Maternal low-protein diet affects epigenetic regulation of hepatic mitochondrial DNA transcription in a sex-specific manner in newborn piglets associated with GR binding to its promoter

Abstract

Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in energy homeostasis by controlling electron transfer and ATP generation. Maternal malnutrition during pregnancy affects mitochondrial (mt) DNA-encoded OXPHOS activity in offspring, yet it is unknown whether epigenetic mechanism is involved in the transcriptional regulation of mtDNA-encoded OXPHOS genes. In this study, 14 primiparous purebred Meishan sows were fed either standard- (SP, 12% crude protein) or low-protein (LP; 6% crude protein) diets throughout gestation, and the hepatic expression and transcriptional regulation of mtDNA-encoded OXPHOS genes were analyzed in newborn piglets. Maternal low protein diet decreased hepatic mtDNA copy number in males, but not in females. LP male piglets had significantly higher hepatic AMP concentration and low energy charge, which was accompanied by enhanced mRNA expression of NADH dehydrogenase subunits 6, cytochrome c oxidase subunit 1, 2, 3 and cytochrome b, as well as increased cytochrome c oxidase enzyme activity. In contrast, LP female piglets showed significantly lower hepatic AMP concentrations and higher energy charge with no alterations in OXPHOS gene expression. Moreover, LP males demonstrated higher glucocorticoid receptor (GR) binding to the mtDNA promoter compared with SP males, which was accompanied by lower cytosine methylation and hydroxymethylation on mtDNA promoter. Interestingly, opposite changes were seen in females, which showed diminished GR binding and enriched cytosine methylation and hydroxymethylation on mtDNA promoter. These results suggest that maternal low protein diet during pregnancy causes sex-dependent epigenetic alterations in mtDNA-encoded OXPHOS gene expression, possibly GR is involved in mtDNA transcription regulation.

Figure 1. The mtDNA copy number in liver. Values are mean ± SEM, Means without a common letter differ, P<0.05.

Filled bar, standard protein diet; blank bar, low-protein diet. mtDNA, mitochondrial DNA.

Figure 2. Expression of mtDNA-encoded genes and COX enzyme activity in liver.

(A) Expression of mtDNA-encoded genes in liver of male newborn piglets. (B) Expression of mtDNA-encoded genes in liver of female newborn piglets. (C) COX enzyme activity in liver of both male and female newborn piglets. Filled bar, standard protein diet; blank bar, low-protein diet. Values are mean ± SEM, * P<0.05 vs. SP of the same sex. Means without a common letter differ, P<0.05.

Figure 3. The levels of GR mRNA and protein in liver.

(A) Hepatic GR gene expression in both male and female newborn piglets. (B) The content of hepatic GR protein in both male and female newborn piglets. Filled bar, standard protein diet; blank bar, low-protein diet. Values are mean ± SEM, * P<0.05 vs. SP of the same sex.

Figure 4. The levels of GR binding and 5(h)mC modifications on mtDNA promoter in the liver of newborn piglets.

(A) Schematic structure of pig mtDNA promoter. (B) GR binding to mtDNA promoter. (C) DNA methylation status of the CpG island on mtDNA promoter. (C) DNA hydroxymethylation status of the CpG island on mtDNA promoter. Values are mean ± SEM, Means without a common letter differ, P<0.05. Filled bar, standard protein diet; blank bar, low-protein diet. GREs, glucocorticoid response elements; HSP, heavy strand promoter; LSP, light strand promoter.