The Effects of Intestine Specific Knockout of Acsl5 in Mice Fed a High-Fat Diet on the Regulation of the Gut Microbiota

Abstract

Background Obesity and its associated metabolic complications are reaching epidemic proportions. A major contributor to the development of obesity is an excessive intake of calories and fat. The critical step for the absorption of dietary fat is the acylation of free fatty acid to acyl-CoA by enterocyte acyl-CoA long-chain synthetase 5 (ACSL5). Our preliminary studies demonstrated that mice with intestine conditional knockout of Acsl5 (ACSL5^int-/-) fed a high-fat diet (HFD) were protected against diet-induced obesity (DIO) secondary to a reduced HFD intake. Since the composition of the intestinal microbiome has been demonstrated to modulate DIO and metabolism, the objective of this thesis work is to explore 1) the short-term effect of chow and HFD on regulating the composition of the intestinal microbiome in Acsl5 conditional knockout mice and 2) the relative role of reduced HFD intake versus conditional knockout of Acsl5 on the composition of the host microbiome.

Methods A short-term feeding study was conducted in which ACSL5^int-/- and floxed littermates ACSL5^loxP/loxP were fed either a chow diet or an HFD ad libitum for two weeks. Cecal contents were collected for 16S rRNA gene sequencing. Qiime2 was used for the sequencing data analysis. Alpha- and Beta-diversity analysis were used to compare the microbial compositions between groups. A long-term pair-feeding study was also conducted in which one group of ACSL5^int-/- mice and one group of ACSL5^loxP/loxP mice received the HFD for 16 weeks ad libitum. At the same time, a second group of ACSL5^loxP/loxP mice was pair-fed with the same amount of food ingested by ACSL5^int-/- littermates. In vivo glucose homeostasis was evaluated by intraperitoneal glucose tolerance test and insulin tolerance test. Cecal contents were collected for shotgun metagenomics sequencing. BioBakery workflow was used for the bacterial species and functional pathway analysis. Intestine inflammatory cytokine markers and antimicrobial peptides synthesis were evaluated by gene expression.

Results Short-term HFD fed ACSL5^int-/- mice possess a significantly distinct microbiome structure compared to ACSL5^loxP/loxP mice with enriched potential beneficial taxa including Akkermansia. However, there is no difference of microbiota between the groups after chow diet feeding. During pair-feeding, we observed that ACSL5^int-/- mice consumed ~20% less energy compared to ACSL5^loxP/loxP when fed an HFD. When pair-fed with the same food consumption as the ACSL5^int-/- mice, the ACSL5^loxP/loxP pair-fed mice have a similar metabolic phenotype including body weight, fat mass, glucose tolerance and insulin tolerance. Using metagenomic sequencing, we found that the microbiota structure of ACSL5^loxP/loxP pair-fed group is shifting intermediate between the ACSL5^loxP/loxP and ACSL5^int-/- mice. ACSL5^int-/- mice have a significantly different microbial functional profile compared to both ACSL5^loxP/loxP ad libitum and pair-feeding groups, as well as a downregulation of ileal IL-6, IL-10 and an upregulation of colonic antimicrobial peptide expression.

Conclusions The intestine conditional knockout of acsl5 resulted in a distinct gut microbiota with enriched beneficial taxa compared to ACSL5loxP/loxP within two weeks, but only after HFD feeding. After long-term HFD feeding, reduced 20% of HFD food intake protects mice against the development of HFD-induced obesity with an improved metabolic profile. Only a portion of microbiota structure and microbial functional profile in ACSL5^int-/- can be described to diminished food intake implicating other metabolic regulatory pathways intrinsic to the conditional knockout of acsl5, including intestinal inflammation status and antimicrobial peptide secretion.