Abstract

Atherosclerosis is an inflammatory disease caused by lipids, cells, and debris accumulating in arterial walls. Despite their popularity for effective weight loss, low-carbohydrate/high-protein diets (LCHP) are more atherogenic than Western diets and are associated with adverse long-term cardiovascular outcomes. However, the mechanisms of this atherogenicity are not currently known. Therefore, the objectives of this work were to determine if circulating lipid profiles, inflammatory lipid mediators, and/or gut dysbiosis contributed to the higher atherogenicity of the LCHP diet in ApoE KO mice during the early development of plaques. Secondly, it was hypothesized that supplementation of two probiotics: Lactobacillus helveticus Lafti 10® and Bifidobacterium bifidum Rosell 71® could reduce diet-induced atherogenicity during the early stages of the disease due to their antilipemic, anti-inflammatory and antioxidant properties.
In order to accurately observe the changes in lipid and inflammatory pathways over a 6-week longitudinal study in mice, an optimization of analytical methods was necessary to ensure appropriate lipid stability during analytical handling and compatibility with low blood volumes (30-50 μL). First, the impacts of short exposure to room temperatures and the effect of the addition of a lipase inhibitor, Orlistat, during lipidomics sample preparation were investigated using liquid chromatography-mass spectrometry analysis. Human plasma samples were prepared using isopropanol protein precipitation at room temperature, 4°C, and -80°C with, or without, Orlistat. Subtle changes in the lipidome were found even for short (<2 hr) exposure to room temperature, so maintaining a strictly controlled temperature of 4°C throughout the sample preparation process is recommended to reduce residual enzymatic activity. Room temperature exposure during sample preparation impacted the levels of 19% of the lipids but the addition of Orlistat reduced this impact to less than 2.4% of measured lipids and improved method precision across all temperatures. To enable oxylipin determination from 15 μL of plasma, solid-phase extraction – liquid chromatography – high-resolution mass spectrometry method was further optimized. This optimization maintained good coverage, acceptable recovery (71 to 100%) and excellent precision (RSD < 11%), thus making the method suitable for longitudinal monitoring of oxylipin status in mice using small blood volumes collected by tail bleeding.
Next, the effects of diet and probiotic supplementation on lipid and gut dysbiosis were investigated. ApoE knockout mice were fed LCHP, W, or standard chow diet for 6 weeks, with or without anti-inflammatory probiotic supplementation (Lactobacillus helveticus Lafti 10® and Bifidobacterium bifidum Rosell 71®) at low- or high-dose. Targeted and untargeted analyses using liquid chromatography – high-resolution mass spectrometry was used to study dyslipidemia and inflammatory pathways, whereas gut microbiome analysis was performed using 16S rRNA sequencing. When comparing the effects of the diet, the time course study showed that the microbiota in the fecal pellets of the mice given W and LCHP diets were globally similar. However, gut dysbiosis occurred faster for the mice fed the LCHP diet compared to the W diet. In addition, mice fed the LCHP diet had a higher abundance of deleterious families such as Sutterellaceae Parasutterella and beneficial families such as Muribaculaceae Muribaculum, Bacteroidaceae Bacteroides or Erysipelotrichaceae Dubosiella than the W diet. Furthermore, the relative abundance of beneficial species such as Bifidobacteriaceae and Eurobacterium Coprostanoligenes and deleterious families such as Erysipelotrichaceae Turicibacter, Bifidobacterium, Streptococcaceae Streptococcus, Lachnospiraceae, and Oscillospiraceae were decreased compared to the W diet groups. Plasma levels of trimethylamine N-oxide (TMAO), a pro-atherogenic gut-derived metabolite, were unchanged between the LCHP and W groups, indicating that the increased atherogenicity of the LCHP diet is not promoted by this pathway. In terms of lipid dysregulation associated with early stages of atherosclerosis, LCHP and W diets showed similar oxylipin profiles but distinct from the C diet. When comparing the LCHP and W diets, only eicosapentaenoic acid (EPA), which prevents atherosclerotic plaque formation and stabilizes it, was lower in the plasma of the mice fed the LCHP diet compared to the W diet. Circulating oxylipin plasma profiles from mice fed LCHP and W diets showed a similar increase of 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-DiHDPA) and a decrease of arachidonic acid (AA) with time. In addition, 8S,15S-dihydroxy-5Z,9E,11Z,13E-eicosatetraenoic acid (8,15-DiHETE) and 15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid (15-HEPE) increased with the LCHP diet over time. In contrast, only 5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid (5,6-DiHETE) increased with time with the W diet. Overall, the inflammatory pathways were similar in both LCHP and W diets and are unlikely the cause of the higher atherogenicity of the LCHP diet. In addition, the 9-HODE/13-HODE ratio increased with time for the LCHP and W diets indicating a pro-inflammatory profile for both diets compared to the C diet. Plasma levels of sphingolipids, lysophospholipids, phospholipids, alkyl-phospholipids, cholesterol, and cholesteryl esters levels were lower in the LCHP diet compared to the W diet but higher when compared to the C diet. In contrast, glycerolipids were higher in the LCHP diet compared to the W diet.
The time-course study revealed that probiotics had minimal effect on the oxylipin profiles and the 9-HODE/13-HODE ratio was not affected by the probiotic supplementation demonstrating limited anti-inflammatory properties. However, the administered probiotics showed antilipemic properties by reducing highly unsaturated lipids from phosphatidylethanolamine and triglyceride classes. In addition, alkyl phospholipids were elevated with the probiotic administration in combination with the LCHP diet. Furthermore, probiotic supplementation also positively influenced the gut microbiota composition by increasing beneficial families such as Tannerellaceae and Clostridia Vadin BB60 genera, and decreasing harmful bacteria including Streptococcaceae, Erysipelotrichaceae, Lachnospiraceae, and Atopobiaceae. Overall, the effects of probiotic supplementation showed a strong dependence on the diet type indicating strong diet-microbiota interaction. In conclusion, this thesis establishes for the first time systematic changes in circulating lipid levels and gut dysbiosis that are contributing to the atherogenicity of the LCHP diet and examines how probiotic supplementation impacts these adverse changes. These findings are critically important to ensure highly popular weight loss interventions such as LCHP diets do not cause unintended adverse cardiovascular consequences.