Western and heart healthy dietary patterns differentially affect the expression of genes associated with lipid metabolism, interferon signaling and inflammation in the jejunum of Ossabaw pigs

Diet Quality and Statin Therapy

• Diet quality and statin therapy are established modulators of coronary artery disease (CAD) progression, but their effect on the gastrointestinal tract and subsequent sequelae that could affect CAD progression are relatively unexplored.
• To address this gap, Ossabaw pigs (N = 32) were randomly assigned to receive isocaloric amounts of a Western-type diet (WD; high in saturated fat, refined carbohydrate, and cholesterol, and low in fiber) with or without atorvastatin, for 6 months
• RNA sequencing with 100 base pair single end reads on NextSeq 500 platform was conducted in isolated pig jejunal mucosa
• A two-factor edgeR analysis revealed that the dietary patterns resulted in three differentially expressed genes related to lipid metabolism (SCD, FADS1, and SQLE).
• The expression of these genes was associated with cardiometabolic risk factors and atherosclerotic lesion severity
• Subsequent gene enrichment analysis indicated the WD, compared to the HHD, resulted in higher interferon signaling and inflammation

Coronary artery disease (CVD)

• Approximately one in three deaths in the United States is attributed to cardiovascular diseases
• Adopting a heart healthy dietary pattern is the primary evidence-based lifestyle recommendation to prevent, treat, or reverse CAD
• This dietary pattern emphasizes intake of vegetables, fruits, nuts, whole grains, and fish, while limiting intake of processed meats, refined carbohydrates, and sugar
• Numerous studies have assessed the relation between individual nutrients or food items and atherosclerosis, but the synergic effects contributed by the combined components of a heart health dietary pattern has for the most part been underinvestigated
• Individuals at elevated risk for CAD and demonstrating inadequate response to dietary modification are frequently treated with statins
• In addition to their LDL cholesterol-lowering effect, statins have pleiotropic effects including antiproliferative, anti-inflammatory, and nitric oxide promoting actions
• The similarities between pig and human GIT anatomical structure and barrier defense mechanisms make the animal a good experimental model to study the relation

Study design and animals

• Thirty-two Ossabaw pigs (16 boars + 16 gilts) were purchased from Indiana University School of Medicine, Indianapolis, IN, USA
• At 5-8 weeks of age, the pigs were transferred to the Beltsville Agricultural Research Center (BARC) and randomly allocated into four groups using a 2 × 2 factorial design
• WD-S, WD+S, HHD-S
• HHD+S.
• Each group consisted of four boars and four gilts
• After 1 month of acclimatization to a "grower diet" and another month of a gradual shift to the experimental diets
• Pigs were fed their respective diets in isocaloric amounts, with a gradual increase in total energy to meet growth requirements

Diets and atorvastatin therapy

• Both diets contained 38% of energy (E) as fat, 47% of E as carbohydrate, and 15% E as protein
• The major differences between the WD and HHD were the types of carbohydrate and fat, and amount of fiber, cholesterol, fruits, vegetables, and fish oil.

Sample collection

• At the end of the intervention period, the pigs were euthanized with an intravenous injection of Euthasol (50 mg sodium pentobarbital/kg body weight)

Isolation of jejunal mucosa and RNA extraction

• Frozen jejunum segments were incubated in pre-chilled RNAlater-ICE at −20°C for 24 h, opened longitudinally, and the mucosa layer was cleanly separated from the muscle layer using a scalpel and tweezers.
• Total RNA was extracted using TRI Reagent according to the manufacturer’s instructions.

RNA sequencing:

• Illumina TruSeq RNA Sample Preparation Kit v2.5.
• AMPure XP beads were used to construct the sample libraries following manufacturers' protocols
• Constructed libraries were quantified by KAPA Library Quantification kit
• Fragment size of libraries was determined by Experion DNA 1K Analysis kit (Bio-Rad, Hercules, CA, USA)

Characterizing jejunal mucosal cell types and sample homogeneity

• One sample in the WD+S group was identified as an outlier and showed a high enrichment of preadipocytes and T-helper 1 cells, suggesting an error during sample collection.
• Hence, this sample was not included in subsequent analysis, resulting in a final sample size of 29 pigs.

Differential expression analysis of RNA-seq data and gene enrichment analysis

• EdgeR, a two-factor model design matrix, was used to identify differential gene expression attributable to dietary patterns, atorvastatin therapy, and their interaction
• Genes with a false discovery rate (FDR) <0.2 and absolute log fold change (logFC) ≥0.6 (absolute fold change ≥1.5) were considered differentially expressed
• An exploratory pathway analysis was conducted
• A Z score was calculated indicating up- or down-regulation of pathways or functional annotations by matching the dataset to IPA Knowledge Base

Gene expression

• A panel of annotated genes primarily related to GIT permeability were selected, including genes encoded for claudin proteins, tight-junction-associated MARVEL proteins, scaffolding proteins, and mucus formation.

Estimated desaturase activity

• Fatty acid profiles were measured in fasting serum collected from the pigs at the end of the 6-month intervention period, as previously described
• The activity of stearoyl-CoA desaturases (SCD, enzyme encoded by SCD gene) was estimated using product/precursor ratios of serum fatty acids expressed in mol%, SCD16 - palmitoleate/palmitate (16:1n-7/16:0), and SCD18 - oleate/stearate (18:1m-9/18:0)
• delta-5 desatur enzyme activity was calculated based on the serum arachidonic acid/dihomo-gamma-linoleic acid (20:4n-6/20:3 n-6) ratio
• Differences were considered significant when P ≤.05

Statistical Analysis

• To evaluate the association of genes expressed in jejunal mucosa with atherosclerotic lesions severity and cardiometabolic risk factors, pigs from the four groups were pooled (n = 29).
• Included in this analysis were three differentially expressed genes (SCD, FADS1, and SQLE), eight genes involved in altered IPA biological pathways (interferon signaling, phospholipase), and 17 genes identified in "inflammation of organ" functional annotation by IPA with certain predictive directions ("increased" or "decreased," but not "affected").

Dietary patterns

• Three differentially expressed genes in the jejunal mucosa attributable to a dietary pattern effect were identified (Table 1).
• Pigs fed the WD, compared to HHD, had higher expression of stearoyl-CoA desaturase (SCD, logFC = 1.70, FDR = 0.14), and lower expression of fatty acid desaturasing enzyme-1 (FADS1), both of which were found in the WD diet.

Atorvastatin therapy

• Decrease in Folate hydrolase 1B gene expression compared to nontreated pigs
• This difference appeared to be driven by two pigs in the HHD-S group that had expression of this gene that was 20times higher than the average expression in the other pigs.
• A reversed trend was observed by excluding these two samples.

Gene enrichment analysis

• One hundred and forty-three genes that differed by dietary patterns with absolute log fold change of ≥0.6 were included in IPA gene enrichment analysis.
• Two significant biological pathways were identified that differed between the two dietary patterns (Table 2).
• The pigs fed WD exhibited lower phospholipase pathway than the pigs in the HHD groups, attributable to four genes (lipase C hepatic type [LIPC], lipase G endothelial type, lipid hydrolysis of lipid, hydrolytic reduction of triglycerides, phosphorylation of cholesterol, inflammation of organ)
• Diseases & functions analysis identified 10 significant functional annotations up-regulated and 7 down-regulated in pigs fed the WD, when compared to HHD (Table 3). The most significant functional annotation module was higher "inflammation of organ" (Z = 3.15, P <.001) in the WD group, compared to the pigs fed HHD.

Estimated desaturase activity

• The activity of SCD16 was significantly higher in the WD- than HHD-fed pigs (P <.0001, Table 4).
• In contrast, the estimated SCD18 activity was similar between the HHD-, WD- and WD-fed animals (P =.23).

Association of gene expression with atherosclerotic lesion severity and cardiometabolomic risk factors

• Among the differentially expressed genes (Table 5), the expression of SCD gene in the jejunal mucosa was positively associated with a person’s risk of developing a lesion, serum LDL and HDL cholesterol concentrations, and serum TNF-α concentrations.
• FADS1 gene expression was negatively associated with the same three variables, as was SQE, which negatively correlated with the levels of cholesterol, LDL, and triglyceride concentrations in the blood.

Sex-specific trends

• The impact of dietary patterns on pathways and functional annotation was similar in boars and gilts (Supplementary Fig. S3.1).

Discussion

• This study assessed the effects of two dietary patterns, designed to mimic a WD and a HHD, with and without atorvastatin therapy, on gene expression in the jejunal mucosa of Ossabaw pigs and relate these data to atherosclerotic lesion severity.
• The study found that dietary patterns altered gene expression associated with lipid metabolism, phospholipase pathway, interferon signaling pathway, and inflammation, and altered estimated enzyme activity or expression of FADS1, SCD, FADS16, and SQLE, among others.

Reference

 10.1016/j.jnutbio.2020.108577