Dietary Beta-Hydroxy Beta-Methyl Butyrate Supplementation Alleviates Liver Injury in Lipopolysaccharide-Challenged Piglets

Abstract

The current study was performed to investigate whether dietary β-hydroxy-β-methylbutyrate (HMB) could regulate liver injury in a lipopolysaccharide- (LPS-) challenged piglet model and to determine the mechanisms involved. Thirty piglets (21 ± 2 days old, 5.86 ± 0.18 kg body weight) were randomly divided into the control (a basal diet, saline injection), LPS (a basal diet), or LPS+HMB (a basal diet + 0.60% HMB-Ca) group. After 15 d of treatment with LPS and/or HMB, blood and liver samples were obtained. The results showed that in LPS-injected piglets, HMB supplementation ameliorated liver histomorphological abnormalities induced by LPS challenge. Compared to the control group, the activities of serum aspartate aminotransferase and alkaline phosphatase were increased in the LPS-injected piglets (P < 0.05). The LPS challenge also downregulated the mRNA expression of L-PFK, ACO, L-CPT-1, ICDH β, and AMPKα1/2 and upregulated the mRNA expression of PCNA, caspase 3, TNF-α, TLR4, MyD88, NOD1, and NF-κB p65 (P < 0.05). However, these adverse effects of the LPS challenge were reversed by HMB supplementation (P < 0.05). These results indicate that HMB may exert protective effects against LPS-induced liver injury, and the underlying mechanisms might involve the improvement of hepatic energy metabolism via regulating AMPK signaling pathway and the reduction of liver inflammation via modulating TLR4 and NOD signaling pathways.

Figure 1

Histological examination of the liver samples of piglets injected with lipopolysaccharide (LPS) or saline. Sections were obtained with haematoxylin and eosin (×200). CON: control; HMB: β-hydroxy-β-methylbutyrate.

Figure 2

Effects of dietary supplementation of HMB on serum biochemical parameters of piglets injected with LPS or saline. (a) Aspartate aminotransferase (ASAT); (b) alanine aminotransferase (ALAT); (c) alkaline phosphatase (AKP); (d) glutamyl transpeptidase (GGT). Values are means, with their standard errors represented by vertical bars (n = 10). ^a,bMean values with different letters were considered to be significantly different (P < 0.05). CON: control; HMB: β-hydroxy-β-methylbutyrate; LPS: lipopolysaccharide.

Figure 3

The mRNA expression of energy metabolism-related genes in the liver of piglets injected with LPS or saline. Carbohydrate metabolism-related genes: Hexok2: hexokinase 2; L-PFK: phosphofructokinase; PK: pyruvate kinase; PDH: pyruvate dehydrogenase. Fatty acid oxidation-related genes: ACO: acyl-coenzyme A oxidase; L-CPT-1: liver carnitine palmitoyltransferase I. Tricarboxylic acid cycle-related genes: CS: citrate synthase; isocitrate dehydrogenase β; ICDH γ: isocitrate dehydrogenase γ. Values are means, with their standard errors represented by vertical bars (n = 10). ^a,bMean values with different letters were considered to be significantly different (P < 0.05). CON: control; HMB: β-hydroxy-β-methylbutyrate; LPS: lipopolysaccharide.

Figure 4

The mRNA expression of AMPKα1/2, Sirt1, and PGC-1α in the liver of piglets injected with LPS or saline. Values are means, with their standard errors represented by vertical bars (n = 10). ^a,bMean values with different letters were considered to be significantly different (P < 0.05). AMPKα1/2: AMP-activated protein kinase α 1/2; CON: control; HMB: β-hydroxy-β-methylbutyrate; LPS: lipopolysaccharide; PGC-1α: peroxisome proliferator-activated receptor-g coactivator-1α; SIRT1: silent information regulator transcript 1.

Figure 5

The mRNA expression of proliferation cell nuclear antigen (PCNA), caspase-3, tumor necrosis factor-α (TNF-α), and heat shock protein 70 (HSP70) in the liver of piglets injected with LPS or saline. Values are means, with their standard errors represented by vertical bars (n = 10). ^a,bMean values with different letters were considered to be significantly different (P < 0.05). CON: control; HMB: β-hydroxy-β-methylbutyrate; LPS: lipopolysaccharide.

Figure 6

The mRNA expression of toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain protein (NODs) and their downstream signaling molecules in the liver of piglets injected with LPS or saline. Values are means, with their standard errors represented by vertical bars (n = 10). ^a,bMean values with different letters were considered to be significantly different (P < 0.05). CON: control; HMB: β-hydroxy-β-methylbutyrate; IRAK1: IL-1 receptor-associated kinase 1; LPS: lipopolysaccharide; MyD88: myeloid differentiation factor 88; RIPK2: receptor-interacting serine/threonine-protein kinase 2; TRAF6: TNF-α receptor-associated factor 6.

Figure 7

Western blot analysis of AMPKα phosphorylation (a) and claudin (b) expression in the liver of piglets injected with LPS or saline. Values are means, with their standard errors represented by vertical bars (n = 10). ^a,b,cMean values with different letters were considered to be significantly different (P < 0.05). CON: control; HMB: β-hydroxy-β-methylbutyrate; LPS: lipopolysaccharide.