Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

Abstract

Background: Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls.

Results: A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p < 0.05 for the overall physiologic state effect (lactation vs. control), and a within tissue pairwise comparison of p < 0.01. The proportion of false positives, an estimate of the ratio of false positives in the list of differentially expressed genes, was calculated for each tissue. The number of differentially expressed genes was 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum. The list of differentially expressed genes was in turn analyzed by Ingenuity Pathways Analysis (IPA) to detect biological pathways that were overrepresented. In all tissues, sterol regulatory element binding protein (Srebp)-regulated genes involved in cholesterol synthesis showed increased mRNA expression, with the fewest changes detected in the jejunum. We detected increased Scap mRNA in the liver only, suggesting an explanation for the difference in response to lactation between the liver and small intestine. Expression of Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC) and Adenosine Triphosphate Binding Cassette (ABC) superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175.

Conclusions: We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and jejunum) was also noted, as well as differential expression of transporters that likely play a key role in nutrient uptake.

Figure 1

T-cell signaling in lactating jejunum and ileum. Downregulation of T-cell signaling in the jejunum (top, p = 1.68 × 10^-7) and ileum (bottom, p = 4.7 × 10^-4). Green shading indicates downregulation of the corresponding gene during lactation.

Figure 2

Plot of Mean Intensities for Known Cholesterol Biosynthetic Enzymes. The mean untransformed intensities were plotted against tissue/physiologic state combination. C and L indicate the mean of Control and Lactating samples, respectively, within each tissue. Abbreviations for genes are as follows. Dhcr7: 7-dehydrocholesterol reductase; Fdft1: farnesyl-diphosphate farnesyltransferase 1; Sc4mol: sterol-C4-methyl oxidase-like; Idi1: isopentenyl-diphosphate delta isomerase 1; Mvd: mevalonate (diphospho) decarboxylase; Hmgcs: 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1; Hmgcr: HMG Coenzyme A reductase; Lss: lanosterol synthase; Cyp51: cytochrome P450 family 51; Sqle: Squalene Epoxidase. A red line between Control and Lactating symbols for a given gene indicates p > .0.01 for the tissue pairwise comparison within the tissue from the mixed models repeated measures ANOVA on the log₂ transformed intensities; the change was not considered significant for the purposes of determining a list of differentially expressed genes. A black line between C and L symbols indicates p < 0.01 as described above.