Effect of Morinda citrifolia (Noni)-Enriched Diet on Hepatic Heat Shock Protein and Lipid Metabolism-Related Genes in Heat Stressed Broiler Chickens

Abstract

Heat stress (HS) has been reported to alter fat deposition in broilers, however the underlying molecular mechanisms are not well-defined. The objectives of the current study were, therefore: (1) to determine the effects of acute (2 h) and chronic (3 weeks) HS on the expression of key molecular signatures involved in hepatic lipogenic and lipolytic programs, and (2) to assess if diet supplementation with dried Noni medicinal plant (0.2% of the diet) modulates these effects. Broilers (480 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to two environmental conditions (heat stress, HS, 35°C vs. thermoneutral condition, TN, 24°C) and fed two diets (control vs. Noni) in a 2 × 2 factorial design. Feed intake and body weights were recorded, and blood and liver samples were collected at 2 h and 3 weeks post-heat exposure. HS depressed feed intake, reduced body weight, and up regulated the hepatic expression of heat shock protein HSP60, HSP70, HSP90 as well as key lipogenic proteins (fatty acid synthase, FASN; acetyl co-A carboxylase alpha, ACCα and ATP citrate lyase, ACLY). HS down regulated the hepatic expression of lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (LIPC), but up-regulated ATGL. Although it did not affect growth performance, Noni supplementation regulated the hepatic expression of lipogenic proteins in a time- and gene-specific manner. Prior to HS, Noni increased ACLY and FASN in the acute and chronic experimental conditions, respectively. During acute HS, Noni increased ACCα, but reduced FASN and ACLY expression. Under chronic HS, Noni up regulated ACCα and FASN but it down regulated ACLY. In vitro studies, using chicken hepatocyte cell lines, showed that HS down-regulated the expression of ACCα, FASN, and ACLY. Treatment with quercetin, one bioactive ingredient in Noni, up-regulated the expression of ACCα, FASN, and ACLY under TN conditions, but it appeared to down-regulate ACCα and increase ACLY levels under HS exposure. In conclusion, our findings indicate that HS induces hepatic lipogenesis in chickens and this effect is probably mediated via HSPs. The modulation of hepatic HSP expression suggest also that Noni might be involved in modulating the stress response in chicken liver.

Keywords: chicken; heat stress; lipogenesis; lipolysis; liver; noni; quercetin.

Figure 1

Effects of HS and Noni-enriched diet on feed intake and growth in broilers. HS depressed cumulative (A) and individual (B) feed intake which in turn results in a reduction of body weight (BW, C), and BW gain (D). Data are presented as mean ±SEM (n = 160). ≠ denote significant difference between TN and HS conditions at P < 0.05. ^*Indicate significant effect of time at P < 0.05.

Figure 2

Effect of Noni supplementation on hepatic HSP expression in heat-stressed broilers. (A–D) acute HS and (E–H) chronic HS. HSP levels were determined by Western blot and their relative expression was presented as a normalized ratio to housekeeping GAPDH protein. Data are presented as mean ± SEM (n = 8/group). Different letters indicate significant difference at P < 0.05.

Figure 3

Effect of Noni supplementation on hepatic expression of lipogenic markers in acute heat-stressed broilers. Phosphorylated and pan levels of ACCα as well as FASN and ACLY were determined using Western blot (A) and their relative expression was presented as normalized ratio of phosphorylated/total target protein (B–D). Relative abundance of ACCα (E), ACLY (F), FASN (G), ME (H), and SCD-1 (I) mRNA was measured by real-time RT-PCR. Data are presented as mean ± SEM (n = 8/group). Different letters indicate significant difference at P < 0.05.

Figure 4

Effect of Noni supplementation on hepatic expression of lipogenic markers in chronic heat-stressed broilers. Phosphorylated and pan levels of ACCα as well as FASN and ACLY were determined using Western blot (A) and their relative expression was presented as normalized ratio of phosphorylated/total target protein (B–D). Relative abundance of ACCα (E), ACLY (F), FASN (G), ME (H), and SCD-1 (I) mRNA was measured by real-time RT-PCR. Data are presented as mean ± SEM (n = 8/group). Different letters indicate significant difference at P < 0.05.

Figure 5

Effect of Noni on hepatic expression of lipogenesis-related transcription factors in heat stressed broilers. (A–C) acute HS and (D–F) chronic HS. Relative expression of SREBP-1 (A,D), SREBP-2 (B,E), and SCAP (C,F) was measured by real-time RT-PCR. Data are presented as mean ± SEM. D, diet; E, environment; TN, thermoneutral; HS, heat stress. Different letters indicate significant difference at p < 0.05.

Figure 6

Effect of Noni supplementation on hepatic expression of lipolytic pathway in heat-stressed broilers. (A–D) acute HS, (E–H) chronic HS. Relative expression of LPL (A,E), ATGL (B,F), LIPC (C,G), and CD36 gene (D,H) was measured by real-time RT-PCR. Data are presented as mean ± SEM (n = 8/group). Different letters indicate significant difference at P < 0.05.

Figure 7

Effect of QCT on lipogenesis-related signatures and SREBP-1 gene expression in heat stressed sim-CEL cells. Phosphorylated and pan levels of ACCα as well as FASN and ACLY were determined using Western blot and their relative expression was presented as normalized ratio of phosphorylated/total target protein (A–D). Immunofluorescence staining (E) shows that HS induce HSP 60 and 70, and reduces ACCα and FASN protein levels in sim-CEL cells. SREBP-1 mRNA abundance was measured by real-time RT-PCR (F). Data are presented as mean ±SEM (n = 6/groups). Different letters indicate significant differences at P < 0.05. Western blotting figure is representative of 3 replicates.

Figure 8

Schematic representation of the effect of heat stress and Noni supplementation on hepatic lipid metabolism in broilers (from protein expression analysis). Heat stress up-regulated the hepatic expression of heat shock proteins (HSP60, HSP70, and HSP90) as well as key lipogenic proteins (FASN, ACCα, and ACLY). HS down-regulated the hepatic expression of LPL and LIPC, but up-regulated ATGL. Noni supplementation down-regulated the hepatic expression of HSPs, ACCα, and FASN but it down regulated ACLY. ↑ and “+” denote induction, “−“ indicates inhibition. HSL, hormone sensitive lipase; FFA, free fatty acids; VLDL, very low density lipoprotein.