Dietary choline activates the Ampk/Srebp signaling pathway and decreases lipid levels in Pacific white shrimp (Litopenaeus vannamei)

Abstract

An 8-week feeding trial was conducted in Pacific white shrimp (Litopenaeus vannamei) to evaluate the effects of dietary choline supplementation on choline transport and metabolism, hepatopancreas histological structure and fatty acid profile, and regulation of lipid metabolism. Six isonitrogenous and isolipidic diets were formulated to contain different choline levels of 2.91 (basal diet), 3.85, 4.67, 6.55, 10.70 and 18.90 g/kg, respectively. A total of 960 shrimp (initial weight, 1.38 ± 0.01 g) were distributed randomly into twenty-four 250-L cylindrical fiber-glass tanks, with each diet assigned randomly to 4 replicate tanks. The results indicated that dietary choline significantly promoted the deposition of choline, betaine and carnitine (P < 0.05). The diameters and areas of R cells, total lipid and triglyceride contents in hepatopancreas, and triglyceride and non-esterified fatty acid contents in hemolymph were negatively correlated with dietary choline level. The contents of functional fatty acids in hepatopancreas, the activity of acetyl-CoA carboxylase (Acc), and the mRNA expression of fas, srebp and acc were highest in shrimp fed the diet containing 4.67 g/kg choline, and significantly higher than those fed the diet containing 2.91 g/kg, the lowest level of choline (P < 0.05). The number of R cells, content of very low-density lipoprotein (VLDL), activities of carnitine palmitoyl-transferase (Cpt1), lipoprotein lipase and hepatic lipase, and the mRNA expression levels of cpt1, fabp, fatp, ldlr, and ampk in hepatopancreas increased significantly as dietary choline increased (P < 0.05). In addition, hepatopancreas mRNA expression levels of ctl1, ctl2, oct1, badh, bhmt, ck, cept, and cct were generally up-regulated as dietary choline level increased (P < 0.01). In conclusion, dietary choline promoted the deposition of choline and its metabolites by up-regulating genes related to choline transport and metabolism. Moreover, appropriate dietary choline level promoted the development of hepatopancreas R cells and maintained the normal accumulation of lipids required for development, while high dietary choline not only promoted hepatopancreas lipid export by enhancing VLDL synthesis, but also promoted fatty acid β-oxidation and inhibited de novo fatty acid synthesis by activating the Ampk/Srebp signaling pathway. These findings provided further insight and understanding of the mechanisms by which dietary choline regulated lipid metabolism in L. vannamei.

Keywords: Ampk signaling pathway; Choline; Fatty acid; Histology; Lipid; Pacific white shrimp.

Fig. 1

Effects of dietary choline (g/kg diet) on contents of (A) choline, (B) betaine and (C) PC in hepatopancreas, (D) ACH in hemolymph, and carnitine in (E) hepatopancreas and (F) muscle of Litopenaeus vannamei fed the experimental diets. PC = phosphatidylcholine; ACH = acetyl choline. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a–d Bars with different superscript letters differ significantly (P < 0.05).

Fig. 2

Effect of dietary choline (g/kg diet) on mRNA expression levels of genes related to choline transport and metabolism in the hepatopancreas of Litopenaeus vannamei. ctl1 = CTL-like protein 1; ctl2 = CTL-like protein 2; oct1 = organic cation transporter 1-like; badh = betaine aldehyde dehydrogenase; bhmt = betaine-homocysteine S-methyltransferase; ck = choline kinase; cept = choline/ethanolamine phosphotransferase; cct = CTP:phosphocholine cytidylyltransferase. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a–d Bars with different superscript letters differ significantly (P < 0.05).

Fig. 3

Effects of dietary choline (g/kg diet) on (A) total lipid contents in hepatopancreas and muscle, (B) hepatopancreas lipid class contents, and (C) hemolymph biochemical parameters of Litopenaeus vannamei. TG = triglyceride; CHO = cholesterol; VLDL = very low-density lipoprotein; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol; NEFA = non-esterified fatty acid. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a–d Bars with different superscript letters differ significantly (P < 0.05).

Fig. 4

Effects of dietary choline at (A) 2.91, (B) 3.85, (C) 4.67, (D) 6.55, (E) 10.70 and (F) 18.90 g/kg diet, respectively, on hepatopancreas histological structure of Litopenaeus vannamei (400× magnification). Scale bar, 50 μm. Data on R cells are provided in panels (G) numbers, (H) diameters, and (I) areas of R cell (n = 40). Bc = blasenzellen cell; Bm = basal membrane; Ec = embryonic cell; Fc = fibrous cell; L = lumen structure; R = restzellen cell; V = vacuole. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a–c Bars with different superscript letters differ significantly (P < 0.05).

Fig. 5

Effects of dietary choline (g/kg diet) on fatty acid profile of hepatopancreas of Litopenaeus vannamei. (A) Fatty acid profile of hepatopancreas, (B) hierarchical cluster analysis (HCA), and (C) heat map visualization of hepatopancreas fatty acid composition. ARA = arachidonic acid; EPA = 20:5n-3; DHA = 22:6n-3; DHA/EPA = ratio of 22:6n-3 to 20:5n-3; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; n-6 PUFA = n-6 polyunsaturated fatty acids; n-3 PUFA = n-3 polyunsaturated fatty acids; n-3/n-6 PUFA = ratio of n-3 polyunsaturated fatty acids to n-6 polyunsaturated fatty acids; LC-PUFA = long-chain polyunsaturated fatty acids. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a,b Bars with different superscript letters differ significantly (P < 0.05).

Fig. 6

Effects of dietary choline (g/kg diet) on hepatopancreas activities of lipid metabolic related enzymes of Litopenaeus vannamei. Fas = fatty acid synthase; Acc = acetyl-CoA carboxylase; Cpt1 = carnitine palmitoyl-transferase; Lpl = lipoprotein lipase; Hl = hepatic lipase. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a–c Bars with different superscript letters differ significantly (P < 0.05).

Fig. 7

Effects of dietary choline (g/kg diet) on mRNA expression levels of hepatopancreas lipid metabolic related genes of Litopenaeus vannamei, related to (A) lipid anabolism, (B) lipid catabolism, (C) transport of fatty acid and lipids, and (D) regulatory factors. fas = fatty acid synthase; acc = acetyl-CoA carboxylase; cpt1 = carnitine palmitoyl-transferase; aco = acyl-CoA oxidase; fabp = fatty acid binding protein; fatp = fatty acid transport protein; ldlr = low-density lipoprotein receptor; ampkα = 5′-AMP-activated protein kinase subunit alpha; ampkβ = 5′-AMP-activated protein kinase subunit beta; ampkγ = 5′-AMP-activated protein kinase subunit gamma; srebp = sterol regulatory element-binding protein. Mean values are based on 4 replicates, and standard errors are represented by vertical bars. ^a–d Bars with different superscript letters differ significantly (P < 0.05).