The microbiome affects liver sphingolipids and plasma fatty acids in a murine model of the Western diet based on soybean oil

 The gut microbiome is a variable in fat metabolism in mice on a soybean oil diet. Here, we addressed whether the microbiome plays a role in diet-induced obesity in mice fed a high-fat, high-saturated fat diet. The results confirm that absorbed fatty acids are mainly a reflection of the diet and that microbial colonization influences liver sphingolipid pools.

Abbreviations ALA alpha-linolenic acid

• ANOVA
• Analysis Of Variance
• Cer ceramide, ClnA conjugated linoleic acid, DGLA dihomo-gamma, LGLA, DH dihydro, EPA, and So sphingosine
• Principal Coordinate Analysis
• qPCR quantitative polymerase chain reaction

The microbiome has emerged as central to the health of an organism

• Germfree animals, animals devoid of a microbiome, often serve as controls to understand a variety of host phenotypes, including metabolism, the development of the immune system, the structure and function of gut cells, and animal behavior
• Early studies indicated that germfree mice are protected from diet induced obesity.

Mouse experiments

• All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Usage Committee of Cornell University protocol 2010-0065.
• Conventional mice were maintained in the Accepted Pathogen Facility at Cornell University with filter top cages and the germfree mice in flexible, plastic ("bubble") isolators. All animals within a given germfree study were maintained within the same isolator at the same time
• Animals in all studies were maintained under a 12-h light cycle.

FA extraction and detection from plasma

• We added 125 mg of heptadecanoic acid (C17:0, 99+% pure, Sigma Chemicals, St. Louis, MO, USA) to the plasma as an internal standard for absolute quantification of extracted FAs
• The amount of plasma used in the extraction was 200 μl or the maximum of plasma acquired
• FAs were converted to FA methyl esters (FAMEs) and measured by gas chromatography (GC)
• Peak areas were measured using PeakSimple software
• These peak areas were corrected using an equal weight mixture of known FAs measured multiple times throughout the GC run

Sphingolipid extraction from liver tissue

• Liver samples were defrosted on ice and homogenized in 1 mL of PBS in tubes containing 1 mm zirconium beads (OPS Diagnostics, Lebanon, NJ, USA) on a Mini Bead Beater homogenizer (BioSpec products, Bartlesville, OK, USA).
• Protein concentrations of the liver PBS homogenate were determined using the Lowry method.
• 900 μL of 1:1 dichloromethane:methanol was added to each sample and gently mixed on a rotating shaker for an hour. After shaking, the samples were transferred to a new 96-deep well plate and stored at -20°C prior to analysis by high performance liquid chromatography-mass spectrometry (HPLC-MS).

Quantitative real-time PCR

• using primers targeting the 16S rRNA gene
• extracted DNA from fecal pellets taken from mice at weeks 9 to 11 on the SBO diets.
• One conventional mouse sample was run in all qPCR runs to serve as an internal standard and 16S RNA gene copy numbers from a given run are reported relative to this sample in that run.

Statistical analyses on FA and sphingolipid profiles

• We utilized adonis (PERMANOVA) to investigate how the full set of FAs in the conventional or germfree mouse studies were influenced by the technical and experimental variables.
• To determine the specific FAs altered by different experimental conditions (diet, gavage, and microbial status), we first reduced the list of FA to only those present in two of the three conventional studies and one of the two germfree studies, then we utilized a linear mixed model to determine which of the FA amounts were significantly affected by the experimental conditions as compared to a null model.

Fat mass is affected by diet SBO content and microbial status, while microbial status dominates effects on body weight

• To assess the effect of the presence of microbes and soybean oil consumption on adipogenicity, lipid absorption and processing of a high linoleic diet, we fed conventional (CNV) and germfree (GF) mice one of two paired Soybean oil diets.
• Mice were fed one of the two diets for 10 weeks beginning at weaning
• Visually, GF1 and GF2 study mice appeared germfree, as evidenced by their grossly enlarged cecal size and over-abundance of bile following oil gavage
• Assuming the blanks had no bacteria, GF2 fecal pellets are estimated to have 104 - 105 bacterial cells per extracted fecal pellet, three orders of magnitude lower than that in the conventional animals (ANOVA, Tukey HSD, P<.0001, Fig. 1B), which have an estimated ~108 bacterial cells
• CNV mice on either diet had greater relative body weights and greater relative fat pad masses compared to their LG and GF counterparts
• In performing an ANOVA on relative body weight as a function of diet, microbial status (MS), and their interaction, we observed that MS explained most of the variation

Microbial status is a minor effector on plasma FA profile

• The animal's diet has a greater effect
• Hepatic lipids, in contrast to plasma FAs, represent lipid processing influenced by multiple organs. They have been previously observed to be dependent on microbial colonization.
• To determine if hepatic sphingolipids were affected by microbial colonization in our SBO fed mice, we measured them in livers of mice gavaged with PBS only.

Specific plasma FAs and hepatic sphingolipids affected by diet, gavage, and/or microbial status

• After removing FAs not present in two of the three conventional studies and one of the two germfree studies (LG and GF mice), we considered 38 distinct FA peaks. Five of these 38 represent more than one FA that could not be adequately separated.
• We identified 17 FAs with P<.05 after a Bonferroni correction significantly impacted by diet/gavage (Table 6).
• The interaction of diet and gavage was important for three FAs
• Only for the LF diet was C20:5n-3 present in higher amounts.

Discussion

• The present study isolated the effects on body fat, plasma fatty acids, and hepatic sphingolipids by the dominant oil in the Western diet consumed in the USA, where SBO makes up roughly 7% of calories
• Fat gain is associated with increased caloric intake, while body weight gain is primarily driven by microbial load
• Our data demonstrate that germfree animals are not resistant to diet-induced fat gain on an SBO diet.
• Moreover, germfree mice acquire less adiposity and weight compared to conventional animals
• Differences in hepatic and serum lipid profiles are diet and microbiota dependent.

Reference

10.1016/j.jnutbio.2021.108808