| Cholesterol homeostasis is necessary for the maintenance of normal life activities. Liver is for the place where cholesterol is synthesized, transported, secreted and transformated, thus liver takes an irreplaceable role in cholesterol homeostasis. Hepatic cholesterol metabolism differs between breeds, yet the molecular mechanism is unclear. Nutrients, such as lipids and sterols in diets, are reported to effect on hepatic cholesterol transportation and transformation, while the effect of dietary protein is rarely reported. Recent study on rats show that maternal dietary protein restriction could change cholesterol homeostasis by epigenetic modification of key enzymes for cholesterol transportation and transformation in offspring livers, while the epigenetic programming on cholesterol synthesis in the liver is not explored yet. Our study divided into two parts. The first part took fat Erhualian and lean Large White as models to study the differences in hepatic cholesterol metabolism between breeds and the mechanism involved. The second part was done on Meishan pigs to study the effects of maternal protein (traditional low-protein diet and standard protein diet of NRC) during pregnancy and lactation on hepatic cholesterol metabolism of offspring and the epigenetic mechanisms.1. The differences on hepatic cholesterol metabolism between Erhualian and Large White pigletsLarge White and Erhualian piglets (at birth and25-day-old) were used in the study,6each time point per breed. Animals were weighted, liver and serum were collected for the analysis. Cholesterol concentration and other lipid parameters were measured by commercial kits, hepatic cholesterol metabolism-related gene expressions were tested by Real-time PCR. The protein of the key regulatory factors and enzyme were investigated by Western blotting. These results suggest that Erhualian piglets had significantly lower body and liver weight compared with Large White (P<0.01), but the liver/body weight ratio showed no difference between the two breeds, no matter which time point tested. On the birth piglets, Erhualian had significantly higher serum total cholesterol (Tch) as well as higher density lipoprotein cholesterol (HDL-C) and liver cholesterol content (P<0.01), but significantly lower serum low density lipoprotein cholesterol (LDL-C) concentration compared with Large White; serum and liver triglyceride (TG) concentrations were similar between breeds (P>0.05) together with significantly lower CYP7A1mRNA in the Erhualian piglets. On the25-day-old piglets, cholesterol and triglyceride concentrations in serum (P<0.01) and liver (P<0.05) were significantly higher in Erhualian than those in Large White, with significantly higher SREBP2mRNA and protein levels in Erhualian.In conclusion, Erhualian had significantly higher liver and serum cholesterol than Large White. The lower CYP7A1expression in birth Erhualian indicated a lower conversion rate from cholesterol to bile acids in Erhualian birth, while the higher content of SREBP2in liver result in a stronger cholesterol synthesis in Erhualian of25-day-old, indicating that the age-depended of the mechanism effect on cholesterol concentration in breed-specific in this study.2. Effects of maternal protein on liver cholesterol, cholesterol metabolism related gene and protein expression in g Little Meishan weanin piglets14primiparous Little Meishan sow were randomly divided into two groups, treated with standard protein (SP) and low-protein (LP) during pregnancy and lactation respectively. Crude protein concentration in SP, according to NTC, was12%during pregnancy and14%during lactation and those of LP were6%and7%, respectively; the energy content was balanced to the same. The piglets were under35-day weaning program, slaughtered at day35. Serum and liver were collected for measurement of hormones, serum lipid parameters and liver triglyceride and cholesterol analysis. Hepatic cholesterol metabolism-related gene expressions were tested by Real-time PCR. The protein of the key regulatory factors and enzyme were investigated by Western blotting. The activities of key enzymes in liver cholesterol synthesis were evaluated by commercial kits. The results indicated that body weight and liver weight of weaning piglets in LP group were significantly lower than that of SP group (P<0.05), while the liver/body weight ratio were comparable. Seum T3and T4were not changed by maternal protein, but circulating insulin, cholesterol and liver cholesterol were significantly lower in piglets from LP fed sows(P<0.05), with a tendency to down of liver TG(P=0.08). The serum HDL, LDL and HDL/LDL ratio were not changed. Piglets from LP fed sows had significant higher mRNA and protein of SREBP2, higher transcription of Insig and HMGCR and lower levels of CYP7al transcript(P<0.05). These results indicated that by feeding Little Meishan sows with low-protein diet during pregnancy and lactation, had lower serum and liver cholesterol level, accompanied with change on cholesterol synthesis-and transformation-related genes and/or proteins, suggesting that the liver cholesterol metabolism in the offspring was programmed by maternal diet protein through changing functional gene and/or protein expression.3. Maternal dietary protein level affects offspring hepatic cholesterol synthesis through epigenetic regulation of gene expressionHMGCR is the rate-limiting enzyme in hepatic cholesterol synthesis and its promoter region is well displaied, so it is an ideal target to explore the epigenetic modifications involved in the programming mechanism on the offsprings.Methylation level of HMGCR promoter region was measured by DNA immunoprecipitation by using5-methylcytosine antibody. Anti-histone H3acetylation, histone H3K9monomethyl, histone H3K27trimethyl and histone H3K4trimethyl antibodies were used to measure histone H3acetylation and methylation in HMGCR promoter region by chromatin immunoprecipitation. In addition, expression of miRNAs targeting HMGCR was measured by Real-time PCR. Results indicated methylation in HMGCR promoter region and histone H3K9me enrichment in the LP group was significantly lower than that in SP group (P<0.01), together with significantly higher histone H3acetylation (P<0.01) but lower histone H3K27me3(P=0.08) enrichment on HMGCR promoter region. The expression of liver miRNAs targeting HMGCR showed no significant difference between LP and SP (P>0.05). In summary, the low-protein diet during pregnancy and lactation period of Little Meishan sows could enhance HMGCR expression in the offspring livers through reducing DNA methylation of HMGCR promoter region, increasing acetylation of histone H3, and down regulation epigenetic modifications, such as H3K9me and H3K27me3. |
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