Multidrug Resistance Like Protein 1 Activity in Malpighian Tubules Regulates Lipid Homeostasis in Drosophila

Abstract

Multidrug resistance proteins (MRPs), members of the ATP-binding cassette transporter (ABC transporter) family, are pivotal for transporting endo- and xenobiotics, which confer resistance to anticancer agents and contribute to the clearance of oxidative products. However, their function in many biological processes is still unclear. We investigated the role of an evolutionarily conserved MRP in metabolic homeostasis by knocking down the expression of Drosophila multidrug-resistance like protein 1 (MRP) in several tissues involved in regulating metabolism, including the gut, fat body, and Malpighian tubules. Interestingly, only suppression of MRP in the Malpighian tubules, the functional equivalent to the human kidney, was sufficient to cause abnormal lipid accumulation and disrupt feeding behavior. Furthermore, reduced Malpighian tubule MRP expression resulted in increased Hr96 (homolog of human pregnane X receptor) expression. Hr96 is known to play a role in detoxification and lipid metabolism processes. Reduced expression of MRP in the Malpighian tubules also conveyed resistance to oxidative stress, as well as reduced normal levels of reactive oxygen species in adult flies. This study reveals that an evolutionarily conserved MRP is required in Drosophila Malpighian tubules for proper metabolic homeostasis.

Keywords: ABCC1; kidney; lipid metabolism; oxidative stress; xenobiotic.

Figure 1

Crossing MRP RNAi with four different tissue-specific drivers. Graphs are presented in box plots, showing starvation resistance of both experimental and control groups. All Gal4 drivers were crossed with a UAS-MRP RNAi line: (A) 48Y-GAL4 (the mid-gut driver), (B) c601-GAL4 (the hind-gut driver), (C) ppl-GAL4 (the fat body driver), and (D) Uro-GAL4 (the Malpighian tubule driver). Error bar represents the max and min values, and 32 male flies were used for each individual crossed strain; ns, not significant compared to either w¹¹¹⁸ > UAS-MRP RNAi or both of the controls; ^# p < 0.01, compared to GAL4-driver > w ¹¹¹⁸; ^### p < 0.001, compared to Uro-GAL4 > w¹¹¹⁸; * p < 0.01, compared to w¹¹¹⁸ > UAS-MRP RNAi flies; one-way ANOVA with Tukey’s post hoc test was performed.

Figure 2

MRP knockdown in the Malpighian tubules affects lipid content. Flies for the carbohydrate assay were either fed ad libitum or starved for 24 h before the assay. Uro-GAL4 > UAS-MRP RNAi flies exhibited no effects on their (A) circulating glucose, (B) circulating trehalose, (C) stored trehalose, and (D) glycogen. However, MRP knockdown in the Malpighian tubules induced a significant increase in (E) triacylglyceride (TAG) content. The mRNA expression of the xenobiotic sensing receptor, Hr96, was notably increased (F). All graphs are presented as mean ± SEM; 10–15 male flies for carbohydrate assay and 6 male flies for TAG assay were used per sample, and 4–6 biological samples were prepared for each assay; ns, not significant compared to either of the control groups; ** p < 0.01 and * p < 0.05, compared to Uro-GAL4 > w¹¹¹⁸; ^### p < 0.001, compared to w¹¹¹⁸ > UAS-MRP RNAi flies; one-way ANOVA with Tukey’s post hoc test was performed.

Figure 3

MRP knockdown in the Malpighian tubules affects feeding behavior. Male flies 5–7 days of age were used, and the flyPAD was employed to assess the feeding behavior. (A) The number of sips, which represents the total food intake; (B) the number of feeding bouts, which represents any interaction with food drop; and (C) the number of feeding bursts, which represents the number of meals, were all significantly decreased in the experimental group. However, both (D) the sips per burst and (E) bout duration were not affected. The feeding burst (F) duration was slightly increased in the experimental group. All graphs are presented as mean ± SEM, n = 32 males per group; ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA with Tukey’s post hoc test was performed; the experimental group was compared to each of the control groups.

Figure 4

Malpighian tubule specific MRP knockdown conferred oxidative stress resistance. Flies 5–7 days of age were used. (A) Thirteen to fifteen flies were placed in a vial with a filter paper soaked with paraquat–sucrose solution. The death of flies was registered 3 times per day. The Uro-GAL4 > UAS-MRP RNAi flies exhibited more resistance to paraquat than both of the control groups (6–8 replicates were used for each group of flies; data are represented as percentage survival ± SEM; p values were shown on the plot; Kaplan–Meier log-rank test was performed). (B) ROS production of the Uro-GAL4 > UAS-MRP RNAi flies was significantly less than that of both of the controls, which indicates diminished oxidative stress level. Six flies were used for each replicate and 6 replicates were prepared for each group. The data were normalized to the Uro-GAL4 > w¹¹¹⁸ group. (C) The expression of ss, cnc, and Keap1 were tested using qPCR. Although no difference was observed in cnc and Keap1 expression, ss expression levels increased significantly in the experimental flies. The graph represents as mean ± SEM; * p < 0.05, ** p < 0.01; one-way ANOVA with Tukey’s post hoc test was performed.