Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting.

Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We find that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.

Anthropometric and dietary assessment of patients with glycogenosis type i / Avaliação antropométrica e dietética de pacientes com glicogenose tipo i

ABSTRACT Objective: To perform anthropometric and dietary evaluation of patients with glycogenosis type Ia and Ib. Methods: This cross-sectional study is composed of a sample of 11 patients with glycogenosis divided into two subgroups according to the classification of glycogenosis (type Ia=5 and type Ib=6), aged between 4 and 20 years. The analyzed anthropometric variables were weight, height, body mass index, and measures of lean and fat body mass, which were compared with reference values. For dietary assessment, a food frequency questionnaire was used to calculate energy and macronutrients intake as well as the amount of raw cornstarch consumed. Mann-Whitney U test and Fisher's exact test were performed, considering a significance level of 5%. Results: Patients ingested raw cornstarch in the amount of 0.49 to 1.34 g/kg/dose at a frequency of six times a day, which is lower than recommended (1.75-2.50 g/kg/dose, four times a day). The amount of energy intake was, on average, 50% higher than energy requirements; however, carbohydrate intake was below the adequacy percentage in 5/11 patients. Short stature was found in 4/10 patients; obesity, in 3/11; and muscle mass deficit, in 7/11. There were no statistical differences between the subgroups. Conclusions: In patients with glycogenosis type I, there was deficit in growth and muscle mass, but no differences were found between the subgroups (Ia and Ib). Although the diet did not exceed the adequacy of carbohydrates, about 1/3 of the patients presented obesity, probably due to higher energy intake.

RESUMO Objetivo: Realizar avaliação antropométrica e dietética de pacientes com glicogenose tipos Ia e Ib. Métodos: Estudo transversal composto de uma amostra de 11 pacientes com glicogenose divididos em dois subgrupos de acordo com a classificação da glicogenose (tipo Ia=5; tipo Ib=6), com idades entre 4 e 20 anos. As variáveis antropométricas analisadas foram peso, estatura, índice de massa corporal e medidas de massa magra e gorda, que foram comparadas com valores de referência. Para avaliação dietética, foi utilizado um questionário de frequência alimentar para cálculo de ingestão de energia e macronutrientes, além da quantidade de amido cru ingerida. Realizaram-se testes U de Mann-Whitney e exato de Fisher, com nível de significância de 5%. Resultados: Os pacientes ingeriram amido cru na quantidade de 0,49 a 1,34 g/kg/dose na frequência de seis vezes ao dia, inferior à dosagem preconizada (1,75-2,50 g/kg/dose quatro vezes ao dia). A quantidade de energia consumida foi, em média, 50% a mais que as necessidades, contudo o consumo de carboidratos foi abaixo da porcentagem de adequação em 5/11 pacientes. Baixa estatura ocorreu em 4/10 pacientes, obesidade em 3/11 e déficit de massa muscular em 7/11. Não houve diferença estatística entre os subgrupos. Conclusões: Em pacientes com glicogenose tipo I, houve déficit de crescimento e de massa muscular, mas não diferença significante entre os subgrupos (Ia e Ib). Embora a dieta não tenha ultrapassado a adequação de carboidratos, 1/3 dos pacientes apresentou obesidade, provavelmente pela maior ingestão de energia.

Structural and functional differentiation of a fat body-like tissue adhering to testis follicles facilitates spermatogenesis in locusts.

The fat body is distributed throughout the body of insects, playing the essential role in intermediary metabolism and nutrient storage. However, the function of differentiation of fat bodies adhering to different tissues remains largely unknown. Here, we identified a fat body-like tissue (FLT) surrounding testis follicles and described its features at morphological, cellular and molecular levels. The FLT is morphologically distinguished with the abdominal fat body (FB) and dominated by diploid cells instead of polyploid cells. The transcriptomic analysis demonstrated that the FLT and FB have dramatically different gene expression profiles. Moreover, genes in the cell cycle pathway, which include both DNA replication- and cell division-related genes, were successively active during development of the FLT, suggesting that FLT cells possibly undergo a mitotic cycle rather than an endocycle. Deprivation of the FLT resulted in distortion of the testis follicles, disappearance of sperm bundles, reduction of total sperm number and increase of dead sperm, indicating a critical role of the FLT in the spermatogenesis in testis follicles. The special functional differentiation of the two similar tissues suggested that FLT-FB cells are able to establish a promising system to study mitotic-to-endocycle transition.

Cathepsin L-like protease can regulate the process of metamorphosis and fat body dissociation in Antheraea pernyi.

Cathepsins are a group of protease, located in lysosome and play a vital role in physiological process. Here, we reported cathepsin L-like protease (Ap-cathL), which contained an open reading frame of 1155 bp and encoding 385 amino acid residues protein. The I29 inhibitor domain and peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) putative conserved domains were detected in Ap-cathL. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ap-cathL highly expressed in the fat body and midgut. The high expression during the molting stage, pupal stage and following 20E (20-hydroxyecdysone) treatment indicated that it maybe involved in the process of molting and metamorphosis. In addition, depletion of Ap-cathL influenced the expression of apoptosis pathway related genes. The protease inhibitor and RNA interference experiments showed that Ap-cathL was involved in the fat body dissociation of A. pernyi. These results suggest that Ap-cathL may involve in the process of metamorphosis and fat body dissociation of A. pernyi.

Suppressor of cytokine signaling 6 can enhance epidermal growth factor receptor signaling pathway in Bombyx mori (Dazao).

The SOCS (Suppressor of cytokine signaling) family members are a potential negative regulator of cytokine signaling pathway and play a key role to maintain immunological functions in animals. SOCS-6 is an important member of the SOCS family, however the functions of this gene have rarely been explored among eukaryotes. Herein, we cloned and expressed SOCS-6 gene from Bombyx mori (Dazao) (BmSOCS-6), and anti-rabbit antibodies were prepared using purified recombinant BmSOCS-6 protein. Under normal physiological conditions, the BmSOCS-6 expression was observed at varied levels in six tissues, with most greatly expressed in fat body and hemocytes. After immune challenge with viral, fungal and bacterial pathogens, the BmSOCS-6 showed distinctly varied expression patterns in tissue, time and microbe dependent manner. By contrast, recombinant BmSOCS-6 protein strongly enhanced the expression of epidermal growth factor receptor (EGFR) pathway related genes, while the depletion of BmSOCS-6 by double stranded RNA suppressed their production. Altogether we concluded that BmSOCS-6 may improve the efficiency of EGFR signaling pathway in B. mori (Dazao).

Correlations of condition factor and gonadosomatic, hepatosomatic and lipo-somatic relations of Leptodactylus macrosternum (ANURA: Leptodactylidae) in the Brazilian Semi-arid

ABSTRACT The objective of this study was to assess variations of the condition factor (K1) in relation to the gonadosomatic- RGS and energy reserves (hepatosomatic - RWL and liposomatic - RFB relations) of Leptodactylus macrosternum and their relationship to climate variation in the Northeast of Brazil, Caatinga area, state of Paraiba. The animals were captured fortnightly through active collecting, between January and December 2013. Significant differences were observed in the monthly variations of K1, RGS and RFB indices in male and female L. macrosternum over the months of collection. In males, K1 showed no significant relationship with the other variables. In females, RGS values only show notable correlations with RWF and K1 values. K1 values showed significant correlations with all other weight and length ratios. Climate change in the HFOB region showed significant relationships with the variation of the indexes evaluated, with the exception of RWF. The variation of K1, RGS, RWL and RFB values over the months of collection as well as their relation with the local climatic variation, showed a brief reproductive activity for the species.

Tissue-specific insulin signaling mediates female sexual attractiveness.

Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Little is known about why specific traits are used for mate quality assessment nor about how they reflect it. We have previously shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in the fruit fly, Drosophila melanogaster. Here we demonstrate that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, here we describe a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells.

The physiological role of fat body and muscle tissues in response to cold stress in the tropical cockroach Gromphadorhina coquereliana.

Protective mechanisms against cold stress are well studied in terrestrial and polar insects; however, little is known about these mechanisms in tropical insects. In our study, we tested if a tropical cockroach Gromphadorhina coquereliana, possesses any protective mechanisms against cold stress. Based on the results of earlier studies, we examined how short-term (3 h) cold (4°C) influences biochemical parameters, mitochondrial respiration activity, and the level of HSPs and aquaporins expression in the fat body and leg muscles of G. coquereliana. Following cold exposure, we found that the level of carbohydrates, lipids and proteins did not change significantly. Nevertheless, we observed significant changes in mitochondrial respiration activity. The oxygen consumption of resting (state 4) and phosphorylating (state 3) mitochondria was altered following cold exposure. The increase in respiratory rate in state 4 respiration was observed in both tissues. In state 3, oxygen consumption by mitochondria in fat body was significantly lower compared to control insects, whereas there were no changes observed for mitochondria in muscle tissue. Moreover, there were cold-induced changes in UCP protein activity, but the changes in activity differed in fat body and in muscles. Additionally, we detected changes in the level of HSP70 and aquaporins expression. Insects treated with cold had significantly higher levels of HSP70 in fat body and muscles. On the other hand, there were lower levels of aquaporins in both tissues following exposure to cold. These results suggest that fat body play an important role in protecting tropical insects from cold stress.

Changes in the Plasma Proteome of Manduca sexta Larvae in Relation to the Transcriptome Variations after an Immune Challenge: Evidence for High Molecular Weight Immune Complex Formation.

Manduca sextais a lepidopteran model widely used to study insect physiological processes, including innate immunity. In this study, we explored the proteomes of cell-free hemolymph from larvae injected with a sterile buffer (C for control) or a mixture of bacteria (I for induced). Of the 654 proteins identified, 70 showed 1.67 to >200-fold abundance increases after the immune challenge; 51 decreased to 0-60% of the control levels. While there was no strong parallel between plasma protein levels and their transcript levels in hemocytes or fat body, the mRNA level changes (i.e.I/C ratios of normalized read numbers) in the tissues concurred with their protein level changes (i.e.I/C ratios of normalized spectral counts) with correlation coefficients of 0.44 and 0.57, respectively. Better correlations support that fat body contributes a more significant portion of the plasma proteins involved in various aspects of innate immunity. Consistently, ratios of mRNA and protein levels were better correlated for immunity-related proteins than unrelated ones. There is a set of proteins whose apparent molecular masses differ considerably from the calculatedMr's, suggestive of posttranslational modifications. In addition, some lowMrproteins were detected in the range of 80 to >300 kDa on a reducing SDS-polyacrylamide gel, indicating the existence of highMrcovalent complexes. We identified 30 serine proteases and their homologs, 11 of which are known members of an extracellular immune signaling network. Along with our quantitative transcriptome data, the protein identification, inducibility, and association provide leads toward a focused exploration of humoral immunity inM. sexta.

Serpin-15 from Bombyx mori inhibits prophenoloxidase activation and expression of antimicrobial peptides.

Serine protease inhibitors (SPIs) play a key role in physiological responses by controlling protease activities. In this study, we studied the biochemical functions of serpin-15, an SPI, from Bombyx mori (Bmserpin-15). Recombinant Bmserpin-15 was expressed in Escherichia coli cells and used to raise rabbit anti-Bmserpin-15 polyclonal antibodies. Bmserpin-15 mRNA and protein expression was detected in all tested tissues, particularly in the fat body and silk gland. After challenge with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus and B. mori nuclear polyhedrosis virus), the expressions of Bmserpin-15 mRNA and protein were induced significantly, particularly by B. bassiana and M. luteus. Recombinant Bmserpin-15 inhibited prophenoloxidase activation, but did not affect phenoloxidase activity, in B. mori hemolymph. Injection of recombinant Bmserpin-15 into B. mori larvae reduced significantly the transcript levels of antimicrobial peptides in fat body. Our results suggested that Bmserpin-15 plays an important role in the innate immunity of B. mori.

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster.

BACKGROUND: SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB. RESULTS: SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. CONCLUSIONS: Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV.

Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila.

BACKGROUND: Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized. RESULTS: Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, ß or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α. CONCLUSIONS: We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity.

Cuerpo adiposo bucal, su utilización en cirugía oral / Buccal fat body, its use in oral surgery

El cuerpo adiposo bucal es de gran utilidad en el tratamiento de defectos intraorales. La técnica descrita ha demostrado ser más sencilla,mejor tolerada y con menor tasa de complicaciones, que otras técnicas descriptas anteriormente. Por estos motivos es importante considerar a este elemento anatómico al momento de cerrar comunicaciones bucosinusales. El presente trabajo presenta la utilizacióndel mismo en tres casos de comunicación buco sinusal, todas de etilogía odontógena.

Fat body bucal is very useful when treating intraoral defects. The technique has proven to be simpler, more tolerable and shows a lowercomplication rate than the utilization of other techniques previously described. This is why it's important to consider this anatomic element when closing communication between oral and nasal cavities. We present the utilization of said element in three cases of communication between cavities, all of them of odontogenic etiology.

Cuticular encystment of Autographa nigrisigna eggs by epidermal cell migration.

It was previously established that Autographa nigrisigna loopers form cuticular cysts at the dorsal site of the 9th (penultimate) abdominal segment after parasitization by the solitary endoparasitoid Campoletis chlorideae and get rid of the parasitoid egg with the old cuticle at ecdysis. The cuticular cyst consists of a space between the old cuticle and new cuticle formed by the epidermis to enclose the parasitoid egg. The fact that A. nigrisigna loopers exclude the oviposited egg from the hemocoel using a cuticular cyst raises the question how the parasitoid egg passes through the epidermis. To exclude the endoparasitoid eggs from the hemocoel, the epidermis is required to move the location of the parasitoid egg. In the current study, we investigated the morphological process of cuticular cyst formation. First, the oviposited egg drifted to the 9th abdominal segment located at the open end of the dorsal vessel as a result of force generated by the hemolymph current from the oviposition site, and formed contacts with the integument containing the fat body (FB). The epidermis, in contact with the egg, then began to move along with the basement membrane formed on the surface of the FB, and settled under the egg, thus altering its location. This inversion was duplicated in vitro using integument from the 9th abdominal segment when parasitoid eggs were inserted between the epidermis and FB. When the integument, without the FB, was incubated on an agar plate, the epidermal cells migrated on the plate. Integument without eggs showed no signs of migration from their original sites. When the actin polymerization inhibitor latrunculin B was added to the cultures, the epidermal cells remained in their original location.

Hormonal and nutritional regulation of insect fat body development and function.

The insect fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. Similar to other larval organs, fat body undergoes a developmental "remodeling" process during the period of insect metamorphosis, with the massive destruction of obsolete larval tissues by programmed cell death and the simultaneous growth and differentiation of adult tissues from small clusters of progenitor cells. Genetic ablation of Drosophila fat body cells during larval-pupal transition results in lethality at the late pupal stage and changes sizes of other larval organs indicating that fat body is the center for pupal development and adult formation. Fat body development and function are largely regulated by several hormonal (i.e. insulin and ecdysteroids) and nutritional signals, including oncogenes and tumor suppressors in these pathways. Combining silkworm physiology with fruitfly genetics might provide a valuable system to understand the mystery of hormonal regulation of insect fat body development and function.

Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila.

The vertebrate Apolipoprotein D (ApoD) is a lipocalin secreted from subsets of neurons and glia during neural development and aging . A strong correlation exists between ApoD overexpression and numerous nervous system pathologies as well as obesity, diabetes, and many forms of cancer . However, the exact relationship between the function of ApoD and the pathophysiology of these diseases is still unknown. We have generated loss-of-function Drosophila mutants for the Glial Lazarillo (GLaz) gene , a homolog of ApoD in the fruit fly, mainly expressed in subsets of adult glial cells. The absence of GLaz reduces the organism's resistance to oxidative stress and starvation and shortens male lifespan. The mutant flies exhibit a smaller body mass due to a lower amount of neutral lipids stored in the fat body. Apoptotic neural cell death increases in aged flies or upon paraquat treatment, which also impairs neural function as assessed by behavioral tests. The higher sensitivity to oxidative stress and starvation and the reduced fat storage revert to control levels when a GFP-GLaz fusion protein is expressed under the control of the GLaz natural promoter. Finally, GLaz mutants have a higher concentration of lipid peroxidation products, pointing to a lipid peroxidation protection or scavenging as the mechanism of action for this lipocalin. In agreement with Walker et al. (, in this issue of Current Biology), who analyze the effects of overexpressing GLaz, we conclude that GLaz has a protective role in stress situations and that its absence reduces lifespan and accelerates neurodegeneration.

Protein tyrosine phosphatases of Toxoneuron nigriceps bracovirus as potential disrupters of host prothoracic gland function.

The genomic sequence of the bracovirus associated with the wasp Toxoneuron nigriceps (Hymenoptera, Braconidae) (TnBV), an endophagous parasitoid of the tobacco budworm larvae, Heliothis virescens (Lepidoptera, Noctuidae), contains a large gene family coding for protein tyrosine phosphatases (PTPs). Here we report the characterization of cDNAs for two of the viral PTPs isolated by screening a cDNA library from haemocytes of parasitized host larvae. The two encoded proteins show 70% amino acid identity and are expressed in the fat body of parasitized hosts. In addition, one was expressed in inactivated prothoracic glands (PTGs), 24 h after parasitoid oviposition. The rapid block of ecdysteroidogenesis does not appear to be due to inhibition of general protein synthesis, as indirectly indicated by the unaltered S6 kinase activity in the cytosolic extracts of basal PTGs from parasitized host larvae. Rather, TnBV PTP over-expression in inactivated host PTGs suggests that gland function may be affected by the disruption of the phosphorylation balance of key proteins regulating points upstream from the ribosomal S6 phosphorylation in the PTTH signaling cascade.

Induction of anhydrobiosis in fat body tissue from an insect.

The larva of the African chironomid Polypedilum vanderplanki can withstand complete desiccation. Our previous reports revealed that even when the larva is dehydrated without a brain, it accumulated a great amount of trehalose and successfully went into anhydrobiosis. In this paper we determined the viability after rehydration in tissues from the larvae followed by complete dehydration. Only fat-body tissues that were the main producer of trehalose could be preserved in a dry state at room temperature for an extended period of more than 18 months in a viable form. Thus we have confirmed that the central nervous system is not involved in the induction of anhydrobiosis, even in this complex multicellular organism.

A role for adenosine deaminase in Drosophila larval development.

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.

Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm (Manduca sexta).

Two major pathways of programmed cell death (PCD)--the apoptotic and the autophagic cell death--were investigated in the decomposition process of the larval fat body during the 5th larval stage of Manduca sexta. Several basic aspects of apoptotic and autophagic cell death were analyzed by morphological and biochemical methods in order to disclose whether these mechanisms do have shared common regulatory steps. Morphological examination revealed the definite autophagic wave started on day 4 followed by DNA fragmentation as demonstrated by agarose gel electrophoresis and TUNEL assay. By the end of the wandering period the cells were filled with autophagic vacuoles and protein granules of heterophagic origin and the vast majority of the nuclei were TUNEL-positive. No evidence was found of other aspects of apoptosis, e.g. activation of executioner caspases. Close correlation was disclosed between the onset of autophagy and the nuclear accumulation of the ubiquitin-proteasome system.