β-carotene genetically-enriched lyophilized orange juice increases antioxidant capacity and reduces β-amyloid proteotoxicity and fat accumulation in Caenorhabditis elegans

Abstract

Citrus sinensis orange juice is an excellent dietary source of β-carotene, a well-known antioxidant. However, β-carotene concentrations are relatively low in most cultivars. We developed a new orange through metabolic engineering strategy (GS) with 33.72-fold increase in β-carotene content compared to its conventional counterpart (CV). Using Caenorhabditis elegans, we found that animals treated with GS showed a greater reduction in intracellular reactive oxygen species (ROS) which is associated with a greater resistance to oxidative stress and induction of the expression of antioxidant genes. Moreover, animals treated with GS orange showed a more effective protection against β-amyloid proteotoxicity and greater hypolipidemic effect under high glucose diet compared to animals treated with CV. These data demonstrate that the increased amount of β-carotene in orange actually provides a greater beneficial effect in C. elegans and a valuable proof of principle to support further studies in mammals and humans.

Fig. 1

Effect of lyophilized orange juice (LOJ) on C. elegans ROS production, stress resistance and longevity. A) ROS was quantified by measuring H₂DCFDA fluorescence levels. For standard condition, L1 stage wild-type animals were treated with 2 % of either CV or GS juices for 48 h. **** p < 0.0001 compared to not treated (NT) control by one-way ANOVA. ^#p = 0.0088 comparing 2 % GS to 2 % CV using two-tailed Student’s t-test. For stress condition, after the worms were treated with 2 % LOJ for 48 h, they were exposed to 10 mM TBHP for 1 h to induce oxidative stress. * p = 0.0130 and *** p = 0.0003 compared to NT control under stress condition by one-way ANOVA. B) Stress resistance assay. L1 stage wild-type animals were treated with 2 % LOJ for 48 h and then incubated with TBHP to induce oxidative stress. Survival fractions were scored every-three hours at 20 °C. **** p < 0.0001 compared to not treated (NT) control and ^#p = 0.0038 comparing LOJ GS to CV by log rank (Mantel-Cox) test. (b) Lifespan assay. Wild-type animals were treated with 2 % LOJ for 48 h starting at L1 stage. Survival fractions were scored daily at 25 °C. **** p < 0.0001 compared to not treated (NT) control by log rank (Mantel-Cox) test.

Fig. 2

Expression levels of antioxidant and stress-related genes after treatment with lyophilized oranges juices (LOJ). L1 stage transgenic worms expressing (A) gcs-1::GFP, (B) gst-4::GFP, (C) sod-3::GFP and (D) hsp-4::GFP were treated with 2 % of LOJ for 48 h until L4 stage. Images were taken using a microscope fluorescent and the measure levels fluorescent were using NIH ImageJ software. **** p < 0.0001 compared control not treated (NT) and ^#p < 0.03 comparing 2 % GS to 2 % CV by one-way ANOVA.

Fig. 3

Effect of lyophilized orange juice (LOJ) in C. elegans neuromuscular parameters. A) Pharyngeal pumping rate. L1 stage wild-type animals were treated with LOJ for 48 h until L4 stage. Pharyngeal pumping rate was scored by counting the movements of the pharynx terminal bulb using a microscope in 40x objective. **** p < 0.0001 compared to not treated (NT) control by one-way ANOVA. B) Body bending rate. L1 stage wild-type animals were treated with LOJ for 48 h until L4 stage. Body bending score was obtained through the counting of the animal’s body movements. No statistical difference was found.

Fig. 4

Effect of lyophilized orange juice (LOJ) on C. elegans proteotoxicity. A) Paralysis-induced by β-amyloid accumulation. L1 stage transgenic worms were treated with 2 % LOJ for 48 h until L4 stage. Analyses were made by scoring paralyzed animals every 1 h at 35 °C. **** p < 0.0001 compared not treated (NT) control and ^# p = 0.0003 comparing 2 % GS to 2 % CV by log rank (Mantel-Cox) test. B) Quantification of proteasome activity. L1 stage wild-type animals were treated with LOJ for 48 h until L4 stage. Proteasome activity was calculated as the difference between the total activity and the activity remaining in the presence of 20 µM MG-132. Experiment performed in duplicate. No statistical difference was found.

Fig. 5

Effect of lyophilized orange juice (LOJ) on C. elegans lipid distribution. A) Oil Red O staining. L1 stage wild-type animals were treated with 2 % LOJ on either NGM plates or 4 % glucose NGM glucose plates for 48 h until L4 stage. Animals were fixed with 40 % isopropanol and lipid droplets were stained with Oil Red O. Images were captured using microscope in 10x objective. B) Quantification of lipid distribution was done by measuring Oil Red O dye using NIH ImageJ software. **** p < 0.0001 compared to control NT by one-way ANOVA and ^# p = 0.0005 comparing 2 % GS-glucose to 2 % CV-glucose by one-way ANOVA. C) Quantification of lysosome related organelles (LRO). L1 stage wild-type animals were treated with 2 % LOJ on NGM plate containing Red Nile dye for 72 h until 1-day old. Images were captured using fluorescent microscope and fluorescence levels were analyzed using NIH ImageJ software. **** p < 0.0001 comparing either CV or GS to NT animals and ^#p = 0.0264 comparing GS to CV treated animals by one-way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)