A Drosophila model targets Eiger/TNFα to alleviate obesity-related insulin resistance and macrophage infiltration

Abstract

Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, we observed significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. We found that attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, we ameliorated both phenotypes. Our data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. Our model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease.

Keywords: Drosophila model; Adipose chronic inflammation; Eiger/TNFα signaling; Insulin resistance; Lipid metabolism; Metabolic disorders; Obesity.

Fig. 1.

Hemocyte infiltration in the fat body (FB) of larvae fed a high-fat diet (HFD) or mutants for brummer phenocopy low-grade chronic inflammation associated with obesity in humans. (A) Percentage of hemocytes infiltrating the FB of wild-type (w¹¹¹⁸) animals fed an HFD or corn meal (CM) and in animals mutant for brummer (bmm¹) measured at 5 days after egg laying (AEL). (B) Hemocytes in the FB measured at 5 days AEL in control P0206-w¹¹¹⁸ and in P0206-NOC1, herein called OBL (see Fig. S1), at 5 and 12 days AEL. Quantification in the FB of hemocytes marked by Hml-DsRed expressed as a percentage of DsRed cells from the total number of cells quantified by nuclear staining using Hoechst. (C) Quantification of apoptosis in cells of the FB analyzed using anti-caspase 3 antibody in OBL animals at 12 days AEL. The dots in A and B indicate the number of experiments (at least ten animals were used for each time point and genotype), and data in C represent one of two experiments done, with the dots representing the number of animals used in the experiment. (D,E) Confocal images of FB from OBL animals at 12 days AEL showing the hemocytes marked by Hml-DsRed expression (Makhijani et al., 2011). (F-H) Confocal images of FB from w¹¹¹⁸ animals at 5 days (F) and of FB from OBL animals at 12 days AEL (G,H), showing the hemocytes, stained using anti-SPARC antibody [panel G is from Valenza et al. (2018); image reproduced under the terms of the CC-BY 4.0 license]. F highlights the crown-like structure formed by the hemocytes surrounding a fat cell. The inset in H shows hemocytes (arrow) with the typical macrophage-like morphology at higher magnification. Scale bars: 50 µm (D,E,H), 25 µm (F,G). Statistical analysis was performed using unpaired two-tailed Student's t-test. Error bars indicate s.d. ns, not significant; **P<0.01, ***P<0.001 and ****P<0.0001.

Fig. 2.

OBL animals increase fatty acid synthesis and accumulate triglycerides (TGs) and free fatty acids (FFAs), accumulating lipids in FB and pericardial cells. (A-C) qRT-PCT showing the level of brummer, Fatty acid synthase, and Plin2 mRNA in whole larvae of the indicated genotype collected 2, 3 and 5 days AEL. (D,E) Photographs of control w¹¹¹⁸ or OBL larvae at 5 or 12 days AEL (D) and relative weight (E). (F) TG content in whole larvae at the indicated time (days AEL). (G) FFAs from whole larvae collected at the indicated time (days AEL). (H) Lipidomic analysis of fats from whole larvae of the indicated genotype at 2, 5 and 12 days AEL. (I) Confocal images of cells from the FB of larvae of the indicated genotype; fat is stained with Nile Red and nuclei with Hoechst. (J) Size analysis of the cells from the FB of animals of the indicated genotype; analysis was performed using ImageJ from confocal images. (K) Schematic of the heart tube (cardiac tube) in embryos, showing the relative cell types. The heart tube contains openings called muscular ostia (Ostia) surrounded by muscle cells; the ostia function as valves to allow the entry of hemolymph into the heart tube. As the heart tube contracts, the hemolymph is propelled forward, filtered and pushed out of the heart tube into the aorta, the main artery of the circulatory system in flies. The cardiovascular valve (CV) ensures the unidirectional hemolymph flow from the heart tube to the aorta, preventing backflow. Myocardial cells (MC) and pericardial cells (PC) are shown, and expression is also detected in the lymph glands (lg). Anterior is to the left. (L) Photograph of the cardiac tube of a third-instar (L3) larva; Hand-GFP expression visualizes the cardiac cells. (M) Analysis of cardiac contraction in larvae of the indicated genotype at 5 days AEL. Dots in the graphs show the number of experiments performed. (N,O) Higher magnifications of the cardiac cells from third-instar larvae in M; cells are marked by the expression of Hand-GFP and stained for lipid contents using Nile Red. Scale bars: 1 mm (D), 100 µm (L), 50 µm (I) and 20 µm (N,O). Statistical analysis was performed using unpaired two-tailed Student's t-test, except for data in H, for which P-values were calculated using one-way ANOVA with Tukey multiple comparisons. Error bars indicate s.d. ns, not significant; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Fig. 3.

OBL larvae are hyperglycemic, have impaired systemic insulin signaling and exhibit insulin resistance in the FB. (A) Hemolymph glucose analysis from animals of the indicated genotypes. At least ten larvae were used for each genotype. (B) DILP2 expression was measured by immunofluorescence using anti-DILP2 antibody in the insulin-producing cells (IPCs) from third-instar larvae raised in corn meal food (fed) or starved for 24 h in PBS (stv). The integrated density of fluorescence was quantified using confocal images as described previously (Parisi et al., 2013). P-values were calculated using unpaired two-tailed Student's t-test from n=10 larvae for each time point and genotype and from at least four independent experiments. Error bars indicate s.d. ns, not significant; *P<0.05, and ****P<0.0001. (C-H) Confocal images of brains from larvae of the indicated age and genotypes raised in normal food (C-E) or kept overnight in PBS (F-H), showing DILP2 expression (red) in the IPCs. The inset in C shows a drawing of the larval brain indicating the IPC position. OL, optic lobe; VNC, ventral nerve cord. Scale bars: 50 µm. (I) Quantification of tGPH-GFP expression in the membrane of the fat cells from control and OBL larvae; the FBs were incubated ex vivo with 1 mM insulin for the indicated time. (J-O) Confocal images of FBs expressing the tGPH-GFP reporter from larvae in I, untreated (J-L) treated with insulin for 10 min (M-O). Scale bars: 50 µm. (P) Western blot of lysates from FBs of animals of the indicated age and genotype upon treatment of insulin, showing the level of phosphorylation of Akt at Ser505. Vinculin was used as a control for loading.

Fig. 4.

Eiger signaling in OBL animals contributes to the infiltration of hemocytes, ROS production and insulin resistance. (A-C) Confocal images showing Eiger-GFP expression in the FB from animals of the indicated age and genotype. Scale bars: 50 µm. (D) Quantification of Eiger-GFP expression was measured as integrated density in the FB of animals at the indicated time and genotype. (E-G) Confocal images of FB showing the presence of hemocytes expressing Hml-DsRed, and of Eiger-GFP. The inset in F highlights hemocytes (red) near FB cells expressing many vesicles containing Eiger-GFP. In G, aggregates of hemocytes (red) co-expressing Eiger-GFP are surrounding cells of the FB from OBL animals at 12 days AEL. Scale bars: 50 µm. (H) Quantification of Eiger-GFP measured in the hemocytes migrating in the FB; GFP is quantified as integrated density in FBs of animals at the indicated time and genotype. (I) Quantification of the number of hemocytes infiltrating the FB from OBL animals at 5 and 12 days AEL or in OBL animals heterozygous for egr¹, egr^1AG or grnd at 12 days AEL. (J) Dihydroethidium (DHE) staining in FBs. Scale bars: 50 µm. (K) Quantification of DHE staining measured as integrated density in the FBs from animals of the indicated days and genotype. (L) Quantification of the number of hemocytes infiltrating the FB in animals treated with anthocyanins (ACN). (M) Western blot using lysates from dissected FBs from animals of the indicated genotypes showing the level of Akt Ser505 phosphorylation upon treatment with insulin for the indicated time. Actin was used as a control for loading; western blot analysis was repeated twice using 20 FBs for each time point. (N,O) Quantification of Eiger-GFP expression (N) and the number of hemocytes in the FB (O), without metformin (−M) and after feeding the larvae 1 mM metformin (+M). P-values were calculated using unpaired two-tailed Student's t-test from n=10 larvae for each time point and genotype. Error bars indicate s.d. ns, not significant; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Fig. 5.

Spaetzle and Relish are involved in the mechanisms controlling hemocyte infiltration in the FB in OBL animals. (A-C) Confocal images of FB, showing activation of the reporter for Diptericin (Dpt-lacZ) at 12 days AEL in the FB of OBL animals. Scale bars: 20 µm. (D-F) Confocal images of FB showing the expression of GFP-positive cells representing activation of the reporter for Drosomycin using the Drs-GFP reporter line in control and OBL animals at the indicated time of development. Nuclei are stained with Hoechst. Scale bars: 50 µm. (G,H) Graphs indicating the percentage of hemocytes in the FB from the indicated genotypes. Dots indicate the independent experiments in which at least ten animals were used for each time and genotype. Statistical analysis was performed using unpaired two-tailed Student's t-test. Error bars indicate s.d. *P<0.05, **P<0.01.