Transcriptomics, metabolomics and histology indicate that high-carbohydrate diet negatively affects the liver health of blunt snout bream (Megalobrama amblycephala)

Abstract

Background: Global trend of the introduction of high levels of relatively cheap carbohydrates to reduce the amount of costly protein in the aquatic animal feed production has affected the aquaculture of an economically important cyprinid fish, blunt snout bream (Megalobrama amblycephala). This dietary shift has resulted in increased prevalence of metabolic disorders, often causing economic losses. High dietary intake of carbohydrates, associated with obesity, is one of the major causes of non-alcoholic fatty liver disease (NAFLD) in humans.

Results: We have conducted an eight-week feeding trial to better understand how a high-carbohydrate diet (HCBD) affects the liver health in this fish. Hepatosomatic index and lipid content were significantly (P < 0.05) higher in the HCBD group. Histology results also suggested pathological changes in the livers of HCBD group, with excessive lipid accumulation and indication of liver damage. Metabolomics and serum biochemistry analyses showed that a number of metabolites indicative of liver damage were increased in the HCBD group. This group also exhibited low levels of betaine, which is a metabolite crucial for maintaining the healthy liver functions. Transcriptomic and qPCR analyses indicated that HCBD had a strong impact on the expression of a large number of genes associated with the NAFLD and insulin signalling pathways, which may lead to the development of insulin resistance in hepatocytes, pathological liver changes, and eventually the NAFLD.

Conclusions: Transcriptomics, metabolomics and histology results all indicate early symptoms of liver damage. However whether these would actually lead to the development of NAFLD after a longer period of time, remains inconclusive. Additionally, a very high number of upregulated genes in the HCBD group associated with several neurodegenerative diseases is a strong indication of neurodegenerative changes caused by the high-carbohydrate diet in blunt snout bream. This suggests that fish might present a good model to study neurodegenerative changes associated with high-carbohydrate diet in humans.

Keywords: Aquaculture; Fish; Hepatosomatic index; NAFLD; Neurodegenerative diseases.

Fig. 1

Photomicrographs of representative hematoxylin- and eosin-stained (a-d) and Oil red O-stained (E and F) histological liver sections of blunt snout bream fed control diet (a, c and e) and high-carbohydrate diet (b, d and f). Arrows indicate examples of a: normal hepatocytes with regular, round nuclei; b: swollen hepatocytes with large diffused lipid vacuoles, c: absent nucleus and d: abnormal nucleus. CV and IF labels indicate examples of central liver vein with abnormal endothelial cells and inflammatory infiltrate respectively. In the Oil red O-staining section, to evaluate lipid content in livers (e and f), lipids are stained red. Magnification ×400 (a, b, e and f) and ×1000 (c and d)

Fig. 2

Serum biochemistry parameters associated with liver damage. * above bar indicates significant difference (P < 0.05) and ** indicates highly significant difference (P < 0.01). Results were analyzed by t-test

Fig. 3

Metabolites in plasma and liver extracts significantly different between the control and HCBD diet groups. a Plasma metabolomics and b Liver metabolomics. Results were analyzed by t-test. * above bars indicates significant difference (P < 0.05) and ** indicates highly significant difference (P < 0.01)

Fig. 4

Transcriptome profiles of the HCBD and control groups. a GO annotation of all unigenes and DEG (differentially expressed) unigenes; b MA plot of differential gene expression levels in the two groups, where expression intensity is on the x-axis (log2 FPKM) and differences in the gene expression levels (fold change) are on the y-axis (log2 FC), each dot represents one gene, the log2 (FC) is plotted against the mean expression level for each gene, red dots represent genes whose abundance is significantly up-regulated, green dots - down-regulated, and black dots unchanged (or non-significantly changed) regulation; c COG annotation of and DEGs; d KEGG annotation of and DEGs

Fig. 5

Expression of 13 DEGs from the transcriptome analysis associated with NAFLD, studied by qPCR. Data were normalized to 18 s rRNA and Rpl13a as reference genes and presented as a fold change between the control and HCBD groups (mean ± SE). HCBD is the high-carbohydrate diet group. Results were analyzed by t-test. (*) above bar indicates significant (P < 0.05) and (**) highly significant differences (P < 0.01)

Fig. 6

A hypothetical mechanism through which the observed changes in transcriptome, serum biochemistry, and serum and liver metabolomics caused by high-carbohydrate diet can lead to fatty liver disease