Abstract
This study investigates the mechanism by which maternal protein restriction induces hepatic autophagy-related gene expression in the offspring of rats. Pregnant Sprague-Dawley rats were fed either a control diet (C, 18 % energy from protein) or a low-protein diet (LP, 8·5 % energy from protein) during gestation, followed by the control diet during lactation and post-weaning. Liver tissue was collected from the offspring at postnatal day 38 and divided into four groups according to sex and maternal diet (F-C, F-LP, M-C and M-LP) for further analysis. Autophagy-related mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. In addition, chromatin immunoprecipitation (ChIP) was performed to investigate the interactions between transcription factors and autophagy-related genes. Protein levels of p- eukaryotic translation initiation factor 2a and activating transcription factor 4 (ATF4) were increased only in the female offspring born to dams fed the LP diet. Correlatively, the mRNA expression of hepatic autophagy-related genes including Map1lc3b, P62/Sqstm1, Becn1, Atg3, Atg7 and Atg10 was significantly greater in the F-LP group than in the F-C group. Furthermore, ChIP results showed greater ATF4 and C/EBP homology protein (CHOP) binding at the regions of a set of autophagy-related genes in the F-LP group than in the F-C group. Our data demonstrated that a maternal LP diet transcriptionally programmed hepatic autophagy-related gene expression only in female rat offspring. This transcriptional programme involved the activation of the eIF2α/ATF4 pathway and intricate regulation by transcription factors ATF4 and CHOP.
Highlights
A gestational LP diet could alter the expression of a set of hepatic genes and induced an altered metabolic phenotype in the liver of offspring[6,7], which may provide a mechanism for the impaired lipid and carbohydrate metabolism induced by maternal protein restriction observed in later life[8,9]
The expression of Atf3 and Chop can be induced by activating transcription factor 4 (ATF4), upregulating the amino acid response (AAR) pathway, which in turn affects the protein metabolism that helps to adapt to the LP environment[14]
From the chromatin immunoprecipitation results, we found that: [1] ATF4 but not C/ EBP homology protein (CHOP) bound at the regions of Becn1 (P < 0·001), Atg3 (P = 0·01) and Map1lc3b (P < 0·001) (Fig. 6); [2] both ATF4 and CHOP bound to P62/ Sqstm1 and Atg7 (Fig. 7); and [3] CHOP bound in the regions of Atg5 (P < 0·001) and Atg10 (P < 0·001) (Fig. 8)
Summary
Timed-pregnant Sprague-Dawley rats were purchased from Charles River Laboratories and were individually housed in standard polycarbonate cages in a humidity- and temperature-controlled room with free access to food and water on a 12-h light/ 12-h dark cycle. They were fed either a control diet (C group, 18 % energy from protein) or a LP diet (LP group, 9 % energy from protein) (Bioserve) from gestation day 2 during gestation. MRNA levels, protein levels and chromatin immunoprecipitation data were analysed using twoway ANOVA to look at the effects of maternal diets, sex and their interactions on the hepatic responses in the offspring, specially changes in the mechanisms that regulate autophagy-related gene expression (F-C and F-LP, M-C and M-LP, sample size n 8 for each). The sample size was calculated based on AAR gene expression data from a previous study[16] to achieve 80 % statistical power and 0·05 two-sided significance level
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