Dietary sodium acetate and sodium butyrate attenuate intestinal damage and improve lipid metabolism in juvenile largemouth bass (Micropterus salmoides) fed a high carbohydrate diet by reducing endoplasmic reticulum stress

Abstract

High-carbohydrate (HC) diets decrease the intestinal levels of sodium acetate (SA) and sodium butyrate (SB) and impair the gut health of largemouth bass; however, SA and SB have been shown to enhance immunity and improve intestinal health in farmed animals. Thus, the present study was to investigate the effects of dietary SA and SB on HC diet-induced intestinal injury and the potential mechanisms in juvenile largemouth bass. The experiment set five isonitrogenous and isolipidic diets, including a low-carbohydrate diet (9% starch) (LC), a high carbohydrate diet (18% starch) (HC), and the HC diet supplemented with 2 g/kg SA (HCSA), 2 g/kg SB (HCSB) or a combination of 1 g/kg SA and 1 g/kg SB (HCSASB). The feeding experiment was conducted for 8 weeks. A total of 525 juvenile largemouth bass with an initial body weight of 7.00 ± 0.20 g were used. The results showed that dietary SA and SB improved the weight gain rate and specific growth rate (P < 0.05) and ameliorated serum parameters (alkaline phosphatase, acid phosphatase, glutamate transaminase, and glutamic oxaloacetic transaminase) (P < 0.05). And, importantly, dietary SA and SB repaired the intestinal barrier by increasing the expression levels of zonula occludens-1, occludin, and claudin-7 (P < 0.05), reduced HC-induced intestinal damage, and alleviated intestinal inflammation and cell apoptosis by attenuating HC-induced intestinal endoplasmic reticulum stress (P < 0.05). Further results revealed that dietary SA and SB reduced HC-induced intestinal fat deposition by inhibiting adipogenesis and promoting lipolysis (P < 0.05). In summary, this study demonstrated that dietary SA and SB attenuated HC-induced intestinal damage and reduced excessive intestinal fat deposition in largemouth bass.

Keywords: Endoplasmic reticulum stress; High carbohydrate diet; Intestine health; Lipid metabolism; Sodium acetate; Sodium butyrate.

Fig. 1

Effect of dietary sodium acetate (SA) and sodium butyrate (SB) on serum biochemical parameters of largemouth bass fed a high carbohydrate diet. LC represents a low carbohydrate diet; HC represents a high carbohydrate diet; HCSA, HCSB, and HCSASB represent a high carbohydrate diet supplemented with 2 g/kg SA, 2 g/kg SB, and a combination of 1 g/kg SA and 1 g/kg SB, respectively. (A) Alkaline phosphatase (AKP). (B) Acid phosphatase (ACP). (C) Glutamic oxaloacetic transaminase (GOT). (D) Glutamate transaminase (GPT). (E) Immunoglobulin (IgM). (F) Fish complement protein 4 (C4). Data were expressed as means ± SEM (n = 6). Asterisk indicates statistical differences between LC and HC groups (∗P < 0.05, ∗∗P < 0.01). ^a–cMeans without a common superscript differ significantly (P < 0.05).

Fig. 2

Dietary sodium acetate (SA) and sodium butyrate (SB) improved intestinal morphology and attenuated barrier damage in largemouth bass fed a high carbohydrate diet. LC represents a low carbohydrate diet; HC represents a high carbohydrate diet; HCSA, HCSB, and HCSASB represent a high carbohydrate diet supplemented with 2 g/kg SA, 2 g/kg SB, and a combination of 1 g/kg SA and 1 g/kg SB, respectively. (A) H&E staining images of intestinal sections (scale bars, 500 μm). (B) Transmission electron microscopy images of intestinal microvilli (scale bars, 500 nm). (C) Intestinal villi height and width. (D) Height of intestinal microvilli. (E) Expression levels of tight junction-related genes. Data were expressed as means ± SEM (n = 6). Asterisk indicates statistical differences between LC and HC groups (∗P < 0.05, ∗∗P < 0.01). ^a–cMeans without a common superscript differ significantly (P < 0.05). ZO-1 = zonula occludens-1.

Fig. 3

Dietary sodium acetate (SA) and sodium butyrate (SB) alleviated high carbohydrate (HC)-induced intestinal endoplasmic reticulum stress in largemouth bass. LC represents a low carbohydrate diet; HC represents a high carbohydrate diet; HCSA, HCSB, and HCSASB represent a high carbohydrate diet supplemented with 2 g/kg SA, 2 g/kg SB, and a combination of 1 g/kg SA and 1 g/kg SB, respectively. (A) Expression levels of intestinal endoplasmic reticulum stress related genes. (B) Intestinal GRP78 immunohistochemistry (the tail of the arrow represent positive expression. Scale bars, 20 μm). (C-E) Expression levels of XBP-1 and Eif2α protein. Data were expressed as means ± SEM (n = 6). Asterisk indicates statistical differences between LC and HC groups (∗P < 0.05, ∗∗P < 0.01). ^a–cMeans without a common superscript differ significantly (P < 0.05). ATF6 = activating transcription factor 6; IRE1 = inositol-requiring enzyme 1; XBP-1 = x-box binding protein 1; GRP78 = glucose related protein 78; CHOPα = C/EBP homologous protein alpha; Eif2α = eukaryotic initiation factor 2 alpha.

Fig. 4

Electron micrographs of largemouth bass guts (scale bars, 1 μm). The red pentagons represent mitochondrial damage. LC represents a low carbohydrate diet; HC represents a high carbohydrate diet; HCSA, HCSB, and HCSASB represent a high carbohydrate diet supplemented with 2 g/kg SA, 2 g/kg SB, and a combination of 1 g/kg SA and 1 g/kg SB, respectively. SA = sodium acetate; SB = sodium butyrate; Mi = mitochondria; ER = endoplasmic reticulum; LYP = lysosome; LD = lipid droplet.

Fig. 5

Dietary sodium acetate (SA) and sodium butyrate (SB) ameliorated high carbohydrate-induced apoptosis and inflammation in largemouth bass. LC represents a low carbohydrate diet; HC represents a high carbohydrate diet; HCSA, HCSB, and HCSASB represent a high carbohydrate diet supplemented with 2 g/kg SA, 2 g/kg SB, and a combination of 1 g/kg SA and 1 g/kg SB, respectively. (A) TUNEL (green) stained images of intestinal sections (scale bars, 50 μm). (B) Relative apoptosis rates in intestinal sections. (C) Expression levels of intestinal apoptosis related genes. (D) Expression levels of intestinal NF-κB and p38-MAPK genes and pro-inflammatory related genes. (E to G) Expression levels of intestinal bax and p38-MAPK proteins. Data were expressed as means ± SEM (n = 6). Asterisk indicates statistical differences between LC and HC groups (∗P < 0.05, ∗∗P < 0.01). ^a–cMeans without a common superscript differ significantly (P < 0.05). bax = bcl2-associated x protein; bcl2 = b-cell lymphoma-2; caspase3 = cysteine aspastic acid-specific protease 3; caspase8 = cysteine aspastic acid-specific protease 8; caspase9 = cysteine aspastic acid-specific protease 9; NF-κB = nuclear factor kappa-B; p38-MAPK = mitogen-activated protein kinases; COX2 = cyclooxygenase 2; CD80 = CD80 molecule; IL-8 = interleukin 8.

Fig. 6

Dietary sodium acetate (SA) and sodium butyrate (SB) ameliorated high carbohydrate-induced intestinal lipid deposition in largemouth bass. LC represents a low carbohydrate diet; HC represents a high carbohydrate diet; HCSA, HCSB, and HCSASB represent a high carbohydrate diet supplemented with 2 g/kg SA, 2 g/kg SB, and a combination of 1 g/kg SA and 1 g/kg SB, respectively. (A) Oil-red O staining images of intestinal sections (scale bars, 500 μm). (B) Relative oil-red O area in intestinal sections. (C) Intestinal triglycerides (TG) levels. (D) Expression levels of adipogenic related genes. (E) Expression levels of lipolytic related genes. (F, G) Expression level of PPARγ protein. Data were expressed as means ± SEM (n = 6). Asterisk indicates statistical differences between low carbohydrate (LC) and high carbohydrate (HC) groups (∗P < 0.05, ∗∗P < 0.01). ^a–cMeans without a common superscript differ significantly (P < 0.05). FAS = fatty acid synthase; ACCA = acetyl-CoA carboxylase; SCD1 = stearoyl-CoA desaturase-1; PPARγ = peroxisome proliferator-activated receptor γ; CPT1α = carnitine palmitoyl transferase-1 alpha; HSL = hormone-sensitive lipase; AMPKα = adenosine 5-monophosphate (AMP)-activated protein kinase alpha; PGC1α = peroxisome proliferators-activated receptor γ coactivator l alpha; PPARα = peroxisome proliferator-activated receptor alpha.