Monkeys eating two distinct protein diets were studied to delineate the effects of soy protein in animals initially fed standard monkey chow (which contains soy) or a casein-based diet (). These monkeys had been consuming their initial diets for a minimum of two years before the current study, and as such, some phenotypic differences were observed between the two groups at baseline (). For this reason, the two diet arms were analyzed separately. Insulin resistance, as determined by HOMA-IR, was significantly higher in animals eating the initial high-fructose diet compared to animals eating chow (4.33 versus 3.00 HOMA units; p
0.005). In addition, proinsulin levels were higher in the high fructose group (131.13 pmol/L versus 42.5 pmol/L, p
0.025) and total plasma cholesterol was lower (91.75 mg/dL versus 102.25 mg/dL, p
0.031). Over the course of the study, there were no significant changes in the clinical characteristics of monkeys in Diet Arm 1 (). In Diet Arm 2, fasting insulin levels were lower after changing from the TAD-casein to the TAD-soy diet (35.75 µU/ml versus17 µU/ml, p
0.01). Related to the insulin levels, lower insulin resistance was also observed with the TAD-soy diet (2.67 versus 6.03 HOMA units, p
0.02). No other clinical or physiologic parameters changed significantly between animals eating the different diets.
Characteristics of monkeys on each diet at baseline and after diet changes.
We performed DNA methylation analysis using the HumanMethylation27 BeadChip (Illumina, Inc.). Using the “detection p-value” metric in GenomeStudio (which evaluates the probe intensity for each CpG site compared to a negative control), 80.6% of the assays generated a sufficient signal in at least one sample from cynomolgus monkeys. Using methylation data from all probes with a detection p-value less than 0.05, PCA analysis was performed with all monkeys, on each diet. Four clusters were observed, predominately defined by tissue type (). Samples from blood provided the most compact cluster, indicating that the DNA methylation pattern from blood was highly similar among all samples. Six liver samples were outside of the liver cluster (three of these were from the same monkey), but all other samples formed easily observable clusters based on the tissue type (i.e., blood, liver, fat, or muscle). Using all animals and each diet condition, a total of 95 samples (one sample was unacceptable for analysis) were analyzed.
PCA analysis of DNA methylation levels from all monkeys eating each diet.
DNA methylation levels varied by diet and tissue type, and overall DNA methylation levels increased in liver and muscle after transitioning from the TAD-soy to the TAD-casein diet (, top). The levels in blood remained the same while levels in fat decreased slightly. In liver, the proportion of overall DNA methylation increased from 0.175 to 0.209 (a 19% increase) and in muscle the increase was from 0.165 to 0.186 (a 13% increase) with the TAD-soy to TAD-casein change. Methylation also increased in liver and muscle after switching from the high-fructose casein diet to the high-fat TAD-casein diet (, bottom). The proportion in liver increased from 0.181 to 0.212 (a 17% increase) and muscle increased from 0.162 to 0.197 (a 22% increase).
Overall proportion of DNA methylation in each tissue, by diet.
Gene-specific analyses were performed with a focus on the transition between the two TAD-based diets. Separate tissue-specific analyses were performed to identify changes at the p<0.01 and p<0.001 levels. Genes with p<0.01 were used to identify Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were over-represented with each transition (). Significant pathways were identified from blood only with the TAD-soy to TAD-casein transition with Diet Arm 1; all tissues except muscle revealed significant pathways with the TAD-casein to TAD-soy change with Diet Arm 2.
Pathways detected after TAD diet changes.
DNA methylation levels for seven individual genes changed significantly (False Discovery Rate, FDR, p<0.20) after switching diets – four with the TAD-soy to TAD-casein transition and three with the TAD-casein to TAD-soy diet change (). These genes included homeobox genes (HOXA5, HOXA11, HOXB1) and ATP-binding cassette, sub-family, member 5 (ABCG5). The sites for HOXA5 and HOXA11 are separated by ~43 kb, suggesting that this entire genomic region may be epigenetically marked. No genomic sequence for cynomolgus macaques is currently available, so to further investigate these findings the region around ten of the 50bp probes from these seven genes was sequenced to determine the identity between the human and cynomolgus sequences. Identities between human and cynomolgus DNA sequences ranged from 0.94 to 1.0, suggesting that these specific probes were assaying the correct CpG loci of the respective human sites.
Genes with significant DNA methylation changes (FDR P<0.20) after the diet switch.
To validate the HumanMethylation27 BeadChip data, we performed pyrosequencing of four of the top seven CpG sites with high-quality sequence data from cynomolgus monkeys. Data from three of these sites (ABCG5
, and HOXB1
) were highly correlated (). For sites in ABCG
, the data were virtually identical between the two assays (r2
0.97 for both), which is consistent with the 100% identity between the human and cynomolgus sequences that encompass the 50bp Illumina probes. The correlation was lower for HOXB1
0.89), where a single base difference exists between the human probe and cynomolgus monkey sequences. Since the base difference is a “C” in cynomolgus monkeys and a “T” in humans, this difference has no effect after bisulfite conversion, where both bases would become a “T”. A second sequence difference 2bp 3′ of the probe creates an adjacent CpG in the cynomolgus monkeys that is absent in humans. This change would alter the fluorescent nucleotide extended in the assay and therefore affect the signal. Importantly, this change alters the apparent magnitude of the methylation but does not affect the difference between samples. A fourth site examined was located in HOXA11
, where a significant difference was detected in muscle tissue (). Unfotunately, we did not have sufficient DNA from muscle, so DNA from fat tissue was used for the validation assay. This site had the lowest correlation of the four tested (r2
0.74), most likely due to a single base difference between the cynomolgus monkey and human sequences. This change (a “C” in humans is a “G” in cynomolgus monkeys), which is located at a CpG site within the Illumina probe, would still differ after bisulfite conversion, decreasing the affinity of the probe for the genomic DNA.
Comparison of HumanMethylation27 BeadChip with pyrosequencing.