are expressed as means±SE. Statistical differences were determined using a Student's t-test. * indicates p<0.05.</p

Primers Sequences for Real-time RT-PCR Analyses.

<p>Gene specific primers were designed using Primer Express™ Software (Applied Biosystems, Foster city, CA). Real-time PCR reactions were carried out according to manufacturer's instructions for 2X SYBR green master mix and monitored on a ABI Prism 7500 sequence detection system (Applied Biosystems, Foster city, CA) as described under methods. SIRT: sirtuin; PGC-1α: peroxisome proliferator activated receptor-γ coactivator-1α.</p

High Fat Diet and <i>In Utero</i> Exposure to Maternal Obesity Disrupts Circadian Rhythm and Leads to Metabolic Programming of Liver in Rat Offspring

<div><p>The risk of obesity in adulthood is subject to programming beginning at conception. In animal models, exposure to maternal obesity and high fat diets influences the risk of obesity in the offspring. Among other long-term changes, offspring from obese rats develop hyperinsulinemia, hepatic steatosis, and lipogenic gene expression in the liver at weaning. However, the precise underlying mechanisms leading to metabolic dysregulation in the offspring remains unclear. Using a rat model of overfeeding-induced obesity, we previously demonstrated that exposure to maternal obesity from pre-conception to birth, is sufficient to program increased obesity risk in the offspring. Offspring of obese rat dams gain greater body weight and fat mass when fed high fat diet (HFD) as compared to lean dam. Since, disruptions of diurnal circadian rhythm are known to detrimentally impact metabolically active tissues such as liver, we examined the hypothesis that maternal obesity leads to perturbations of core clock components and thus energy metabolism in offspring liver. Offspring from lean and obese dams were examined at post-natal day 35, following a short (2 wk) HFD challenge. Hepatic mRNA expression of circadian (CLOCK, BMAL1, REV-ERBα, CRY, PER) and metabolic (PPARα, SIRT1) genes were strongly suppressed in offspring exposed to both maternal obesity and HFD. Using a mathematical model, we identified two distinct biological mechanisms that modulate PPARα mRNA expression: i) decreased mRNA synthesis rates; and ii) increased non-specific mRNA degradation rate. Moreover, our findings demonstrate that changes in PPARα transcription were associated with epigenomic alterations in H3K4me3 and H3K27me3 histone marks near the PPARα transcription start site. Our findings indicated that offspring from obese rat dams have detrimental alternations to circadian machinery that may contribute to impaired liver metabolism in response to HFD, specifically via reduced PPARα expression prior to obesity development.</p></div

Indirect calorimetry of offspring from lean and obese rat dams.

<p>(<b>A</b>) EC50 values for energy expenditure (kcal/day) and (<b>B</b>) Respiratory exchange ratio (RER) as shown by PRCF analysis in the offspring of lean and obese rat dams (N = 5 per group) fed either an AIN-93G or high fat diet (45% kcals from fat) <i>ad libitum</i>. EC50 values were also included as means±SE. Different letter superscripts indicate statistical significance (p<0.05). (<b>C</b>) 24-hr averaged values of energy expenditure, (<b>D</b>) RER, (<b>E</b>) total activity counts, and (<b>F</b>) food intake are shown from offspring of lean and obese rat dams (N = 5 per group) on either an AIN-93 diet or high fat diet (45% kcals from fat). Values are expressed as means±SE, different letter superscripts indicate statistical significance (p<0.05).</p

Hepatic mRNA expression of sirtuin 1-7 from offspring of lean and obese dams at PND 21.

<p>Gene expression was assessed via real-time RT-PCR (N = 7 per group). Values are expressed as means±SE. Statistical differences were determined using a Student's t-test. * indicates p<0.05.</p

Hepatic SIRT3 mitochondrial protein content of lean and obese dam offspring.

<p>Representative blot and densitometric quantitation of SIRT3 protein content in the mitochondrial fraction from livers of offspring of lean and obese dams at PND21 by Western blotting (N = 4 pools representing a total of 8 animals/group). Values are expressed as means±SE. Statistical differences were determined using a Student's t-test. * indicates p<0.05.</p

Hepatic mitochondrial protein content and acetylation of LCAD of lean and obese dam offspring.

<p>(<b>A</b>) Representative blot and densitometric quantitation of LCAD in the mitochondrial fraction from livers of offspring from lean and obese dams at PND21 by Western blotting (N = 4 pools representing a total of 8 animals/group). (<b>B</b>) Representative blot and densitometric quantitation of acetylated LCAD in the mitochondrial fraction from fasted livers of offspring at PND21 (N = 3 per group). Immunoprecipitation of LCAD was performed in total liver lysates and immunoblotting was performed using anti-acetylated lysine antibody. Values are expressed as means±SE. Statistical differences were determined using a Student's t-test. * indicates p<0.05.</p

H3K27me3 enrichment on the PPARα promoter.

<p>Chromatin immunoprecipitation for H3K27me3 was carried out and A) upstream (−500 base pairs) and B) downstream (+500 base pairs from TSS) regions of the PPAR-α gene were amplified in pools of liver samples (each pool represents 3–5 separate animals) from offspring of lean or obese dams fed either control or HFD (run in triplicate). Enrichment was determined by real time RT-PCR and normalized to input levels. Data are presented as mean ± SEM. Statistical differences were determined using a two-way ANOVA to examine the effects of maternal obesity and post-weaning HFD (P<0.05). Significant interactions were followed by one way ANOVA and Student-Newman-Keuls <i>post hoc</i> analyses (P<0.05). Bold values represent significant main effects and interactions and values with different letter superscripts are significantly different from each other (P<0.05).</p

H3K4me3 enrichment on the PPARα promoter.

<p>Chromatin immunoprecipitation for H3K4me3 was carried out and A) upstream (−500 base pairs) and B) downstream (+500 base pairs from TSS) regions of the PPAR-α gene were amplified in pools of liver samples (each pool represents 3–5 separate animals) from offspring of lean or obese dams fed either control or HFD (run in triplicate). Enrichment was determined by real time RT-PCR and normalized to input levels. Data are presented as mean ± SEM. Statistical differences were determined using a two-way ANOVA to examine the effects of maternal obesity and post-weaning HFD. Significant interactions were followed by one way ANOVA and Student-Newman-Keuls <i>post hoc</i> analyses (P<0.05). Bold values represent significant main effects and interactions and values with different letter superscripts are significantly different from each other (P<0.05).</p