Physiological and metabolomic consequences of reduced expression of the Drosophila brummer triglyceride Lipase

Abstract

Disruption of lipolysis has widespread effects on intermediary metabolism and organismal phenotypes. Defects in lipolysis can be modeled in Drosophila melanogaster through genetic manipulations of brummer (bmm), which encodes a triglyceride lipase orthologous to mammalian Adipose Triglyceride Lipase. RNAi-mediated knock-down of bmm in all tissues or metabolic specific tissues results in reduced locomotor activity, altered sleep patterns and reduced lifespan. Metabolomic analysis on flies in which bmm is downregulated reveals a marked reduction in medium chain fatty acids, long chain saturated fatty acids and long chain monounsaturated and polyunsaturated fatty acids, and an increase in diacylglycerol levels. Elevated carbohydrate metabolites and tricarboxylic acid intermediates indicate that impairment of fatty acid mobilization as an energy source may result in upregulation of compensatory carbohydrate catabolism. bmm downregulation also results in elevated levels of serotonin and dopamine neurotransmitters, possibly accounting for the impairment of locomotor activity and sleep patterns. Physiological phenotypes and metabolomic changes upon reduction of bmm expression show extensive sexual dimorphism. Altered metabolic states in the Drosophila model are relevant for understanding human metabolic disorders, since pathways of intermediary metabolism are conserved across phyla.

Fig 1. RT-qPCR and lifespan of Ubi > bmm-RNAi flies.

Offspring of Ubi-Gal4 mated with GD control (Ubi > +) or UAS-bmm-RNAi flies (Ubi > bmm-RNAi^V37877 and Ubi > bmm-RNAi^V37880) were used for the assays. Asterisks indicate significant differences at p < 0.05 compared with the appropriate female or male control, following Tukey’s correction for multiple tests. (a) RT-qPCR (n = 9 for females and males of each genotype), relative quantification (RQ) was done using the 2^-ΔΔCt method, and GAPDH was used as an internal standard. (b) Spread wings phenotype in males of Ubi > bmm-RNAi^V37877 (100%, n = 100). (c) Average lifespan for Ubi > + (n = 132 for females and 118 for males), Ubi > bmm-RNAi^V37877 (n = 136 for females and 134 for males) and Ubi > bmm-RNAi^V37880 (n = 137 for females and 132 for males). Error bars are standard errors of the mean (SEM). ANOVA tests are reported in S1 Table.

Fig 2. Locomotor activity in Ubi > bmm-RNAi flies.

Offspring of Ubi-Gal4 mated with GD control (Ubi > +) or UAS-bmm-RNAi flies (Ubi > bmm-RNAi^V37877 and Ubi > bmm-RNAi^V37880) were used for the locomotor activity assay in normal feeding (a-c), normal feeding without wings (d-f) and starvation conditions (g-i). (a, d and g) Average of daily locomotor activity. (b, e and h) Average of locomotor activity during the daytime and nighttime. (c, f and i) Average activity profiles in Zeitgeber (ZG) time. Averages were calculated from 7 days of the behavior assays except for starvation, which was measured over 3 days. For this figure and Fig 3, “n” for normal feeding in females (F) and males (M) were: Ubi > + F (n = 49), Ubi > + M (n = 57), Ubi > bmm-RNAi^V37877 F (n = 54), Ubi > bmm-RNAi^V37877 M (n = 53), Ubi > bmm-RNAi^V37880 F (n = 57) and Ubi > bmm-RNAi^V37880 M (n = 62); “n” in normal feeding without wings were: Ubi > + F (n = 64), Ubi > + M (n = 62), Ubi > bmm-RNAi^V37877 F (n = 56), Ubi > bmm-RNAi^V37877 M (n = 55), Ubi > bmm-RNAi^V37880 F (n = 58) and Ubi > bmm-RNAi^V37880 M (n = 61); “n” in starvation were: Ubi > + F (n = 56), Ubi > + M (n = 42), Ubi > bmm-RNAi^V37877 F (n = 54), Ubi > bmm-RNAi^V37877 M (n = 52), Ubi > bmm-RNAi^V37880 F (n = 63) and Ubi > bmm-RNAi^V37880 M (n = 63). Asterisks indicate significant differences at p < 0.05 compared with the appropriate female or male control, following Tukey’s correction for multiple tests. Error bars are SEM. ANOVA tests are reported in S2 Table.

Fig 3. Sleep behavior in Ubi > bmm-RNAi flies.

Offspring of Ubi-Gal4 mated with GD control (Ubi > +) or UAS-bmm-RNAi flies (Ubi > bmm-RNAi^V37877 and Ubi > bmm-RNAi^V37880) were used for the sleep assay in normal feeding (a-c), normal feeding without wings (d-f) and starvation conditions (g-i). (a, d and g) Average number of sleep bouts. (b, e and h) Average length of sleep bout. (c, f and i) Average sleep during the daytime and the nighttime. Sleep was calculated using the standard definition of a continuous period of inactivity lasting at least 5 minutes, and average was calculated from 7 days of behavior assay except for starvation, which was measured over 3 days, and recalculated to periods of 30 minutes of sleep. Asterisks indicate significant differences at p < 0.05 compared with the appropriate female or male control, following Tukey’s correction for multiple tests. Error bars are SEM. ANOVA tests are reported in S3 Table.

Fig 4. Metabolites with altered abundance levels in Ubi > bmm-RNAi flies.

Offspring of Ubi-Gal4 mated with GD control (Ubi > +) or UAS-bmm-RNAi flies (Ubi > bmm-RNAi^V37877 and Ubi > bmm-RNAi^V37880) were used for metabolome analysis. F1 6-day-old adult females (F) and males (M) were used for the metabolomic profiles (n = 6). ANOVA contrasts were used to identify metabolites that differed significantly between experimental groups and corresponding controls at p ≤ 0.05 (q-value is reported in S1 Dataset). (a) Principal component analysis of variation in the composition of the metabolome of flies in which bmm is downregulated and control flies. (b) Venn diagram of metabolites that significantly changed in Ubi > bmm-RNAi females compared with controls. (c) Number of metabolites that significantly increased (black bars) and decreased (white bars) in Ubi > bmm-RNAi females compared with controls. (d) Venn diagram of metabolites that significantly changed in Ubi > bmm-RNAi males compared with controls. (e) Number of metabolites that significantly increased (black bars) and decreased (white bars) in Ubi > bmm-RNAi males compared with controls.

Fig 5. Compounds of lipid metabolism with altered levels of abundance in Ubi > bmm-RNAi flies.

Heat map of changes in biochemicals of lipid metabolism in flies in which bmm is downregulated (Ubi > bmm-RNAi) compared with control flies (Ubi > +). For this graph and the graphs below, we performed a single comparison of each strain with its corresponding control for females (V37877-F and V37880-F) and males (V37877-M and V37880-M). Also, we performed a comparison of both strains with their corresponding controls for females (V37877-F + V37880-F) and males (V37877-M + V37880-M). Red and dark blue represent the metabolites that increased and decreased respectively at p ≤ 0.05, light red and light blue represent the metabolites that increased and decreased respectively at 0.05 ≤ p ≤ 0.1 (q-value is reported in S1 Dataset). Asterisks indicate compounds that have not been confirmed based on a standard.

Fig 6. Compounds of acyl carnitine/fatty acid metabolism with altered abundance levels in Ubi > bmm-RNAi flies.

(b) Diagram of the acyl carnitine/fatty acid metabolic pathway, highlighting the role of carnitine in facilitating transport of fatty acids into the mitochondria for fatty acid oxidation. (b) Heat map of changes in biochemicals of acyl carnitine/fatty acid metabolism in flies in which bmm is downregulated (Ubi > bmm-RNAi) compared with control flies (Ubi > +). Red and dark blue represent the metabolites that increased and decreased respectively at p ≤ 0.05, light red and light blue represent the metabolites that increased and decreased respectively at 0.05 ≤ p ≤ 0.1 (q-value is reported in S1 Dataset). Asterisks indicate compounds that have not been confirmed based on a standard.

Fig 7. Metabolites of glycolysis and the TCA cycle with altered abundance levels in Ubi > bmm-RNAi flies.

(a) Diagram of the glycolysis pathway and the TCA cycle. Black dots represent intermediates in the pathways and the enzymes in the reactions are marked with arrows and their initials: HK = Hexokinase, PGI = Phosphoglucose isomerase, FBP = Fructose 1,6-biphosphatase, PFK = Phosphofructokinase, ALD = Aldolase, TPI = Triosephosphate isomerase, GAPDH = Glyceraldehyde-3-phosphate dehydrogenase, PGK = Phosphoglycerate kinase, PGLYM = Phosphoglyceromutase, ENO = Enolase, PYK = Pyruvate kinase, PDHE1 = Pyruvate dehydrogenase E1, PDK = Pyruvate dehydrogenase kinase, PDP = Pyruvate dehydrogenase phosphatase, CS = Citrate synthase, mACON = Mitochondrial aconitase, IDH = Isocitrate dehydrogenase, KGD = alpha-ketoglutarate dehydrogenase, SDH = Succinate dehydrogenase, MDH2 = Malate dehydrogenase. Brown dots designate ADP, bright-red dots, ATP, opaque-green dots, NAD+ and bright-green dots, NADH. (b) Heat map of variation in intermediates of carbohydrate metabolism and TCA cycle with altered abundance levels in bmm down-regulated (Ubi > bmm-RNAi) and control flies (Ubi > +). Red and dark blue represent the metabolites that increased and decreased respectively at p ≤ 0.05, light red and light blue represent the metabolites that increased and decreased respectively at 0.05 ≤ p ≤ 0.1 (q-value is reported in S1 Dataset). Asterisks indicate compounds that have not been confirmed based on a standard.