Ilex paraguariensis modulates fat metabolism in Caenorhabditis elegans through purinergic system (ADOR-1) and nuclear hormone receptor (NHR-49) pathways

Abstract

Ilex paraguariensis is a well-known plant that is widely consumed in South America, primarily as a drink called mate. Mate is described to have stimulant and medicinal properties. Considering the potential anti-lipid effects of I. paraguariensis infusion, we used an extract of this plant as a possible modulator of fat storage to control lipid metabolism in worms. Herein, the I. paraguariensis-dependent modulation of fat metabolism in Caenorhabditis elegans was investigated. C. elegans were treated with I. paraguariensis aqueous extract (1 mg/ml) from L1 larvae stage until adulthood, to simulate the primary form of consumption. Expression of adipocyte triglyceride lipase 1 (ATGL-1) and heat shock protein 16.2, lipid accumulation through C1-BODIPY-C12 (BODIPY) lipid staining, behavioral parameters, body length, total body energy expenditure and overall survival were analyzed. Total body energy expenditure was determined by the oxygen consumption rate in N2, nuclear hormone receptor knockout, nhr-49(nr2041), and adenosine receptor knockout, ador-1(ox489) strains. Ilex paraguariensis extract increased ATGL-1 expression 20.06% and decreased intestinal BODIPY fat staining 63.36%, compared with the respective control group, without affecting bacterial growth and energetic balance, while nhr-49(nr2041) and ador-1(ox489) strains blocked the worm fat loss. In addition, I. paraguariensis increased the oxygen consumption in N2 worms, but not in mutant strains, increased N2 worm survival following juglone exposure, and did not alter hsp-16.2 expression. We demonstrate for the first time that I. paraguariensis can decrease fat storage and increase body energy expenditure in worms. These effects depend on the purinergic system (ADOR-1) and NHR-49 pathways. Ilex paraguariensis upregulated the expression of ATGL-1 to modulate fat metabolism. Furthermore, our data corroborates with other studies that demonstrate that C. elegans is a useful tool for studies of fat metabolism and energy consumption.

Fig 1. Ilex paraguariensis effects on ATGL-1::GFP expression in Caenorhabditis elegans VS20 strain.

(A) Visualization of ATGL-1::GFP expression and (B) measurement of ATGL-1::GFP expression on young-adult Caenorhabditis elegans. *p<0.05 and **p<0.01, statistically significant compared with the untreated group by One-Way ANOVA followed by Bonferroni post-test (mean, standard error of the mean [SEM], n = 30 worms per group). The experiments were performed in triplicate.

Fig 2. BODIPY fluorescence decrease induced by Ilex paraguariensis exposition in Caenorhabditis elegans wild-type (N2).

Visualization of lipid droplets evidenced by BODIPY labeling in (A) wild-type young adults, (B) nhr-49(nr2041), and (C) ador-1(ox489) and fluorescence quantification in (D) wild-type young adults, (E) nhr-49(nr2041), and (F) ador-1(ox489). ***p<0.001, statistically significant compared with the untreated group by Student’s t-test (mean, SEM, n = 60 worms per group). The experiment was performed in triplicate.

Fig 3. Caenorhabditis elegans wild-type behavior following Ilex paraguariensis treatment.

Effect of I. paraguariensis on (A) pharyngeal pumping rate, (B) defecation cycle length, (C) body bends in wild-type young adults, (D) egg production and (E) body length in wild-type young-adults. No statistically significant difference was found by Student’s t-test (mean, SEM, n = 30 worms per group). The experiments were performed in triplicate.

Fig 4. Measurement of oxygen consumption rate in C. elegans treated with I. paraguariensis extract.

Oxygen consumption rates in young-adult worms. (A) Wild type, (B) nhr-49(nr2041), and (C) ador-1(ox489). **p < 0.01, statistically significant compared with the untreated group by Student’s t-test (mean, SEM, n = 7). The experiments were performed seven times.

Fig 5. I. paraguariensis on resistance to oxidative stress.

Survival of young-adult worms exposed to 100 μM juglone for 1 h. (A) Wild type, (B) nhr-49(nr2041), and (C) ador-1(ox489). Data are expressed as percentage of worms alive. **p < 0.01, statistically significant compared with the untreated group by Student’s t-test (mean, SEM, n = approximately 500 worms per group).

Fig 6. I. paraguariensis on hsp-16.2p::GFP and hsp-16.2 transgene expression.

(A) Fluorescence expression of hsp-16.2p::GFP worms. Data are expressed as arbitrary fluorescence units (AFU). (B) hsp-16.2 mRNA levels using a ΔΔCq method in wild-type (N2). Student’s t-test, p < 0.05. The experiments were performed five times on different days.