The effects of hempseed meal intake and linoleic acid on Drosophila models of neurodegenerative diseases and hypercholesterolemia

Abstract

Hempseed is rich in polyunsaturated fatty acids (PUFAs), which have potential as therapeutic compounds for the treatment of neurodegenerative and cardiovascular disease. However, the effect of hempseed meal (HSM) intake on the animal models of these diseases has yet to be elucidated. In this study, we assessed the effects of the intake of HSM and PUFAs on oxidative stress, cytotoxicity and neurological phenotypes, and cholesterol uptake, using Drosophila models. HSM intake was shown to reduce H(2)O(2) toxicity markedly, indicating that HSM exerts a profound antioxidant effect. Meanwhile, intake of HSM, as well as linoleic or linolenic acids (major PUFA components of HSM) was shown to ameliorate Aβ42-induced eye degeneration, thus suggesting that these compounds exert a protective effect against Aβ42 cytotoxicity. On the contrary, locomotion and longevity in the Parkinson's disease model and eye degeneration in the Huntington's disease model were unaffected by HSM feeding. Additionally, intake of HSM or linoleic acid was shown to reduce cholesterol uptake significantly. Moreover, linoleic acid intake has been shown to delay pupariation, and cholesterol feeding rescued the linoleic acid-induced larval growth delay, thereby indicating that linoleic acid acts antagonistically with cholesterol during larval growth. In conclusion, our results indicate that HSM and linoleic acid exert inhibitory effects on both Aβ42 cytotoxicity and cholesterol uptake, and are potential candidates for the treatment of Alzheimer's disease and cardiovascular disease.

Fig. 1.. Inhibitory effect of HSM and linoleic acid against hydrogen peroxide toxicity. (A, B) Comparison of survival rates among wild-type flies reared in appropriate media with 3% hydrogen peroxide. (A) HSM media evidenced higher antioxidant ability than cornmeal soybean standard (CTL) media (n ≥ 190). (B) The CTL media contains linoleic acid, which evidenced an effect similar to that of HSM media against hydrogen peroxide toxicity (n ≥ 120). Error bars represent ± SE. CTL, cornmeal-soybean standard media; HSM, hempseed meal media; LI, CTL with 5.76 mg/ml linoleic acid.

Fig. 2.. Suppression of eye degeneration phenotype of AD model flies by dietary intake of HSM. In particular, some com-ponents of HSM (linoleic acid, linolenic acid, and campesterol) affect the eye degeneration phenotype. (A, B) The eye degeneration phenotype caused by Aβ42 overexpression was strongly suppressed by HSM intake. (A) Microscopic images of adult eyes showing the sub-types (mild and severe) of Aβ42-overexpressing flies divided by their phe-notypic severity. (B) Bar graphs represent the ratio of eye phenotypes (n ≥ 16). Phenotypic severity was expressed as the rate of mild (white) and severe (black). (C, D) The effects of the various compo-nents of HSM (C) and dose-dependent effect of linoleic acid (D) on the eye degeneration of Aβ42 overexpressing flies (n ≥ 20). The files were reared in CTL media with linoleic acid, linolenic acid, campesterol, or γ-linolenic acid. All fly strains were maintained at 29℃. CTL, cornmeal-soybean standard media; HSM, hempseed meal media; Linolenic acid, CTL with 2.26 mg/ml of linolenic acid; Linoleic acid, CTL with 5.76 mg/ml of linoleic acid; γ-Linolenic acid, CTL with 400 μg/ml of γ-linolenic acid; Campesterol, CTL with 7.32 μg/ml of campesterol; 1×, CTL with 5.76 mg/ml of linoleic acid; 0.1×, CTL with 0.576 mg/ml of linoleic acid; 0.01×, CTL with 57.6 μg/ml of linoleic acid. The genotype of the sample is GMR-Aβ42; GMR-Aβ42.

Fig. 3.. The effect of HSM intake on the phenotype of PD and HD model flies. (A, B) The effect of HSM intake on the phenotypes of PD model flies. The climbing ability (n ≥ 140) (A) and the longevity (n ≥ 200) (B) of parkin mutant flies were unaffected by HSM intake. Error bars represent ± SE. (C, D) The effect of HSM intake on the phenotype of HD and JNK hyperactivation model flies. Microscopic images of the adult eyes of HD (C) and JNK hyperacti-vation (D) model flies demonstrate that HSM intake do not influence their cytotoxic phenotypes. CTL, cornmeal-soybean standard media; HSM, hempseed meal media; AE, after eclosion. The genotypes of the samples are Dpark¹/Dpark¹, GMR-GAL4/+; UAS-p127Q/+, sev-GAL4/+; UAS- hep^CA/+.

Fig. 4.. Reduction of cholesterol uptake by HSM feeding. The graph represents the relative quantity of sterol in larvae reared in CTL media with/without cholesterol, HSM media with cholesterol, or CTL media with cholesterol and linoleic acid. (^*p < 0.01, n ≥ 3, Student’s t-test). Error bars represent ± SE. CTL, cornmeal-soybean standard media; CTL + Cholesterol, CTL media with 35.1 μg/ml cholesterol; HSM + Cholesterol, hempseed meal media with 35.1 μg/ml of cholesterol; CTL + Cholesterol + Linoleic acid, CTL media with 35.1 μg/ml cholesterol and 5.76 mg/ml linoleic acid.

Fig. 5.. The antagonistic effect of linoleic acid against cholesterol on larval growth. The graph represents pupariation time of larvae reared in the CTL media with/without linoleic acid or CTL media with cholesterol and linoleic acid (n ≥ 300, Student’s t-test). Error bars represent ± SE. CTL, cornmeal-soybean standard media; CTL + Linoleic acid, CTL media with 5.76 mg/ml of linoleic acid; CTL + Cholesterol + Linoleic acid, CTL media with 0.351 μg/ml of cholesterol and 5.76 mg/ml of linoleic acid; AEL, after egg laying.