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1.
Front Physiol ; 14: 1201670, 2023.
Article in English | MEDLINE | ID: mdl-37469565

ABSTRACT

The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial ß-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10 days later, but no changes were observed in the flight muscle. ß-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.

2.
Front Insect Sci ; 2: 885172, 2022.
Article in English | MEDLINE | ID: mdl-38468769

ABSTRACT

Rhodnius prolixus is an obligatory hematophagous insect, vector of Chagas disease. After blood meal, lipids are absorbed, metabolized, synthesized, and accumulated in the fat body. When necessary, stored lipids are mobilized, transported to other organs, or are oxidized to provide energy. Mitochondrial ß-oxidation is a cyclic conserved pathway, where degradation of long-chain fatty acids occurs to contribute to cellular energetic demands. Three of its reactions are catalyzed by the mitochondrial trifunctional protein (MTP), which is composed by hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunits alpha and beta (HADHA and HADHB, respectively). Here, we investigated the role of HADHA in lipid metabolism and reproduction of Rhodnius prolixus females. The expression of HADHA gene (RhoprHadha) was determined in the organs of starving adult insects. The flight muscle and ovary had higher expression levels when compared to the anterior and posterior midguts or the fat body. RhoprHadha gene expression was upregulated by blood meal in the flight muscle and fat body. We generated insects with RNAi-mediated knockdown of RhoprHadha to address the physiological role of this gene. RhoprHadha deficiency resulted in higher triacylglycerol content and larger lipid droplets in the fat body during starvation. After feeding, lifespan of the knockdown females was not affected, but they exhibited a decrease in oviposition, although hatching was the same in both groups. Silenced females showed lower forced flight capacity than the control ones, and their fat bodies had lower gene expression levels of Brummer lipase (RhoprBmm) and long-chain acyl-CoA synthetase 2 (RhoprAcsl2). Taken together, these findings indicate that HADHA is important to guarantee successful reproduction and efficient mobilization of lipid stores during starvation and flight.

3.
Article in English | MEDLINE | ID: mdl-33160077

ABSTRACT

Rhodnius prolixus, a vector of Chagas disease, is a hematophagous insect that feeds exclusively on blood. Each blood meal is digested within the first fourteen days after feeding, providing substrates for lipid synthesis for storage and egg production. These events are precisely regulated and emerging evidence points to a key function of insulin-like peptides (ILPs) in this control. Here we investigated the role of insulin receptor in the regulation of nutrient metabolism in fed adult females. The expression of insulin receptor (RhoprIR) gene was determined in adult organs, and it was highest in ovaries and previtellogenic follicles. We generated insects with RNAi-mediated knockdown of RhoprIR to address the physiological role of this receptor. RhoprIR deficiency improved longevity and reduced triacylglycerol storage in the fat body, whereas blood digestion remained unchanged for seven days after blood meal. The lower lipid content was attributable to decreased de novo lipogenesis as well as reduced incorporation of hemolymph-derived fatty acids into newly synthesized lipids within this organ. Consistent with that, fat bodies from RhoprIR-deficient insects exhibited decreased gene expression levels of lipophorin receptor (RhoprLpR), glycerol-3-phosphate acyltransferase 1 and 4 (RhoprGpat1 and RhoprGpat4), and carnitine palmitoyltransferase 1 (RhoprCpt1). Although hemolymph lipid profile was not affected by RhoprIR disruption, the concentration of circulating vitellogenin was increased. In line with these changes, RhoprIR-deficient females exhibited smaller ovaries and a marked reduction in oviposition. Taken together, these findings support a key role of insulin receptor in nutrient homeostasis, lipid synthesis and egg production following a blood meal.


Subject(s)
Insect Proteins/deficiency , Insect Vectors/physiology , Oogenesis/genetics , Receptor, Insulin/deficiency , Rhodnius/physiology , Animals , Blood , Chagas Disease/parasitology , Chagas Disease/transmission , Fat Body/metabolism , Feeding Behavior , Female , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Hemolymph/chemistry , Humans , Insect Proteins/genetics , Insect Vectors/parasitology , Lipid Droplets/metabolism , Lipogenesis/physiology , Models, Animal , Ovary/metabolism , Rabbits , Receptor, Insulin/genetics , Rhodnius/parasitology , Triglycerides/analysis , Triglycerides/metabolism
4.
Gene ; 596: 27-44, 2017 Jan 05.
Article in English | MEDLINE | ID: mdl-27697616

ABSTRACT

The kissing bug Rhodnius prolixus is both an important vector of Chagas' disease and an interesting model for investigation into the field of physiology, including lipid metabolism. The publication of this insect genome will bring a huge amount of new molecular biology data to be used in future experiments. Although this work represents a promising scenario, a preliminary analysis of the sequence data is necessary to identify and annotate the genes involved in lipid metabolism. Here, we used bioinformatics tools and gene expression analysis to explore genes from different genes families and pathways, including genes for fat breakdown, as lipases and phospholipases, and enzymes from ß-oxidation, fatty acid metabolism, and acyl-CoA and glycerolipid synthesis. The R. prolixus genome encodes 31 putative lipase genes, including 21 neutral lipases and 5 acid lipases. The expression profiles of some of these genes were analyzed. We were able to identify nine phospholipase A2 genes. A variety of gene families that participate in fatty acid synthesis and modification were studied, including fatty acid synthase, elongase, desaturase and reductase. Concerning the synthesis of glycerolipids, we found a second isoform of glycerol-3-phosphate acyltransferase that was ubiquitously expressed throughout the organs. Finally, all genes involved in fatty acid ß-oxidation were identified, but not a long-chain acyl-CoA dehydrogenase. These results provide fundamental data to be used in future research on insect lipid metabolism and its possible relevance to Chagas' disease transmission.


Subject(s)
Insect Proteins/genetics , Lipid Metabolism/genetics , Rhodnius/genetics , Rhodnius/metabolism , Acyl Coenzyme A/genetics , Acyl Coenzyme A/metabolism , Acyl-CoA Dehydrogenase, Long-Chain/genetics , Aldehyde Oxidoreductases/genetics , Animals , Fatty Acids/genetics , Fatty Acids/metabolism , Female , Gene Expression Regulation , Genome, Insect , Insect Proteins/metabolism , Lipase/genetics , Male , Multigene Family , Oxidation-Reduction , Phospholipases A2/genetics
5.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(3): 324-336, 2017 Mar.
Article in English | MEDLINE | ID: mdl-27956137

ABSTRACT

Glycerol-3-phosphate acyltransferases (GPAT) catalyze the initial and rate-limiting step for the de novo synthesis of triacylglycerol (TAG). Four mammalian GPAT isoforms have been identified: the mitochondria-associated GPAT1 and 2, and the endoplasmic reticulum (ER)-associated GPAT3 and 4. In the insect Rhodnius prolixus, a vector of Chagas' disease, we previously predicted a mitochondrial-like isoform (RhoprGPAT1) from genomic data. In the current study, we clone the RhoprGPAT1 coding sequence and identify an ER-associated GPAT (RhoprGPAT4) as the second isoform in the insect. RhoprGPAT1 contributes 15% of the total GPAT activity in anterior midgut, 50% in posterior midgut and fat body, and 70% in the ovary. The RhoprGpat1 gene is the predominant transcript in the midgut and fat body. To evaluate the physiological relevance of RhoprGPAT1, we generate RhoprGPAT1-deficient insects. The knockdown of RhoprGpat1 results in 50% and 65% decrease in TAG content in the posterior midgut and fat body, respectively. RhoprGpat1-deficient insects also exhibits impaired lipid droplet expansion and a 2-fold increase in fatty acid ß-oxidation rates in the fat body. We propose that the RhoprGPAT1 mitochondrial-like isoform is required to channel fatty acyl chains towards TAG synthesis and away from ß-oxidation. Such a process is crucial for the insect lipid homeostasis.


Subject(s)
Fat Body/metabolism , Fatty Acids/metabolism , Glycerol-3-Phosphate O-Acyltransferase/metabolism , Insecta/metabolism , Rhodnius/metabolism , Triglycerides/metabolism , Animals , Endoplasmic Reticulum/metabolism , Lipid Droplets/metabolism , Mitochondria/metabolism , Oxidation-Reduction
6.
Biochim Biophys Acta ; 1861(7): 650-62, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27091636

ABSTRACT

Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 µM), but different Km for arachidonic acid (0.5 and 6 µM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid ß-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for ß-oxidation in the fat body, and is also required for normal reproduction.


Subject(s)
Coenzyme A Ligases/genetics , Fat Body/metabolism , Fatty Acids/metabolism , Oogenesis/genetics , Rhodnius/genetics , Triglycerides/biosynthesis , Amino Acid Sequence , Animals , Coenzyme A Ligases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Gene Expression Regulation , Gene Knockdown Techniques , Insect Proteins , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Oxidation-Reduction , Phylogeny , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reproduction/genetics , Rhodnius/classification , Sequence Alignment , Transcription, Genetic , Triazenes , Zygote/metabolism
7.
J Biol Chem ; 291(25): 12917-29, 2016 Jun 17.
Article in English | MEDLINE | ID: mdl-27129262

ABSTRACT

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.


Subject(s)
Epithelial-Mesenchymal Transition , Protein Processing, Post-Translational , Adenosine Triphosphate/metabolism , Biosynthetic Pathways , Cell Line, Tumor , Enzyme Induction , Glucose/metabolism , Glycogen/metabolism , Glycosylation , Hexosamines/biosynthesis , Humans , Lactic Acid/metabolism , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Pyruvic Acid/metabolism , Transforming Growth Factor beta/physiology
8.
Insect Biochem Mol Biol ; 69: 51-60, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26163435

ABSTRACT

Adipokinetic hormone (AKH) has been associated with the control of energy metabolism in a large number of arthropod species due to its role on the stimulation of lipid, carbohydrate and amino acid mobilization/release. In the insect Rhodnius prolixus, a vector of Chagas' disease, triacylglycerol (TAG) stores must be mobilized to sustain the metabolic requirements during moments of exercise or starvation. Besides the recent identification of the R. prolixus AKH peptide, other components required for the AKH signaling cascade and its mode of action remain uncharacterized in this insect. In the present study, we identified and investigated the expression profile of the gene encoding the AKH receptor of R. prolixus (RhoprAkhr). This gene is highly conserved in comparison to other sequences already described and its transcript is abundant in the fat body and the flight muscle of the kissing bug. Moreover, RhoprAkhr expression is induced in the fat body at moments of increased TAG mobilization; the knockdown of this gene resulted in TAG accumulation both in fat body and flight muscle after starvation. The inhibition of Rhopr-AKHR transcription as well as the treatment of insects with the peptide Rhopr-AKH in its synthetic form altered the transcript levels of two genes involved in lipid metabolism, the acyl-CoA-binding protein-1 (RhoprAcbp1) and the mitochondrial glycerol-3-phosphate acyltransferase-1 (RhoprGpat1). These results indicate that the AKH receptor is regulated at transcriptional level and is required for TAG mobilization under starvation. In addition to the classical view of AKH as a direct regulator of enzymatic activity, we propose here that AKH signaling may account for the regulation of nutrient metabolism by affecting the expression profile of target genes.


Subject(s)
Insect Hormones/metabolism , Oligopeptides/metabolism , Pyrrolidonecarboxylic Acid/analogs & derivatives , Receptors, Cell Surface/genetics , Rhodnius/genetics , Rhodnius/metabolism , Triglycerides/metabolism , Animals , Fat Body/metabolism , Flight, Animal , Food Deprivation , Gene Expression Regulation , Muscle, Skeletal/metabolism , Pyrrolidonecarboxylic Acid/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction
9.
Proc Natl Acad Sci U S A ; 112(48): 14936-41, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26627243

ABSTRACT

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.


Subject(s)
Adaptation, Physiological/genetics , Chagas Disease , Host-Parasite Interactions/genetics , Insect Vectors , Rhodnius , Trypanosoma cruzi/physiology , Animals , Base Sequence , Gene Transfer, Horizontal , Humans , Insect Vectors/genetics , Insect Vectors/parasitology , Molecular Sequence Data , Rhodnius/genetics , Rhodnius/parasitology , Wolbachia/genetics
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