Uridine alleviates high-carbohydrate diet-induced metabolic syndromes by activating sirt1/AMPK signaling pathway and promoting glycogen synthesis in Nile tilapia (Oreochromis niloticus)

Abstract

Carbohydrates have a protein sparing effect, but long-term feeding of a high-carbohydrate diet (HCD) leads to metabolic disorders due to the limited utilization efficiency of carbohydrates in fish. How to mitigate the negative effects induced by HCD is crucial for the rapid development of aquaculture. Uridine is a pyrimidine nucleoside that plays a vital role in regulating lipid and glucose metabolism, but whether uridine can alleviate metabolic syndromes induced by HCD remains unknown. In this study, a total of 480 Nile tilapia (Oreochromis niloticus) (average initial weight 5.02 ± 0.03 g) were fed with 4 diets, including a control diet (CON), HCD, HCD + 500 mg/kg uridine (HCUL) and HCD + 5,000 mg/kg uridine (HCUH), for 8 weeks. The results showed that addition of uridine decreased hepatic lipid, serum glucose, triglyceride and cholesterol (P < 0.05). Further analysis indicated that higher concentration of uridine activated the sirtuin1 (sirt1)/adenosine 5-monophosphate-activated protein kinase (AMPK) signaling pathway to increase lipid catabolism and glycolysis while decreasing lipogenesis (P < 0.05). Besides, uridine increased the activity of glycogen synthesis-related enzymes (P < 0.05). This study suggested that uridine could alleviate HCD-induced metabolic syndrome by activating the sirt1/AMPK signaling pathway and promoting glycogen synthesis. This finding reveals the function of uridine in fish metabolism and facilitates the development of new additives in aquatic feeds.

Keywords: AMPK; High-carbohydrate diet; Metabolism; Nile tilapia; Uridine.

Fig. 1

The effect of uridine on the liver lipid metabolism of Nile tilapia. (A) Total lipid content in liver (n = 12). (B) Oil-red staining of liver. Arrow shows the stained red lipid drops in liver cells. (C) TG in liver (n = 12). (D) TG in serum (n = 12). (E) TC in liver (n = 12). (F) TC in serum (n = 12). (G) AST activity in serum (n = 12). (H) ALT activity in serum (n = 12). (I) mRNA expression of genes related to lipid metabolism (n = 6). Data are represented as mean ± SEM. Significant difference between CON and HCD (#P < 0.05, ##P < 0.01). Significant difference compared with HCD (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). CON = control diet; HCD = high-carbohydrate diet; HCUL = high-carbohydrate diet + 500 mg/kg uridine; HCUH = high-carbohydrate diet + 5,000 mg/kg uridine; LD = lipid drop; scale bars = 100 μm; TG = triglyceride; TC = total cholesterol; AST = aspartate aminotransferase; ALT = alanine aminotransferase; accα = acetyl-CoA carboxylase alpha; fas = fatty acid synthase; dgat2 = diacylglycerol O-acyl-transferase 2; atgl = adipose triglyceride lipase; hsl = hormone-sensitive lipase; cpt1a = carnitine palmitoyltransferase 1a; aco = acyl-coenzyme A oxidase; pparα = peroxisome proliferator activated receptor-alpha.

Fig. 2

The effect of uridine on liver and muscle glucose metabolism of Nile tilapia. (A) Serum glucose (n = 12). (B) Glycogen content in liver (n = 6). (C) Glycogen content in muscle (n = 6). (D) PAS staining of liver and muscle. Arrow shows the stained fuchsia glycogen granule in liver and muscle cells. (E) mRNA expression of genes related to glucose metabolism in liver (n = 6). (F) The activity of liver glycogen synthetase (n = 6). (G) The activity of liver glycogen phosphorylase (n = 6). (H) mRNA expression of ugp2 in liver (n = 6). (I) The activity of muscle glycogen synthetase (n = 6). (J) The activity of muscle glycogen phosphorylase (n = 6). (K) mRNA expression of ugp2 in muscle (n = 6). Data are represented as mean ± SEM. Significant difference between CON and HCD (#P < 0.05, ##P < 0.01). Significant difference compared with HCD (∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001). CON = control diet; HCD = high-carbohydrate diet; HCUL = high-carbohydrate diet + 500 mg/kg uridine; HCUH = high-carbohydrate diet + 5,000 mg/kg uridine; Gn = glycogen; scale bars = 100 μm; gck = glucokinase; pfk = phosphofructokinase; pk = pyruvate kinase; pc = pyruvate carboxylase; pepck = phosphoenolpyruvate carboxykinase; fbpase = fructose-1,6-bisphosphate phosphatase; ugp2 = uridyl diphosphate glucose pyrophosphorylase 2.

Fig. 3

The effect of uridine on activities of enzymes related to uridine metabolism in Nile tilapia. (A) The activity of liver Uck1 (n = 6). (B) The activity of liver Cmpk1 (n = 6). (C) The activity of muscle Uck1 (n = 6). (D) The activity of muscle Cmpk1 (n = 6). Data are represented as mean ± SEM. Significant difference compared with HCD (∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001). Uck1 = uridine–cytidine kinase 1; Cmpk1 = cytidine/uridine monophosphate kinase 1.

Fig. 4

The effects of uridine on the expression of AMPK protein and gene and the expression of sirt1 gene in vivo and in vitro. (A) The protein expression of p-AMPK (n = 3). (B) Quantitation of the levels of p-AMPK was normalized to that of GAPDH (n = 3). (C) mRNA expression of sirt1 in liver (n = 6). (D) Effect of uridine on the expression of sirt1 in primary hepatocytes in the presence of nicotinamide (n = 3). (E) Effect of uridine on the expression of ampkα1 in primary hepatocytes in the presence of nicotinamide (n = 3). (F) Effect of uridine on the expression of ampkα2 in primary hepatocytes in the presence of nicotinamide (n = 3). Data are represented as mean ± SEM. (B–C): significant difference compared with HCD (∗P < 0.05, ∗∗P < 0.01); (D–F): significant difference between CON and UR (#P < 0.05, ##P < 0.01); significant difference compared with UR (∗P < 0.05, ∗∗P < 0.01). CON = control diet; HCD = high-carbohydrate diet; HCUL = high-carbohydrate diet + 500 mg/kg uridine; HCUH = high-carbohydrate diet + 5,000 mg/kg uridine; UR = hepatocytes treated with 500 μM uridine; UNL = hepatocytes treated with 500 μM uridine and 0.5 μM niacinamide; UNM = hepatocytes treated with 500 μM uridine and 1 μM niacinamide; UNH = hepatocytes treated with 500 μM uridine and 2 μM niacinamide. AMPK = adenosine 5-monophosphate-activated protein kinase; sirt1 = sirtuin 1; ampkα1 = AMP-activated protein kinase alpha 1; ampkα2 = AMP-activated protein kinase alpha 2.

Fig. 5

The mechanism of uridine on glucose and lipid metabolism in Nile tilapia. Uridine intake by Nile tilapia activated sirt1 and then enhanced AMPK phosphorylation, which subsequently activated lipolysis, inhibited lipid synthesis, and promoted glycolysis, resulting in a decrease in liver lipid and serum glucose content. On the other hand, the increased synthesis of UTP after the intake of uridine in Nile tilapia further promotes the synthesis of glycogen, which helps to lower serum glucose content. sirt1 = sirtuin 1; AMPK = adenosine 5-monophosphate-activated protein kinase; UTP = uridine triphosphate.