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. 2024 Aug 13;22(1):171.
doi: 10.1186/s12915-024-01973-3.

Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development

Yan-Xue Li #  1 , Qiao Yan #  1 , Tian-Wen Liu  1 , Jin-Xing Wang  1 , Xiao-Fan Zhao  2
Affiliations

Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development

Yan-Xue Li et al. BMC Biol. .

Abstract

Background: Free fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study.

Results: This study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling.

Conclusions: The differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.

Keywords: 20-Hydroxyecdysone; Forkhead box O; Juvenile hormone; Lipase; Methoprene-tolerant 1.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Phylogenetic tree of lipases in H. armigera. Pink font: lipases in the H. sapiens genome; black font: lipases in the H. armigera genome. Inner red ring: lipases related to human pancreatic lipase (PTL); inner blue ring: lipases related to lipases in human adipose tissue; inner yellow ring: lipases related to human acid lipases. Outer orange ring: 32 lipases are highly expressed in the midgut at the feeding stage; outer green ring: 5 lipases are highly expressed in the fat body at the metamorphosis stage. Red dot: lipase member H-A-like (LHA-like). Blue dot: monoacylglycerol lipase ABHD12-like (ABHD12-like). The phylogenetic tree was made by MEGA 7
Fig. 2
Fig. 2
The expression profiles and hormone induction of Lha-like and Abhd12-like. A The mRNA levels of Lha-like in the epidermis, midgut, fat body, and brain were measured using qRT‒PCR. 5F: fifth instar feeding larvae; 5M: fifth instar molting larvae; 6th-6 h to 120 h represent sixth instar larvae at different stages. P2 to P8: 2- to 8-day-old pupae. F: feeding; M: molting; MM: metamorphosis molting; P: pupae. n = 3. B The expression of Lha-like in the midgut under stimulation with different concentrations of JH III for 12 h. DMSO was used as the control. n = 9. C Time course of Lha-like expression in the midgut after JH III (500 ng/larva) induction. n = 9. D qRT‒PCR analysis of Abhd12-like mRNA levels in the epidermis, midgut, fat body, and brain. n = 3. E The expression of Abhd12-like in the fat body under stimulation with different concentrations of 20E for 12 h. DMSO was used as the control. n = 9. F Time course of Abhd12-like expression in the fat body after 20E (500 ng/larva) induction. n = 9. The error bar represents the mean ± SD of three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.01 (two-tailed Student's t test). n indicates the number of data points and Additional file 2 for individual data values
Fig. 3
Fig. 3
JH III upregulated the expression of Lha-like via MET1. A Alignment of JHRE of Et from A. aegypti and Lha-like from H. armigera. B The expression of Lha-like was measured after Met1 knockdown in the midgut, followed by stimulation with JH III (500 ng/larva) for 12 h. The fifth instar 20 h larvae were injected with the dsRNA three times, 24 h apart, and JH III was injected after 12 h the third dsRNA injection. n = 9. C Schematic diagram of the pLha-LUCI-GFP-His reporter plasmid. D Western blotting showed the difference in reporter gene expression levels of pLha-LUCI-GFP-His under different treatments. ACTB was used as a loading control. The ratio of the reporter protein to ACTB band density was statistically calculated by ImageJ. The mRNA levels of Met1 in HaEpi cells under different treatments were measured by qRT‒PCR. Cells were incubated with 2 μM JH III for 24 h. n = 3. E Transcriptional activity was detected by dual-luciferase reporter assay. The MET1-RFP-His or RFP-His with pLha-LUCI-GFP-His vector and the reference pRL-TK vector were cotransfected into HaEpi cells. The pLha-LUCI-GFP-His vector carries firefly luciferase. The reference pRL-TK vector carries renilla luciferase. The relative luciferase activity was defined as the reporter firefly luciferase level/the reference renilla luciferase level. Fluc: Firefly luciferase; Rluc: Renilla luciferase. n = 3. F ChIP assay showing that JH III upregulated Lha-like expression via MET1 binding to JHRE, as measured by qRT‒PCR. Primer JHRE is the sequence containing JHRE. The primer sequence of Lha-like was used as a non-JHRE control targeting the Lha-like open reading frame (ORF). n = 3. The error bar represents the mean ± SD of three biological replicates. ***p < 0.01 (two-tailed Student's t test). The comparison between multiple sets of data was analyzed by ANOVA. The different lowercase letters show significant differences. n indicates the number of data points and Additional file 2 for individual data values
Fig. 4
Fig. 4
EcR and FOXO were involved in 20E up-regulation of Abhd12-like expression. A Sequence alignment of FOXOBE in the 5′ upstream sequence of Brz7 and Abhd12-like from H. armigera. B The mRNA levels of Abhd12-like after knockdown of Foxo in the fat body. The sixth instar 6 h larvae were injected with dsFoxo or dsGfp three times at intervals of 24 h, and 20E was injected after the last dsRNA injection 12 h. C Sequence alignment of EcRE in the 5′ upstream sequence of Hr3 and Abhd12-like from H. armigera. D The mRNA levels of Abhd12-like after knockdown of Ecr in the fat body. The sixth instar 6 h larvae were injected with the dsRNA three times, 24 h apart, and 20E was injected after the third dsRNA injection 12 h. The error bar represents the mean ± SD of three independent experiments. Statistically significant differences were calculated using two‑tailed Student's t tests (*p < 0.05, **p < 0.01). n = 9 for all graphs and see Additional file 2
Fig. 5
Fig. 5
20E upregulated the expression of Abhd12-like via EcR and FOXO. A Western blotting showed the difference in protein levels of LUCI-GFP-His under different treatments, and the mRNA levels of Foxo in HaEpi cells under different treatments were measured by qRT‒PCR. Cells were incubated with 2 μM 20E for 24 h. B Transcriptional activity of the pAbhd12-LUCI-GFP-His reporter plasmid was detected by dual-luciferase reporter assay. The FOXO-RFP-His or RFP-His with pAbhd12-LUCI-GFP-His plasmid and the reference pRL-TK plasmid were cotransfected into HaEpi cells. The pAbhd12-LUCI-GFP-His vector carries firefly luciferase. The reference pRL-TK vector carries renilla luciferase. The relative luciferase activity was defined as the Fluc level/Rluc level. Fluc: Firefly luciferase; Rluc: Renilla luciferase. C ChIP assay showing that 20E promoted Abhd12-like expression via FOXO binding to FOXOBE, as measured by qRT‒PCR. The FOXOBE1 primer is the sequence containing FOXOBE1. The FOXOBE2 primer is the sequence containing FOXOBE2. The Abhd12-like primer was used as a non-FOXOBE control targeting the Abhd12-like ORF. D The expression of LUCI-GFP-His was analyzed by western blotting under different conditions. ACTB was used as the loading control. Cells were incubated with 2 μM 20E for 24 h. The ratio of the reporter protein to ACTB band density was statistically calculated by ImageJ. The error bar represents the mean ± SD of the three biological replicates. ANOVA was used to analyze multiple sets of data. Different letters indicate statistically significant differences (p < 0.05). n = 3 for all graphs and see Additional file 2
Fig. 6
Fig. 6
Knockdown of Lha-like delayed pupation and decreased pupae weight. A The interference efficiency in the midgut after the fourth dsRNA injection. n = 9. B FFA levels in the hemolymph after the fourth dsRNA injection 24 h. n = 3 C The TAG levels in the fat body after fourth dsRNA injection 24 h. n = 3. D Nile red staining showing LDs after knockdown of Lha-like, observed for 66 h and 90 h after the first dsRNA injection. The scale bar represents 50 μm. E The brain morphology, after knockdown of Lha-like, was observed for 66 h, 90 h, and 186 h after the first injection of dsRNA. The bars represent 200 μm. Back view. Directions: A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. F Quantification of the data in (E). n = 3. G Phenotypes after Lha-like knockdown (2 μg/larva at fifth instar 20 h, four times at a 24 h interval). Scale bar: 1 cm. The phenotype images of pupation on time and normal pupae exhibit 2-day-old pupae. The phenotype image of delayed pupation exhibits sixth instar 166 h larva. The phenotype image of small pupae exhibits 1-day-old pupae. H Ratio of phenotypes of delayed pupation. n = 30 × 3 (three replicates, 30 larvae each). I Statistics of pupation time from sixth instar 0 h to pupae. The time point of the fifth instar larvae transforming into the sixth instar larvae after molting, representing the sixth instar 0 h (6th-0 h). 6th-0 h larvae indicate that larvae have just entered the sixth instar stage. In the dsGfp group, n for each experiment was 27, 26, and 27 respectively; in the dsLha-like group, n for each experiment was 25, 26, and 27 respectively. J The percentage of small pupae. n = 30 × 3. K Statistics of average pupae weight in the dsLha-like and dsGfp groups. In the dsGfp group, n for each experiment was 27, 26, and 27 respectively; in the dsLha-like group, n for each experiment was 25, 26, and 27 respectively. The error bar represents the mean ± SD of the three biological replicates, and Student's t test analysis indicated significant differences (*p < 0.05, **p < 0.01, and ***p < 0.001). Multiple sets of data were compared by analysis of variance (ANOVA), and different letters represent significant differences (p < 0.05). n indicates the number of data points and Additional file 2 for individual data values
Fig. 7
Fig. 7
Injection of dsAbhd12-like led to delayed pupation. A The interference efficiency of Abhd12-like in the fat body after the fourth dsRNA injection. n = 9. B FFA levels in the hemolymph after the fourth dsRNA injection 24 h. n = 3. C The TAG levels in the fat body after the fourth dsRNA injection 24 h. n = 3. D Nile red staining showing LDs after Abhd12-like knockdown, observed 90 h and 138 h after the first dsRNA injection. The scale bar represents 50 μm. E Brain morphology observed after the knockdown of Abhd12-like and the first injection of dsRNA for 90 h, 138 h, and 186 h. The scale bars represent 200 μm. Back view. Directions: A, anterior; D, dorsal; L, lateral; P, posterior; V, ventral. F Quantification of the data in (E). n = 3. G Phenotypes after Abhd12-like knockdown (2 μg/larva at sixth instar 6 h, four times at a 24 h interval). Scale bar: 1 cm. The phenotype image of pupation on time exhibits 2-day-old pupae. The phenotype image of delayed pupation exhibits sixth instar 163 h larva. H Ratio of phenotypes of delayed pupation. n = 30 × 3 (three replicates, 30 larvae each). I Statistics of pupation time from 6th-0 h to pupae. In the dsGfp group, n for each experiment was 27, 27, and 28 respectively; in the dsAbhd12-like group, n for each experiment was 25, 25, and 27 respectively. J Statistics of average pupae weight in the dsAbhd12-like and dsGfp groups. In the dsGfp group, n for each experiment was 27, 27, and 28 respectively; in the dsAbhd12-like group, n for each experiment was 25, 25, and 27 respectively. Statistical analysis was conducted using Student's t test (*p < 0.05, **p < 0.01, and ***p < 0.001) or ANOVA, and different letters represent significant differences (p < 0.05). The error bar represents the mean ± SD of the three biological replicates. n indicates the number of data points and Additional file 2 for individual data values
Fig. 8
Fig. 8
A diagram illustrating that JH and 20E regulate FFA homeostasis. JH upregulates the transcription of Lha-like via the intracellular receptor MET1. LHA-like contains signaling peptides, which are secreted to midgut to hydrolyze dietary TAG to produce hemolymph FFAs and fat body TAG (1). Whereas, 20E upregulates the expression of Abhd12-like via FOXO. ABHD12-like promotes the hydrolysis of fat body TAG to compensate the FFAs in hemolymph during metamorphosis (2), thus maintaining FFA homeostasis in larval hemolymph at the feeding and metamorphic stages for brain development (3)
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