TRPg mutants exhibit altered carbohydrate, lipid, and protein levels. (A) TAG level measurement in the whole-body extract from control (w1118) and TRP channel mutant lines (n=9). (B) Protein level measurement in the whole-body extract from control (w1118) and TRP channel mutant lines (n=6). (C) Measurement of total glucose and trehalose levels (µg/mg) in the whole-body extracts of control (w1118) and trpg1 adult males (n=6). (D) Measurement of tissue glycogen levels (µg/mg) in adult control (w1118) and trpg1 males (n=6). (E) Measurement of Hemolymph glucose + trehalose level in the male flies of control (w1118) and trpγ1 (n=6). (F) Measurement of TAG level in adult males to test for rescue of the TAG defect in trpg flies with the UAS-trpg and the trpgG4 or with the trpg genomic transgene (n=6-10). (G) Rescue experiments showing the measurement of protein levels in adult flies with the indicated genotypes (n=6).

Comparisons between multiple experimental groups were conducted via single-factor ANOVA coupled with Scheffe’s post hoc test. The asterisks indicate significant differences from the controls (**P<0.01).

Dh44 neurons are essential for regulating lipid tissue levels. (A) Tissue TAG level measurement in whole-body extracts of adult male flies after silencing of Dh44-GAL4 and dILP2-GAL4 with UAS-Kir2.1 (n=6). (B) Measurement of tissue protein level in whole-body extracts of adult male flies. Brain-specific Dh44-GAL4 and dILP2-GAL4 neurons were ablated using UAS-Kir2.1 (n=6). (C) Measurement of TAG from whole-body extract of adult male flies in the indicated genotypes (n=6). (D) Measurement of tissue protein level from the whole-body extracts of adult males (n=6). (E) TAG levels in whole-body extracts after RNAi knockdown of trpγ mutants under control of the Dh44-GAL4 (n=6). (F) Measurement of area (µm2) of LDs in adult fat body across the indicated genotypes involved the selection of the 30 largest LDs, choosing the top 10 LDs from each sample for analysis (n=3). (G–L) Nile red stating of fat body from the male of indicated genotypes. The scale bar represents 50 µm.

All values are reported as means ± SEM. Comparisons between multiple experimental groups were conducted via single-factor ANOVA coupled with Scheffe’s post hoc test. The asterisks indicate significant differences from the controls (*P<0.05, **P<0.01). Each dot indicates the distribution of individual sample values. (+) and (-) indicate the presence or absence of the indicated transgenes, respectively.

trpγ1 have deficits in the lipolytic pathway under starvation conditions. (A) Survival assay to measure the total survival time (days) of control (w1118) and trpγ1 male flies fed with a normal corn meal diet (n=8). (B) Survival assay to measure the survival time (h) of the indicated genotypes with male flies under starvation conditions (n=4). (C) TAG level measurement in control (w1118) and trpγ1 adult male flies in both sated (0 h starvation) and starved (24 h starvation) conditions (n=8). (D–E) Nile red staining of the LDs extracted from FB of (D) w1118 and (E) trpγ1 flies under sated (D1 and E1) and starved (D2 and E2) conditions, respectively. The scale bar represents 50 µm. (F) Measurement of area (µm2) of the LDs extracted from the FBs of w1118and trpγ1 flies under sated (0 h starvation) and starved (24 h starvation) conditions (n=3). (G) Measurement of TAG level with controls and the flies after expressing UAS-AMPKTD under the control of Dh44-GAL4 in the trpγ1 mutant background (n=4).

All values are reported as means ± SEM. Survival curves in A and B were estimated for each group, using a Kaplan-Meier method and compared statistically using the log-rank tests. Comparisons between multiple experimental groups in C, F, G, and H were conducted via single-factor ANOVA coupled with Scheffe’s post hoc test. The asterisks indicate significant differences from the controls (**P<0.01).

Rescue of starvation susceptibility phenotype using the lipolytic drug metformin. (A) TAG level measurement at 0, 7, and 14 days in the control (w1118) and trpγ1 adult male flies after dietary exposure to 1 mM metformin (n=6). (B) TAG measurement at 0, 7, and 14 days in w1118 and trpγ1 adult male flies after dietary exposure to 5 mM metformin (n=6). (C) Pictures of Nile red staining of LDs after dietary exposure to 1 mM and 5 mM metformin in standard fly food for 7 and 14 days in w1118 and trpγ1 flies. The scale bars represent 50 µm. (D) Measurement of area (µm2) of LDs extracted from the FB of w1118and trpγ1 flies after dietary exposure to 1mM and 5 mM metformin in standard fly food for 7 and 14 days (n=3). (E) Survival assay to measure the survival time (h) of control (w1118) and trpγ1 males after dietary exposure to 1 mM metformin in 1% agar food (n=6). (F) Starvation survival assay of control (w1118) and trpγ1 males after dietary exposure to 5 mM metformin in 1% agar food (n=6).

All values are reported as means ± SEM. Comparisons between multiple experimental groups were conducted via single-factor ANOVA coupled with Scheffe’s post hoc test. Each dot indicates the distribution of individual sample values. Survival curves in E and F were estimated for each group, using a Kaplan-Meier method and compared statistically using the log-rank tests. The asterisks indicate significant differences from the controls (**P<0.01).

Dietary supply of lipase, glycerol, mixed TAG, and free FA to rescue the starvation sensitivity phenotype. (A–D) Nile red staining of LDs in full gut and the magnified R2 region of adult male flies. (A) w1118, (B) trpγ1, (C) trpγ1,UAS-trpγ/trpγG4, (D) trpγ1,UAS-trpγ/trpγ1;Dh44-GAL4/+. The scale bar represents 50 µm. The arrow indicates the orientation of intestine from anterior to posterior. (E) Measurement of area of LDs in the R2 region of trpγ1 (n=10). Note that control and rescued flies have no LD. (F) Survival assays of w1118 and trpγ1 flies under starvation condition by feeding 0.1% lipase or 0.1% denatured lipase mixed into 1% agar food (n=8). (G) Starvation survival assay to measure the survival time (h) of w1118 and trpγ1 male flies after feeding 1% glycerol mixed into 1% agar food (n=4). (H) Survival assay to measure the survival time (h) of w1118 and trpγ1 flies after feeding 0.2% hexanoic acid (HA) supplemented into 1% agar food (n=8). (I) Survival assay to measure the survival time (h) of w1118 and trpγ1 flies after feeding 0.2% mixed (mono-, di-, and tri-) glycerides (n=4).

All values are reported as means ± SEM. Survival curves were estimated for each group, using a Kaplan-Meier method and compared statistically using the log-rank tests. (**P<0.01).

Quantitative analysis of the expression of gluconeogenic, lipogenic, and lipolytic genes and the effect of bmm expression and metformin feeding. (A) qRT-PCR analysis to measure the expression of gluconeogenic genes (fbp, pepck1), lipogenic genes (acc, desat1), and a lipolytic gene (bmm) under sated (0 h starvation) and starved (24 h starvation) conditions in w1118 and trpγ1 flies. The relative fold change in the expression of starvation-induced genes (gluconeogenic genes: fbp and pepck1; lipogenic genes: acc and desat1; and lipolytic gene: bmm) was determined in whole-body samples of male adult w1118 and trpγ1 flies by qRT-PCR. Tubulin was used as an internal control to standardize the samples. Each graph shows the number of evaluated samples (n=3). (B) Fluorescence microscopic imaging of bmm::GFP expression in the FBs of w1118 and trpγ1 under sated (0 h starvation) and starvation (24 h starvation) conditions. The scale bar represent 50 µm. (C) Quantification of intensity level of bmm::GFP in the FBs of w1118and trpγ1 under sated and starvation conditions. (D–H) Nile red staining of lipids in the FB of flies with the indicated genotypes. The scale bar represent 50 µm. (I) Measurement of area of LDs from samples D–H entailed selecting a total of 30 LDs, with the 10 largest LDs chosen from each sample for analysis (n=3). (J) qRT-PCR analysis to measure the expression of acc, desat1, and bmm from the whole body samples of flies after feeding 5 mM metformin for 1 day (n=3).

All values are reported as means ± SEM. Comparisons between multiple experimental groups were conducted via single-factor ANOVA coupled with Scheffe’s post hoc test. The asterisks indicate significant differences from the controls (*P<0.05, **P<0.01).

Functional analysis of Dh44, Dh44R1, and Dh44R2 mutants and their roles in lipid accumulation. (A) Total TAG level (µg TAG/mg fly) measurement in whole body extracts from control (w1118), Dh44Mi, Dh44R1Mi, and Dh44R2Mi mutants (n=5). (B–E) Nile red stains of the fat body from w1118, Dh44Mi, Dh44R1Mi, and Dh44R2Mi respectively. Scale bars represent 50 µm. (F) Area of LDs in each indicated genotype (n=3). (G) The protein (µg protein/mg fly) measurement in the whole-body extracts from control (w1118) and Dh44Mi, Dh44R1Mi, and Dh44R2Mi mutants (n=4). (H) Quantification (qRT-PCR) of lipolytic gene (bmm) expression level in the fat body of w1118, Dh44Mi, Dh44R1Mi, and D44R2Mi flies under sated and starved conditions (n=3). (I) Total TAG level measurement in whole body extracts from the indicated genotypes (n=4). (J–M) Nile red stains of the fat body from (J) w1118, (K) trpγ1 (L) trpγ1,UAS-trpγ1/trpγ1,Dh44R1-GAL4, and (M) trpγ1,UAS-trpγ1/trpγ1,Dh44R2-GAL4. (N) Area of LDs in each indicated genotype (n=3). Scale bar represent 50 µm. (O-P) Expression of Dh44R2 in the brain and intestine. (O) Expression of Dh44R2 in the subesophageal zone (SEZ) of the brain. Scale bars represent 50 µm. (P) Expression of Dh44R2 in the intestine. Scale bars represent 200 µm in full intestine and 500 µm in magnified form.

Means ±SEMs. Single factor ANOVA with Scheffe’s analysis was used as a post hoc test to compare multiple sets of data. The asterisks indicate significance from control (*P < 0.05, **P < 0.01). Each dot indicates distribution of individual sample value.