Double-edged effect of sodium citrate in Nile tilapia (Oreochromis niloticus): Promoting lipid and protein deposition vs. causing hyperglycemia and insulin resistance

Abstract

Citrate is an essential substrate for energy metabolism that plays critical roles in regulating glucose and lipid metabolic homeostasis. However, the action of citrate in regulating nutrient metabolism in fish remains poorly understood. Here, we investigated the effects of dietary sodium citrate on growth performance and systematic energy metabolism in juvenile Nile tilapia (Oreochromis niloticus). A total of 270 Nile tilapia (2.81 ± 0.01 g) were randomly divided into three groups (3 replicates per group, 30 fish per replicate) and fed with control diet (35% protein and 6% lipid), 2% and 4% sodium citrate diets, respectively, for 8 weeks. The results showed that sodium citrate exhibited no effect on growth performance (P > 0.05). The whole-body crude protein, serum triglyceride and hepatic glycogen contents were significantly increased in the 4% sodium citrate group (P < 0.05), but not in the 2% sodium citrate group (P > 0.05). The 4% sodium citrate treatment significantly increased the serum glucose and insulin levels at the end of feeding trial and also in the glucose tolerance test (P < 0.05). The 4% sodium citrate significantly enhanced the hepatic phosphofructokinase activity and inhibited the expression of pyruvate dehydrogenase kinase isozyme 2 and phosphor-pyruvate dehydrogenase E1 component subunit alpha proteins (P < 0.05). Additionally, the 4% sodium citrate significantly increased hepatic triglyceride and acetyl-CoA levels, while the expressions of carnitine palmitoyl transferase 1a protein were significantly down-regulated by the 4% sodium citrate (P < 0.05). Besides, the 4% sodium citrate induced crude protein deposition in muscle by activating mTOR signaling and inhibiting AMPK signaling (P < 0.05). Furthermore, the 4% sodium citrate significantly suppressed serum aspartate aminotransferase and alanine aminotransferase activities, along with the lowered expression of pro-inflammatory genes, such as nfκb, tnfα and il8 (P < 0.05). Although the 4% sodium citrate significantly increased phosphor-nuclear factor-kB p65 protein expression (P < 0.05), no significant tissue damage or inflammation occurred. Taken together, dietary supplementation of sodium citrate could exhibit a double-edged effect in Nile tilapia, with the positive aspect in promoting nutrient deposition and the negative aspect in causing hyperglycemia and insulin resistance.

Keywords: Insulin resistance; Lipid and protein deposition; Nile tilapia; Sodium citrate.

Fig. 1

Effects of sodium citrate on glucose metabolism in the liver of Nile tilapia. (A) Serum glucose, n = 3; (B) serum insulin, n = 3; (C) liver pyruvate, n = 3; (D) liver lactate, n = 3; (E) liver phosphofructokinase (PFK) activity; (F and G) Serum glucose and insulin of glucose tolerance test, n = 3; (H) mRNA expression of genes related to glucose metabolism, n = 3; (I) mRNA expression of genes related to TCA cycle, n = 3; (J) expression of proteins related to glucose metabolism, n = 3. Values are means ± SEM. Difference from group control: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Independent-samples t test). glut2 = glucose transporter protein 2; glut4 = glucose transporter protein 4; gk = glucokinase; pdhe1a1 = pyruvate dehydrogenase E1 component subunit alpha 1; pdk2 = pyruvate dehydrogenase kinase isozyme 2; pdk4 = pyruvate dehydrogenase kinase isozyme 4; gs = glycogen synthase; fbpase = fructose-1,6-bisphosphatase; pepck = phosphoenolpyruvate carboxykinase; g6pase = glucose-6-phosphatase; cs = citrate synthase; idh = isocitrate dehydrogenase; cicb = citrate carrier; p-Pdhe1a = phosphor-pyruvate dehydrogenase E1 component subunit alpha; Pdk2 = pyruvate dehydrogenase kinase isozyme 2; G6pc = glucose-6-phosphatase catalytic subunit.

Fig. 2

Effects of sodium citrate treatment on lipid metabolism in the liver of Nile tilapia. (A) Liver triglyceride, n = 3; (B) serum cholesterol, n = 3; (C) liver cholesterol, n = 3; (D) liver acetyl-CoA, n = 3; (E and F) mRNA expression of genes related to lipid metabolism, n = 3; (G) expression of proteins related to lipid metabolism, n = 3. Values are means ± SEM. Difference from group control: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Independent-samples t test). fasn = fatty acid synthase; m-Srebp1/srebp1 = (mature) sterol regulatory element-binding protein 1; ppara/pparg = peroxisome proliferator activated receptor a/g; Acly/acly = ATP-citrate lyase; hsl = hormone-sensitive lipase; atgl = adipose triglyceride lipase; Cpt1a/cpt1a = carnitine palmitoyltransferase 1a; t/p-Acc = total/phosphor-acetyl-CoA carboxylase 1.

Fig. 3

Effects of sodium citrate treatment on the protein deposition in the muscle of Nile tilapia. (A) Crude protein in the muscle, n = 3; (B and C) expression of genes (n = 3) and proteins related to protein metabolism (n = 3). Values are means ± SEM. Difference from group control: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Independent-samples t test). gls2 = glutaminase 2; bckdha = branched chain keto acid dehydrogenase E1 subunit alpha; gdh1 = glutamate dehydrogenase 1; glns = glutamine synthetase; asns = asparagine synthetase; t/p-S6 = total/phosphor-ribosomal protein s6; t/p-mTor = total/phosphor-mammalian target of rapamycin; t/p-Ampk = total/phosphor-AMP-activated protein kinase.

Fig. 4

Effects of sodium citrate treatment on the liver health of Nile tilapia. (A) Serum AST activity, n = 3; (B) serum ALT activity, n = 3; (C) H&E staining of the liver; (D and E) expression of genes related to inflammation, n = 3; (F) expression of p-NF-κB p65 (Ser536) protein, n = 3. Values are means ± SEM. Difference from group control: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Independent-samples t test). AST = aspartate aminotransferase; ALT = alanine aminotransferase; nfκb = nuclear factor kappa B; tnfa = tumor necrosis factor alpha; il1b = interleukin 1 beta; il8 = interleukin 8; tgfb = transforming growth factor beta; il10 = interleukin 10.

Fig. 5

Sodium citrate could exhibit a double-edged effect in Nile tilapia, with the positive aspect in promoting nutrient deposition and the negative aspect in causing hyperglycemia and insulin resistance.