Effects of Supplementation with NAD + Precursors on Metabolic Syndrome Parameters: A Systematic Review and Meta-Analysis.

Intracellular levels of NAD + regulate metabolism, among other ways, through enzymes that use NAD + as a substrate, capable of inducing catabolic processes, such as lipid oxidation, glucose uptake, and mitochondrial activity. In several model organisms, administering precursor compounds for NAD + synthesis increases its levels, improves lipid and glucose homeostasis, and reduces weight gain. However, evidence of the effects of these precursors on human patients needs to be better evaluated. Therefore, we carried out a systematic review and meta-analysis of randomized clinical trials that assessed the effects of NAD + precursors on Metabolic Syndrome parameters in humans. We based our methods on PRISMA 2020. Our search retrieved 429 articles, and 19 randomized controlled trials were included in the meta-analysis. We assessed the risk of bias with the Rob 2 algorithm and summarized the quality of evidence with the GRADE algorithm. Supplementation with NAD + precursors reduced plasma levels of total cholesterol and triglycerides in volunteers, but the intervention did not significantly affect the other outcomes analyzed. Three of the included articles presented a high risk of bias. The quality of evidence varied between very low and low due to the risk of bias, imprecision, and indirectness. The number of participants in outcomes other than lipidemia is still generally tiny; therefore, more clinical trials evaluating these parameters will increase the quality of the evidence. On the other hand, quality randomized studies are essential to assess better the effects of NAD + precursors on lipidemia.

Lipophorin: The Lipid Shuttle.

Insects need to transport lipids through the aqueous medium of the hemolymph to the organs in demand, after they are absorbed by the intestine or mobilized from the lipid-producing organs. Lipophorin is a lipoprotein present in insect hemolymph, and is responsible for this function. A single gene encodes an apolipoprotein that is cleaved to generate apolipophorin I and II. These are the essential protein constituents of lipophorin. In some physiological conditions, a third apolipoprotein of different origin may be present. In most insects, lipophorin transports mainly diacylglycerol and hydrocarbons, in addition to phospholipids. The fat body synthesizes and secretes lipophorin into the hemolymph, and several signals, such as nutritional, endocrine, or external agents, can regulate this process. However, the main characteristic of lipophorin is the fact that it acts as a reusable shuttle, distributing lipids between organs without being endocytosed or degraded in this process. Lipophorin interacts with tissues through specific receptors of the LDL receptor superfamily, although more recent results have shown that other proteins may also be involved. In this chapter, we describe the lipophorin structure in terms of proteins and lipids, in addition to reviewing what is known about lipoprotein synthesis and regulation. In addition, we reviewed the results investigating lipophorin's function in the movement of lipids between organs and the function of lipophorin receptors in this process.

Diet supplementation with egg yolk powder fattens the beetle Tribolium castaneum.

Insects have become essential models in studying human metabolic diseases, mainly due to their low maintenance cost and available tools. Both mutations and modified diets induce metabolic states similar to human obesity and diabetes. Here, we explore the effect of a high-calorie, high-fat diet on the metabolism of the beetle Tribolium castaneum. Supplementation of the wheat flour diet with powdered egg yolk for 3 weeks increased the total triacylglycerol and accelerated larval development. In addition, this diet increased the triacylglycerol levels of adult beetles. However, this egg yolk supplementation did not alter the larvae's total glucose levels or lipogenic capacity and ATP citrate lyase activity. The diet also did not change the expression profile of several lipid and carbohydrate metabolism genes and insulin-like peptides. Thus, we conclude that the diet supplemented with egg yolk induces increased fat without causing diabetes phenotypes, as seen in other hypercaloric diets in insects.

Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Effects of vitamin D supplementation on metabolic syndrome parameters in patients with obesity or diabetes in Brazil, Europe, and the United States: A systematic review and meta-analysis.

Plasma 25-dihydroxyvitamin D levels appear reduced in patients with obesity or type 2 diabetes, as reported in several observational studies. However, the association between these reduced hormone levels and metabolic parameters is unclear. In any case, vitamin D supplementation in patients with Metabolic Syndrome is standard. Still, the impacts of this supplementation on conditions such as glycemia, blood pressure, and lipidemia are debatable. Based on this question, we carried out a systematic review and meta-analysis of randomized clinical trials in Brazil, Europe, and the United States that analyzed the effects of vitamin D supplementation on Metabolic Syndrome parameters in patients with obesity or type 2 diabetes. Our search yielded 519 articles and included 12 randomized controlled trials in the meta-analysis. Vitamin D supplementation had no effect on any of the outcomes analyzed (fasting blood glucose and insulinemia, glycated hemoglobin, HOMA index, systolic and diastolic blood pressure, weight, waist circumference, total cholesterol, LDL and HDL, and triglycerides). However, subgroup analyses indicated that using vitamin D up to 2000 IU daily reduced participants' fasting blood glucose and glycated hemoglobin. Furthermore, the intervention reduced diastolic blood pressure only in participants with vitamin D deficiency. At least two studies showed a high risk of bias using the Rob2 protocol. According to the GRADE protocol, the evidence quality varied from moderate to very low. These results indicate that vitamin D supplementation does not improve patients' metabolic parameters and that the association between plasma 25-dihydroxyvitamin D levels and Metabolic Syndrome may not be causal but caused by other confounding characteristics. However, in any case, the quality of evidence is still low, and more randomized clinical trials are essential to clarify these relationships.

Rhodnius prolixus lipophorin: lipid composition and effect of high temperature on physiological role.

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55 °C. When it was pretreated at 75 °C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75 °C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55 °C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75 °C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65 °C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85 °C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.

Trypanosoma cruzi: effects of heat shock on ecto-ATPase activity.

In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.

Use of FGF21 analogs for the treatment of metabolic disorders: a systematic review and meta-analysis.

FGF21 is a hormone produced primarily by the liver with several metabolic functions, such as induction of heat production, control of glucose homeostasis, and regulation of blood lipid levels. Due to these actions, several laboratories have developed FGF21 analogs to treat patients with metabolic disorders such as obesity and diabetes. Here, we performed a systematic review and meta-analysis of randomized controlled trials that used FGF21 analogs and analyzed metabolic outcomes. Our search yielded 236 articles, and we included eight randomized clinical trials in the meta-analysis. The use of FGF21 analogs exhibited no effect on fasting blood glucose, glycated hemoglobin, HOMA index, blood free fatty acids or systolic blood pressure. However, the treatment significantly reduced fasting insulinemia, body weight and total cholesterolemia. None of the included studies were at high risk of bias. The quality of the evidence ranged from moderate to very low, especially due to imprecision and indirection issues. These results indicate that FGF21 analogs can potentially treat metabolic syndrome. However, more clinical trials are needed to increase the quality of evidence and confirm the effects seen thus far.

Alterations in energy metabolism of Rhodnius prolixus induced by Trypanosoma rangeli infection.

Trypanosoma rangeli is a protozoan parasite that infects triatomines and mammals in the Americas, producing mixed infections with Trypanosoma cruzi, the etiological agent of Chagas disease. The former parasite is not pathogenic to humans, but has different levels of pathogenicity, as well as causing physiological and behavioral alterations, to its invertebrate hosts. In this study, we measured locomotory activity, and the glyceride accumulation profile in the hemolymph and fat body, as well as the expression of key genes related to triglyceride metabolism, of Rhodnius prolixus nymphs infected with T. rangeli. We found that the locomotory activity of the insects was correlated with the amount of triglycerides in the fat body. Infected nymphs had increased activity when starved, and also had an accumulation of glycerides in the fat body and hemolymph. These alterations were also associated with a higher expression of the diacylglycerol acyltransferase, lipophorin and lipophorin receptor genes in the fat body. We infer that T. rangeli is able to alter the energetic processes of its invertebrate host, in order to increase the availability of lipids to the parasite, which, in turn modifies the activity levels of the insect. These alterations are discussed with regard to their potential to increase the transmission rate of the parasite.

Gene identification and RNAi-silencing of p62/SQSTM1 in the vector Rhodnius prolixus reveals a high degree of sequence conservation but no apparent deficiency-related phenotypes in vitellogenic females.

Autophagy and the ubiquitin-proteasome system (UPS) are important cellular mechanisms that coordinate protein degradation essential for proteostasis. P62/SQSTM1 is a receptor cargo protein able to deliver ubiquitinated targets to the proteasome proteolytic complex and/or to the autophagosome. In the insect vector of Chagas disease, Rhodnius prolixus, previous works have shown that the knockdown of different autophagy-related genes (ATGs) and ubiquitin-conjugating enzymes resulted in abnormal oogenesis phenotypes and embryo lethality. Here, we investigate the role of the autophagy/UPS adaptor protein p62 during the oogenesis and reproduction of this vector. We found that R. prolixus presents one isoform of p62 encoded by a non-annotated gene. The predicted protein presents the domain architecture anticipated for p62: PB1 (N-term), ZZ-finger, and UBA (C-term) domains, and phylogenetic analysis showed that this pattern is highly conserved within insects. Using parental RNAi, we found that although p62 is expressed in the ovary, midgut, and fat body of adult females, systemic silencing of this gene did not result in any apparent phenotypes under in-house conditions. The insects' overall levels of blood meal digestion, lifespan, yolk protein production, oviposition, and embryo viability were not altered when compared to controls. Because it is known that autophagy and UPS can undergo compensatory mechanisms, we asked whether the silencing of p62 was triggering adaptative changes in the expression of genes of the autophagy, UPS, and the unfolded protein response (UPR) and found that only ATG1 was slightly up regulated in the ovaries of silenced females. In addition, experiments to further investigate the role of p62 in insects previously silenced for the E1-conjugating enzyme (a condition known to trigger the upregulation of p62), also did not result in any apparent phenotypes in vitellogenic females.

Knockdown of carnitine palmitoyltransferase I (CPT1) reduces fat body lipid mobilization and resistance to starvation in the insect vector Rhodnius prolixus.

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.

ATP synthase affects lipid metabolism in the kissing bug Rhodnius prolixus beyond its role in energy metabolism.

ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and ß family, including the α and ß subunits of ATP synthase (RpATPSynα and RpATPSynß), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynß knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynß increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.

Silencing of the 20S proteasomal subunit-α6 triggers full oogenesis arrest and increased mRNA levels of the selective autophagy adaptor protein p62/SQSTM1 in the ovary of the vector Rhodnius prolixus.

The high reproductive rates of insects contribute significantly to their ability to act as vectors of a variety of vector-borne diseases. Therefore, it is strategically critical to find molecular targets with biotechnological potential through the functional study of genes essential for insect reproduction. The ubiquitin-proteasome system is a vital degradative pathway that contributes to the maintenance of regular eukaryotic cell proteostasis. This mechanism involves the action of enzymes to covalently link ubiquitin to proteins that are meant to be delivered to the 26S proteasome and broken down. The 26S proteasome is a large protease complex (including the 20S and 19S subcomplexes) that binds, deubiquitylates, unfolds, and degrades its substrates. Here, we used bioinformatics to identify the genes that encode the seven α and ß subunits of the 20S proteasome in the genome of R. prolixus and learned that those transcripts are accumulated into mature oocytes. To access proteasome function during oogenesis, we conducted RNAi functional tests employing one of the 20S proteasome subunits (Prosα6) as a tool to suppress 20S proteasomal activity. We found that Prosα6 silencing resulted in no changes in TAG buildup in the fat body and unaffected availability of yolk proteins in the hemolymph of vitellogenic females. Despite this, the silencing of Prosα6 culminated in the impairment of oocyte maturation at the early stages of oogenesis. Overall, we discovered that proteasome activity is especially important for the signals that initiate oogenesis in R. prolixus and discuss in what manner further investigations on the regulation of proteasome assembly and activity might contribute to the unraveling of oogenesis molecular mechanisms and oocyte maturation in this vector.

Gene identification and enzymatic properties of a membrane-bound trehalase from the ovary of Rhodnius prolixus.

Trehalose represents the main hemolymph sugar in most insects and its metabolic availability is regulated by trehalase. In this study, trehalase activity associated with the reproductive system was investigated in the insect Rhodnius prolixus, a hematophagous hemipteran vector of Chagas' disease. A single-copy gene that encodes a membrane-bound trehalase (RpTre-2) was identified in the genome of R. prolixus. RpTre-2 deduced amino acid sequence is closely related to other insect membrane-bound trehalases. The expression of this gene was detected in all analyzed organs, including ovary, where total trehalase enzymatic activity was determined, and was highest at day 7 after blood meal. Ovary membranes showed a major trehalase specific activity, which confirmed the presence of a membrane-bound trehalase in this insect. This trehalase activity seemed not to be regulated at transcriptional level, as the expression of RpTre-2 gene in the ovary did not change over the days after feeding. Similarly, ovarian follicles at different developmental stages did not show any variation in the transcription level of this gene. The RpTre-2 kinetic parameters were also investigated. Activity was highest at pH 5.5 and followed Michaelis-Menten kinetics, with an apparent K(m) = 1.42 ± 0.36 mM and Vmax = 167.90 ± 12.91 nmol/mg protein/h. These data reveal the presence of a membrane-bound trehalase in R. prolixus that is active in ovary and probably takes part in the insect carbohydrate metabolism associated with the reproductive process.

Lipid accumulation and utilization by oocytes and eggs of Rhodnius prolixus.

Insect eggs must contain the necessary nutrients for embryonic growth. In this article, we investigated the accumulation of triacylglycerol (TAG) in growing oocytes and its utilization during embryonic development. TAG makes up about 60% of the neutral lipids in oocytes and accumulates as oocytes grow, from 2.2 ± 0.1 µg in follicles containing 1.0 mm length oocytes to 10.2 ± 0.8 µg in 2.0 mm length oocytes. Lipophorin (Lp), the hemolymphatic lipoprotein, radioactively labeled in free fatty acid (FFA) or diacylglycerol (DAG), was used to follow the transport of these lipids to the ovary. Radioactivity from both lipid classes accumulated in the oocytes, which was abolished at 4°C. The capacity of the ovary to receive FFA or DAG from Lp varied according to time after a blood meal and reached a maximum around the second day. (3) H-DAG supplied by Lp to the ovaries was used in the synthesis of TAG as, 48 hr after injection, most of the radioactivity was found in TAG (85.7% of labeling in neutral lipids). During embryogenesis, lipid stores were mobilized, and the TAG content decreased from 16.4 ± 2.1 µg/egg on the first day to 10.0 ± 1.3 µg on day 15, just before hatching. Of these, 7.4 ± 0.9 µg were found in the newly emerged nymphs. In unfertilized eggs, the TAG content did not change. Although the TAG content decreased during embryogenesis, the relative lipid composition of the egg did not change. The amount of TAG in the nymph slowly decreased during the days after hatching.

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence.

Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.

The Fate of Dietary Cholesterol in the Kissing Bug Rhodnius prolixus.

Insects are unable to synthesize cholesterol and depend on the presence of sterols in the diet for cell membrane composition and hormone production. Thus, cholesterol absorption, transport, and metabolism are potential targets for vector and pest control strategies. Here, we investigate the dietary cholesterol absorption and tissue distribution in the kissing bug Rhodnius prolixus using radiolabeled cholesterol. Both the anterior and posterior midguts absorbed cholesterol from the ingested blood, although the anterior midgut absorbed more. We also observed esterified cholesterol labeling in the epithelium, indicating that midgut cells can metabolize and store cholesterol. Only a small amount of labeled cholesterol was found in the hemolymph, where it was mainly in the free form and associated with lipophorin (Lp). The fat body transiently accumulated cholesterol, showing a labeled cholesterol peak on the fifth day after the blood meal. The ovaries also incorporated cholesterol, but cumulatively. The insects did not absorb almost half of the ingested labeled cholesterol, and radioactivity was present in the feces. After injection of 3H-cholesterol-labeled Lp into females, a half-life of 5.5 ± 0.7 h in the hemolymph was determined. Both the fat body and ovaries incorporated Lp-associated cholesterol, which was inhibited at low temperature, indicating the participation of active cholesterol transport. These results help describe an unexplored part of R. prolixus lipid metabolism.

dHNF4 regulates lipid homeostasis and oogenesis in Drosophila melanogaster.

The fly genome contains a single ortholog of the evolutionarily conserved transcription factor hepatocyte nuclear factor 4 (HNF4), a broadly and constitutively expressed member of the nuclear receptor superfamily. Like its mammalian orthologs, Drosophila HNF4 (dHNF4) acts as a critical regulator of fatty acid and glucose homeostasis. Because of its role in energy storage and catabolism, the insect fat body controls non-autonomous organs including the ovaries, where lipid metabolism is essential for oogenesis. The present paper used dHNF4 overexpression (OE) in the fat bodies and ovaries to investigate its potential roles in lipid homeostasis and oogenesis. When the developing fat body overexpressed dHNF4, animals exhibited reduced size and failed to pupariate, but no changes in body composition were observed. Conditional OE of dHNF4 in the adult fat body produced a reduction in triacylglycerol content and reduced oogenesis. Ovary-specific dHNF4 OE increased oogenesis and egg-laying, but reduced the number of adult offspring. The phenotypic effects on oogenesis that arise upon dHNF4 OE in the fat body or ovary may be due to its function in controlling lipid utilization.

Blood meal drives de novo lipogenesis in the fat body of Rhodnius prolixus.

In insects, lipids are stored in the fat body mainly as triacylglycerol. Lipids can be directly provided by digestion and incorporated from the hemolymph, or synthesized de novo from other substrates such as carbohydrates and amino acids. The first step in de novo lipid synthesis is catalyzed by acetyl-CoA carboxylase (ACC), which carboxylates acetyl-CoA to form malonyl-CoA. Rhodnius prolixus is a hematophagous insect vector of Chagas disease and feeds exclusively on large and infrequent blood meals. Adult females slowly digest the blood and concomitantly accumulate lipids in the fat body. In this study, we investigated the regulation of R. prolixus ACC (RhoprACC) expression and de novo lipogenesis activity in adult females at different nutritional and metabolic conditions. A phylogenetic analysis showed that insects, similar to other arthropods and unlike vertebrate animals, have only one ACC gene. In females on the fourth day after a blood meal, RhoprACC transcript levels were similar in the anterior and posterior midgut, fat body and ovary and higher in the flight muscles. In the fat body, gene expression was higher in fasted females and decreased after a blood meal. In the posterior midgut it increased after feeding, and no variation was observed in the flight muscle. RhoprACC protein content analysis of the fat body revealed a profile similar to the gene expression, with higher protein contents before feeding and in the first two days after a blood meal. Radiolabeled acetate was used to follow de novo lipid synthesis in the fat body and it was incorporated mainly into triacylglycerol, diacylglycerol and phospholipids. This lipogenic activity was inhibited by soraphen A, an ACC inhibitor, and it varied according to the insect metabolic status. De novo lipogenesis was very low in starved females and increased during the initial days after a blood meal. The flight muscles had a very low capacity to synthesize lipids when compared to the fat body. Radiolabeled leucine was also used as a substrate for de novo lipogenesis and the same lipid classes were formed. In conclusion, our results indicate that the blood meal induces the utilization of diet-derived amino acids by de novo lipogenesis in the fat body, and that the control of this activity does not occur at the RhoprACC gene or protein expression level.

Expression of acyl-CoA-binding protein 5 from Rhodnius prolixus and its inhibition by RNA interference.

The acyl-CoA-binding proteins (ACBP) act by regulating the availability of acyl-CoA in the cytoplasm and must have essential functions in lipid metabolism. The genome of the kissing-bug Rhodnius prolixus encodes five proteins of this family, but little is known about them. In this study we investigated the expression and function of RpACBP-5. Feeding induced RpACBP-5 gene expression in the posterior midgut, and an increase of about four times was observed two days after the blood meal. However, the amount of protein, which was only detected in this organ, did not change during digestion. The RpACBP-5 gene was also highly expressed in pre-vitellogenic and vitellogenic oocytes. Recombinant RpACBP-5 was shown to bind to acyl-CoA of different lengths, and it exhibited nanomolar affinity to lauroyl-CoA in an isothermal titration assay, indicating that RpACBP-5 is a functional ACBP. RpACBP-5 knockdown by RNA interference did not affect digestion, egg laying and hatching, survival, or accumulation of triacylglycerol in the fat body and oocytes. Similarly, double knockdown of RpACBP-1 and RpACBP-5 did not alter egg laying and hatching, survival, accumulation of triacylglycerol in the fat body and oocytes, or the neutral lipid composition of the posterior midgut or hemolymph. These results show that RpACBP-5 is a functional ACBP but indicate that the lack of a detectable phenotype in the knockdown insects may be a consequence of functional overlap of the proteins of the ACBP family found in the insect.