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. 2015 Mar 30:5:9336.
doi: 10.1038/srep09336.

Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism

Silvia Falcinelli  1 , Simona Picchietti  2 , Ana Rodiles  3 , Lina Cossignani  4 , Daniel L Merrifield  3 , Anna Rita Taddei  5 , Francesca Maradonna  1 , Ike Olivotto  1 , Giorgia Gioacchini  1 , Oliana Carnevali  1
Affiliations

Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism

Silvia Falcinelli et al. Sci Rep. .

Abstract

The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders.

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Figures

Figure 1
Figure 1. Digestive tract bacterial community analysis of 6 dpf zebrafish larvae.
(A) Alpha rarefaction plot of observed species. (B) Relative abundance of reads assigned to phyla. (C) Stacked bar chart representing the relative abundance of bacterial phylum and classes. (D) and (E) Principal coordinates analysis (PCoA) plots using Bray-Curtis metric and weighted Unicfrac distances, respectively. (F) Unique and shared OTUs (at the level of genera) present in the treatment groups.
Figure 2
Figure 2. L. rhamnosus treatment modulates expression of genes involved in lipid metabolism.
qRT-PCR analysis. Relative agpat (A), dgat2 (B), mgll (C), hnf4α (D), fit2 (E), scap (F), cck (G) gene expression normalized against β-act and arp, in pools of 15 zebrafish larvae from control and probiotic groups collected at hatching (H), 96 hpf, 6 dpf and 8 dpf. Assays were performed in triplicate. Values with different superscript letters are significantly different (P < 0.05).
Figure 3
Figure 3. Transmission Electron Microscopy (TEM) shows the ultrastructure of the intestine in control and probiotic treated zebrafish.
Thin section (80 nm) of 6 dpf zebrafish showing the epithelial layer arranged into broad, irregular folds (A). Intact epithelial barrier, lack of cell debris in the lumen and no signs of damage in larvae exposed to probiotic L. rhamnosus (B). Electron micrographs showing uniform, columnar, polarized epithelia, with an apical brush border in control (C) and probiotic treated groups (D). Higher magnification of junctional complexes (E). Microvilli on the apical surface and copious spherical mitochondria in the enterocyte cytoplasm of control (F) and treated larvae (G). Higher magnification of the lipid droplets in the enterocytes of control larvae (H) and in the probiotic treated intestine (I). BB: brush border; L: lumen; M: mitochondria; *: nucleus; arrows: lipid droplets. Scale bar: 5 μm in (A, B); 10 μm in (C, D); 500 nm in (E), 1 μm in (F, G); 2 μm in (H, I). (See also Figure S3).
Figure 4
Figure 4. BODIPY 505/515 staining evidenced an accumulation of non-polar fatty acids in probiotic treated gallbladder.
Representative fluorescent images of live 6 dpf zebrafish soaked in BODIPY 505/515 for 1 hour. Images revealed green fluorescence in the intestine in both control (A–B) and probiotic treated groups (C–D). Probiotic treated larvae exhibited higher fluorescent signal in the gallbladder (D). Red asterisk: gallbladder; red arrow: intestine. Scale bar: 500 μm. (See also Figure S4).
Figure 5
Figure 5. L. rhamnosus administration increased larvae total length and body weight.
Total length (A) and wet weight (B) of zebrafish larvae from control and probiotic groups collected at 96 hpf, 6 dpf and 8 dpf. Data are the mean ± s.d. Asterisk indicates significant differences (P < 0.05).
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