Oligonucleotide microarray analysis of dietary-induced hyperlipidemia gene expression profiles in miniature pigs

Abstract

Background: Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly.

Methodology: Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF) Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD) and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD). Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models.

Principal findings: Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO) based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs.

Conclusions: No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in dietary-induced hyperlipidemia gene expression profiles in miniature pigs.

Figure 1. Subject body weights.

• represents control, ⧫ represents HFCD, and ▴ represents HFCSD. Values correspond to means (SD).

Figure 2. Correlation matrix of dietary-related gene expression profiles of whole blood and white blood cells.

This color-coded correlation matrix illustrates pairwise correlations between the levels of gene expression in individuals. Probe sets with normalized signals (log-transformed and scaled) were used to calculate correlations between 45 arrays using Pearson correlation coefficient; signals flagged as “absent” were excluded. The color scale at the bottom indicates the strengths of the correlations.

Figure 3. Summary of dietary-related correlation coefficients within the same diet groups.

(A) Whole blood after 10 weeks. (B) Whole blood after 19 weeks. (C) Whole blood after 27 weeks. (D) White blood cells after 27 weeks. The bottom and top of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiskers denote the minimum and maximum values of the data. Using Fisher's Z-transform for normalization the correlation distribution, continuous variables were analyzed by one-way factorial ANOVA followed by Tukey-Kramer multiple comparisons test for multiple groups. Correlations were considered to be statistically significant when ANOVA test among all groups and t-test between 2 groups should p<0.05. NS; not significant.

Figure 4. Summary of dietary-related correlation coefficients among different diet groups.

(A) Whole blood after 10 weeks. (B) Whole blood after 19 weeks. (C) Whole blood after 27 weeks. (D) White blood cells after 27 weeks. The bottom and top of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiskers denote the minimum and maximum values of the data. Comparisons of the groups were made with the ANOVA test. NS; not significant.

Figure 5. Correlation coefficients between whole blood and white blood cells within the same diet groups.

Correlation coefficients were calculated between whole blood and white blood cells within the same diet group at 27 weeks feeding period. The bottom and top of the boxes represent the 25th and 75th percentiles respectively. The lower and upper whiskers denote the minimum and maximum values of the data. Comparisons of the groups were made with the ANOVA test. NS; not significant.

Figure 6. Scatter plot of dietary-related correlation coefficients.

(A) Control vs. HFCD. (B) Control vs. HFCSD. The correlation coefficient between whole blood and white blood cells after 27 weeks of each GO tem was plotted for each spot. The solid line represents the regression line. The dashed line represents the slope equal to 1.

Figure 7. The relation of tissue or organ ESTs to the white blood cell contribution indicator.

The X-axis indicates the expression intensity ratio of white blood cells to whole blood for each gene as the white blood cell contribution indicator in our experiments. The Y-axis indicates the liver, adipose tissue, or muscle EST numbers normalized to the blood EST number of each gene in Unigene, an NCBI database of the transcriptome.