Adipokinetic hormone signaling determines dietary fatty acid preference through maintenance of hemolymph fatty acid composition in the cricket Gryllus bimaculatus

Abstract

Adipokinetic hormone (AKH), an analog of mammalian glucagon, functions in supplying the required energy by releasing lipids and carbohydrates from the fat body into the hemolymph. Our previous study showed that AKH receptor (AKHR) knockdown in the two-spotted cricket Gryllus bimaculatus decreased hemolymph lipid levels and increased its feeding frequency. To reveal underlying mechanisms by which AKH signaling modulates lipid homeostasis, we analyzed the fatty acid composition as the lipid structure in the crickets. AKH administration significantly increased the proportion of unsaturated fatty acids (USFAs) to total fatty acids with decrease of the saturated fatty acids (SFAs) in hemolymph, while these proportions were inversely changed in RNA interference-mediated AKHR-knockdowned (AKHR^RNAi) crickets. Interestingly, knockdown of hormone-sensitive lipase (Hsl) by RNAi (Hsl^RNAi) affected the proportion of USFAs and SFAs in a similar manner to that observed in AKHR^RNAi crickets. AKH administration in Hsl^RNAi crickets did not change hemolymph fatty acid composition, indicating that AKH signaling critically altered fatty acid composition in the hemolymph through Hsl. In addition, a choice assay revealed that AKHR^RNAi significantly increases the preference of USFAs. These data indicate that hemolymph lipid level and composition were modulated by AKH signaling with a complementary feeding behavior toward USFAs.

Figure 1

Effects of AKH injection on lipid levels and lipid composition in crickets. Hemolymph lipid levels in AKH-injected adult crickets (A) and fifth instar cricket nymphs (B). The lipid levels after AKH injection were compared with those of non-treated crickets. Also, the lipid levels by AKH injection were compared with those of PBS-injected crickets after 90 min. Fatty acid composition in the hemolymph of fifth instar cricket nymphs (C) and the proportions of SFAs and USFAs to total fatty acids (D). Fatty acid composition in the fat body of fifth instar cricket nymphs (E) and the proportions of SFAs and USFAs to total fatty acids in the hemolymph (F). Values are expressed as means ± SD (A; n = 5, B; n = 5–6, C; n = 5, D; n = 5, E; n = 7, F; n = 7, *P < 0.05 for Tukey’s post hoc test).

Figure 2

Effects of RNAi-mediated AKHR knockdown on lipid levels and lipid composition. The AKHR^RNAi crickets were compared with the experimental control crickets treated with dsRNA encoding DsRed2. The transcriptional level of AKHR in the fat body compared with transcript levels of elongation factor (A). Hemolymph lipid levels in AKHR^RNAi crickets (B). Fatty acid composition in the hemolymph of AKHR^RNAi crickets (C), and the proportions of SFAs and USFAs to total fatty acids (D). Fatty acid composition in the fat body of AKHR^RNAi crickets (E), and the proportions of SFAs and total USFAs (F). Values are expressed as means ± SD (A; n = 8, B; n = 6, C; n = 5, D; n = 5, E; n = 5, F; n = 5, **P < 0.01 and *P < 0.05 for Student’s t-test).

Figure 3

Effects of AKH signaling on the expression of genes involved in lipid metabolism. The relative transcriptional levels of lipogenic and lipolytic genes compared with transcriptional levels of elongation factor in AKHR^RNAi crickets (A–E). The experimental control was performed using crickets treated with dsRNA encoding DsRed2. Values are expressed as means ± SD (n = 5, ***P < 0.001 and *P < 0.05 for Student’s t-test).

Figure 4

Effects of RNAi-mediated Hsl knockdown on fatty acid composition. The fatty acid composition in the hemolymph of Hsl^RNAi crickets with or without AKH injection (A), and the proportions of SFAs and USFAs to total fatty acids (B). Values are expressed as means ± SD (A and B; n = 7). The statistical analyses were performed within the same structure (A) or within same structural group (B). Different letters indicate significant differences over Tukey’s post hoc test (P < 0.05), n.s. indicates not significant.

Figure 5

Effects of RNAi-mediated AKHR knockdown on the feeding preference. Representative feeding behaviors for 9 h (A). Colors represent the duration of time a cricket spent within a particular area with thicker red indicating longer periods of time. The amount of food intake from the USFA or SFA diets (B). PI for the USFA diet (C). Total food intake of SFA and USFA diets (D). Values are expressed as means ± SD (B,C, and D; n = 9, **P < 0.01 and *P < 0.05 for Student’s t-test).