The findings from this study revealed that heat processing of fat led to changes in its effects on metabolic markers and the gut microbiota in
apoe
−/−
mice as compared to unheated fat. Mice fed HT displayed elevated circulating levels of CML and CEL as well as enlarged spleens, to a higher extent than mice fed HF. Enlargement of the spleen has previously been observed even in healthy mice with intra-peritoneal administration of RAGE and this has been suggested to be exerted through stimulation of adaptive immune system in response to inflammation [
34]. Interestingly, the amounts of atherosclerotic plaque were similar in HF and HT, which were both higher than LF. The amount of atherosclerotic plaques was anticipated to be higher in mice fed HT as heat processing of fat would add up the effect of AGEs to the effect of fat on atherosclerosis. The lower epididymal fat-pad weight observed in HT mice, to the same level as in LF mice, could possibly provide an explanation. Chemical modifications of fat by heat-treatment might abate the effect of unheated fat, leaving only the effect of AGEs on atherosclerosis. Thus, HT mice reached similar levels of atherosclerosis as HF mice despite lower adiposity, which may suggest an additive effect of AGEs on atherosclerosis in the HT mice. Heat-treated LF has not been included in this study as the aim was only to compare the effects of fat and heat-treatment of HF (HT), using LF as a baseline control. However, heat-treatment of LF diet may provide more insight into the role of fat and AGEs on atherogenesis.
The results from 16S sequencing of caecal samples showed that HF and HT had an impact on the composition of the gut microbiota in the mice. Both HF and HT lowered the diversity of the gut microbiota, although the change was only significant with HT. The most dominant phyla in all groups were Firmicutes and Bacteroidetes, whose relative abundance were significantly altered with HT, as well as with HF but to a lesser extent. An imbalanced ratio of Firmicutes and Bacteroidetes has been found to be associated with risk factors of obesity in both animals and humans. High dimensional comparison using LEfSe analysis showed that both HF and HT mice differed in their gut microbiota composition and functions, as compared to each other and to LF mice. The LF group had a high diversity of bacterial genera from different phyla. Although most groups of bacteria found enriched in LF are not well known regarding their roles for the host, the predicted gene functions revealed increases of genes involved in a broad range of metabolism pathways including amino acids, enzymes, as well as cofactors and vitamins.
Mucispirillum found enriched in the mice fed HF, is one of the putative mucin degraders [
35] and has been found in a study of Belzer et al., to be involved in the onset of symptomatic colitis in mice [
36]. Interestingly, the lactic acid bacteria,
Lactococcus, found enriched also in the mice fed HF, has been found in a study of Parks et al., to have positive correlation with body fat percentage gain in mice fed high-fat, high-sucrose diet [
37]. Although genes enriched in HF mice were encoded for several metabolism pathways, it is remarkable that most of these were involved in a cascade of a fatty acid synthesis pathway. Some bacteria have been known to synthesize glycerophospholipid on the inner leaflet of the inner membrane and different lipid biosynthetic mechanisms have previously been defined [
38]. These bacterial genera could therefore generate even more fat to the host’s energy metabolism. In addition, the gut microbiota composition has also been shown to play a role in how much energy is harvested from the diet [
39‐
41], and indeed, mice fed HF had significantly higher epididymal fat pad weight, as well as slightly higher body weight than mice fed HT. Interestingly,
Allobaculum which has previously been found to be decreased in mice fed high-fat diet [
37,
42], became enriched in HT mice in this study. Although these studies showed somewhat similar 16S gene results as observed in this current study, it is worth mentioning that some of them were performed on animal faeces as opposed to caecum and these two locations in the gut may harbour different bacterial species. Together with the finding that no bacterial genes encoding for lipid metabolism were enriched in mice fed HT, we hypothesize that heat processing of fat changed the chemical properties of the fat, allowing different substrates to reach the lower gut. However, the exact mechanism of how heat-treated fat appears to be less accessible to the gut microbiota than unheated fat needs further investigation.