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. 2020 Oct 27;9:e56969. doi: 10.7554/eLife.56969

Figure 4. Pi3K/Akt1/TOR signaling acts downstream of PvR in the oenocytes to regulate adiposity.

(A) p4EBP staining (Red) of the oenocytes from control (musts = muscle-Gal4Gal80ts) and flies with muscle-specific knockdown of pvf1 (musts>pvf1-i1). Muscle-specific loss of pvf1 leads to a significant decrease (p<0.01) in p4EBP levels in the oenocytes. N = 10 animals, student t-test, ** denotes p≤0.01, error bars = SEM. (B) p4EBP staining (Green) of oenocytes from control flies (oenots = oenocyte-Gal4Gal80ts) and flies with oenocyte-specific over-expressing tsc1/tsc2 (oenots>tsc1,tsc2), pvrDN (oenots>pvrDN) or inrDN (oenots>inrDN). (C) Quantification of p4EBP staining intensity in the oenocytes for samples shown in Figure 3C. Oenocyte-specific over-expression of tsc1/tsc2 (oenots>tsc1,tsc2) and pvrDN (oenots>pvrDN) led to a significant reduction in p4EBP levels (p<0.0001). Over-expression of inrDN (oenots>inrDN), however, does not affect p4EBP levels significantly. N = 6/7 animals, Student t-test, **** denotes p≤0.0001, error bars = SEM. (D) BODIPY staining showing neutral lipid accumulation in the adipose tissue and oenocytes of control males (oenots) and males with oenocyte-specific over-expression of either pvrDN (oenots>pvrDN) or over-expression of pvrDN along with tsc2 knockdown (oenots>pvrDN,tsc2-i). Flies over-expressing pvrDN in the oenocytes along with an empty UAS construct (oenots>pvrDN,UAS) serve as an additional control to account for any effect of Gal4 dilution on the obesity phenotype. (E) Mean lipid droplet size (≥10 microns in diameter) in the adipose tissue of flies shown in (D). Oenocyte-specific knockdown of tsc2 along with pvrDN over-expression (oenots>pvrDN,tsc2-i) significantly rescues the obesity phenotype observed flies with oenocyte-specific over-expression of pvrDN (p<0.001). N = 5 animals, One-way ANOVA followed by Tukey’s HSD test, *** denotes p≤0.001, **** denotes p≤0.0001, error bars = SEM.

Figure 4.

Figure 4—figure supplement 1. Oenocyte-specific activation of TOR signaling does not affect lipid accumulation.

Figure 4—figure supplement 1.

(A) BODIPY staining showing neutral lipid accumulation in the adipose tissue and oenocytes of males with oenocyte-specific over-expression of either pvrDN (oenots>pvrDN) or over-expression of pvrDN along with over-expression of a constitutively active form of rheb (oenots>pvrDN; rhebAV4). oenots = oenocyte-Gal4Gal80ts. (B) Mean lipid droplet size (≥10 microns in diameter) in the adipose tissue of flies shown in (A). Oenocyte-specific over-expression of rehbAV4 along with pvrDN over-expression (oenots>pvrDN; rhebAV4) significantly rescues the obesity phenotype observed flies with oenocyte-specific over-expression of pvrDN (p<0.001). N = 6 animals, One-way ANOVA followed by Tukey’s HSD test, *** denotes p≤0.001, **** denotes p≤0.0001, error bars = SEM. (C) BODIPY staining showing neutral lipid accumulation in the adipose tissue and oenocytes of control males (oenots and tsc2-i) and males with oenocyte-specific tsc2 knockdown (oenots>tsc2-i). (D) Mean lipid droplet size (≥10 microns in diameter) in the adipose tissue of flies shown in (C). Oenocyte-specific knockdown of tsc2 showed a mild increase in mean lipid droplet size only compared to once control. N = 6 animals, One-way ANOVA followed by Tukey’s HSD test, error bars = SEM.
Figure 4—figure supplement 2. InR signaling or oenocyte size does not affect PvR signaling-mediated effects on systemic lipid accumulation.

Figure 4—figure supplement 2.

(A) BODIPY staining showing neutral lipid accumulation in the adipose tissue and oenocytes of control males (oenots = oenocyte-Gal4Gal80ts and inrDN = UAS-inrDN/+) and males with oenocyte-specific overexpression of pvrDN either by itself (oenots>pvrDN) or along with overexpression of inrDN (oenots>pvrDN; inrDN). Flies over-expressing pvrDN in the oenocytes along with an empty UAS construct (oenots>pvrDN,UAS) serve as an additional control to account for any effect of Gal4 dilution on the obesity phenotype. (B) Mean lipid droplet size (≥10 microns in diameter) in the adipose tissue of flies shown in (A). Oenocyte-specific over-expression inrDN along with pvrDN (oenots>pvrDN; inrDN) rescues lipid accumulation in the oenocytes. However, the flies still show a significant increase in lipid droplet size in the adipose tissue compared to controls. N = 6 animals, One-way ANOVA followed by Tukey’s HSD test, *** denotes p≤0.001, **** denotes p≤0.0001, error bars = SEM. (C) DAPI staining showing the density of nuclei in the oenocytes of control flies (oenots and akt1-i = akt1-i/+) and flies lacking Akt signaling (oenotye-specific knockdown of akt1: oenots>akt1-i), TOR signaling (oenotye-specific overexpression of tsc1 and tsc2: oenots>tsc1, tsc2) and InR signaling (oenocyte-specific overexpression of inrDN: oenots>inrDN). Loss of Akt signaling and InR signaling leads to a significant decrease in oenocyte size as reflected by an increase in oenocyte density per unit area. N = 5/6 animals (about three oenocyte clusters were measured per animal), One-way ANOVA followed by Tukey’s HSD test, *** denotes p≤0.001, **** denotes p≤0.0001, error bars = SEM. Representative images of adipose tissue lipid droplets for each genotype is also shown. Note that the images were drawn from experiments already reported elsewhere in the manuscript and quantification of lipid droplet size is already reported in Figure 3C, E and Figure 4—figure supplement 3B.
Figure 4—figure supplement 3. Additional control showing baseline lipid droplet size in the adipose tissue and lipid accumulation in the oenocytes.

Figure 4—figure supplement 3.

(A) BODIPY staining showing neutral lipid accumulation in the adipose tissue and oenocytes of control males flies. oenots = oenocyte-Gal4Gal80ts. (B) Mean lipid droplet size (≥10 µm in diameter) in the adipose tissue of flies shown in (A). N = 6 animals, One-way ANOVA followed by Tukey’s HSD test, *** denotes p≤0.001, **** denotes p≤0.0001, error bars = SEM.