Choline supplementation prevents diet induced gut mucosa lipid accumulation in post-smolt Atlantic salmon (Salmo salar L.)

Abstract

Background: Various intestinal morphological alterations have been reported in cultured fish fed diets with high contents of plant ingredients. Since 2000, salmon farmers have reported symptoms indicating an intestinal problem, which we suggest calling lipid malabsorption syndrome (LMS), characterized by pale and foamy appearance of the enterocytes of the pyloric caeca, the result of lipid accumulation. The objective of the present study was to investigate if insufficient dietary choline may be a key component in development of the LMS.

Results: The results showed that Atlantic salmon (Salmo salar), average weight 362 g, fed a plant based diet for 79 days developed signs of LMS. In fish fed a similar diet supplemented with 0.4% choline chloride no signs of LMS were seen. The relative weight of the pyloric caeca was 40% lower, reflecting 65% less triacylglycerol content and histologically normal gut mucosa. Choline supplementation further increased specific fish growth by 18%. The concomitant alterations in intestinal gene expression related to phosphatidylcholine synthesis (chk and pcyt1a), cholesterol transport (abcg5 and npc1l1), lipid metabolism and transport (mgat2a and fabp2) and lipoprotein formation (apoA1 and apoAIV) confirmed the importance of choline in lipid turnover in the intestine and its ability to prevent LMS. Another important observation was the apparent correlation between plin2 expression and degree of enterocyte hyper-vacuolation observed in the current study, which suggests that plin2 may serve as a marker for intestinal lipid accumulation and steatosis in fish. Future research should be conducted to strengthen the knowledge of choline's critical role in lipid transport, phospholipid synthesis and lipoprotein secretion to improve formulations of plant based diets for larger fish and to prevent LMS.

Conclusions: Choline prevents excessive lipid accumulation in the proximal intestine and is essential for Atlantic salmon in seawater.

Keywords: Choline; Fish feed; Gut health; LMS; Lipid accumulation; Lipid malabsorption; Lipid transport; Plant ingredients.

Fig. 1

Organ somatic indices of the intestinal sections, pyloric intestine (PI), mid-intestine (MI), distal intestine (DI) and liver (LI). Values are means (PI n = 20 and MI, DI and LI n = 30) with standard errors represented by vertical bars. Significant differences (p <  0.05) between the LF and LFC group are indicated with *. The inclusion of choline resulted in a significant lower organ somatic index for PI, MI and LI (p <  0.05)

Fig. 2

a example of white pyloric caeca with grossly visible of lipid accumulation. Image credit: Vegard Denstadli. Histological appearance of pyloric caeca in fish fed (b) the low fishmeal diet, LF and (c) the choline supplemented diet, LFC. Scale bare = 100 μm

Fig. 3

Contingency charts of the pyloric intestine showing proportions of sampled individuals that scored vacuolation grade “normal”, “moderate” and “marked” (none scored “mild”). Fish fed the low fishmeal diet displayed hyper-vacuolated enterocytes. Choline inclusion resulted in normal epithelium. The differences between the diets were significant (p <  0.05; Chi-squared test)

Fig. 4

Distribution of the lipid classes; free fatty acids (FFA), monoacylglycerol (MAG), diacylglycerol (DAG), triacylglycerol (TAG) and phospholipid (PL) in pyloric caeca tissue. Values are means (n = 10) with standard errors represented by vertical bars. Significant differences (p <  0.05) between the LF and LFC group are indicated with *. The inclusion of choline resulted in a significant lower content of TAG (p <  0.05; T-test)

Fig. 5

Overview of genes involved in lipid digestion and absorption in the intestine of Atlantic salmon and studied in the present study. Arrows indicates steps in the pathways. Studied genes are italicized. Green color indicates genes which were significantly down-regulated and red color indicate up-regulated genes. No color represents genes not significantly affected. Dietary choline (CL) is synthesized by choline kinase (chk) to phosphocholine (P-CL) and after an intermediate step not studied here, choline-phosphate cytidylyltransferase (pcyt1a) to phosphatidylcholine (PC). PC could also be synthesized from endogenous phosphatidylethanolamine (PE) by phosphatidylethanolamine N-methyltransferase (pemt). PC is an important element in the membrane portion of lipoproteins preventing triacylglycerol (TAG) from leaking out. Cholesterol (CH) is transported from the lumen and over the membrane by Niemann-Pick C1-Like1 (npc1l1). Acyl-CoA cholesterol acyltransferase (acat) located in ER, facilitates the esterification of CH to cholesterol esters (CE). ATP-binding cassette G5 (abcg5) returns some of the free cholesterol back to the gut for reuse. Some of the free cholesterol is also shuttled to the basolateral membrane for biogenesis of high-density lipoprotein (HDL) mediated by ATP-binding cassette A1 (abca1). Fatty acids (FA) are transported from the gut lumen over the brush border membrane and into the epithelial cell by cd36 (cluster of differentiation 36). The fatty acid-binding protein 2 (fabp2) shuttles the fatty acids within the epithelial cell and the fatty acid transport protein (fatp) further to the smooth endoplasmic reticulum (ER). Monoacylglycerol (MAG) is esterified by monoacylglycerol acyltransferase (mgat2a), located in ER, to diacylglycerol (DAG) which is further transformed into triacylglycerol (TAG), a step not studied here. Microsomal triglyceride transfer protein (mtp) further facilitates the transport of TAG by assisting in the assembly of the lipoprotein. The three apolipoproteins apoB48, apoAI and apoAIV are important elements for successful production and secretion of the lipoprotein. The formation of lipoproteins is again an essential step for export of lipid to the general circulation and to other organs such as the liver. Excess lipid is stored as lipid droplets in the enterocytes. The lipid droplet structure and formation are regulated by the amphiphilic structural protein, adipophilin/perilipin 2 (plin2)

Fig. 6

Severity of vacuolation (steatosis) of the pyloric caeca tissue, representative for (a) marked (b) moderate (c) mild and (d) normal. Scale bar = 100 μm