Solanaceae Glycoalkaloids Disturb Lipid Metabolism in the Tenebrio molitor Beetle.

Glycoalkaloids (GAs) are produced naturally by plants and affect insect survivability and fertility. These compounds can be considered potential bioinsecticides; however, the mechanisms and effects of their action remain undiscovered. As lipids are essential molecules for the proper functioning of an insect organism, this research aimed to determine the effects of GAs on the lipid metabolism of the Tenebrio molitor beetle. Solanine, chaconine, tomatine, and tomato leaf extract were applied to larvae by injection at two concentrations, 10-8 and 10-5 M. Then, the tissue was isolated after 2 and 24 h to determine the levels of free fatty acids, sterols and esters using the GC-MS technique. Moreover, the triacylglyceride level and the activity of the key ß-oxidation enzyme, 3-hydroxyacyl-CoA dehydrogenase (HADH), were measured. The results indicate that GAs affect the content and composition of lipid compounds in the beetles' haemolymph and fat body. The effects depend on the GA concentrations, incubation time, and kind of tissue. Moreover, the tested compounds decrease HADH activity, especially in the fat body, which may affect energy production. To our knowledge, this is the first study concerning lipid metabolism in T. molitor after GA application. Our results provide some insights into that topic.

Mitochondrial metabolism and oxidative stress in the tropical cockroach under fluctuating thermal regimes.

The cockroach Gromphadorhina coquereliana can survive at low temperatures under extensive periods of cold stress. To assess energy management and insect adaptation in response to cold, we measured mitochondrial activity and oxidative stress in muscle and fat body tissues from G. coquereliana under a fluctuating thermal regime (FTR; stressed at 4°C for 3 h on 3 consecutive days, with or without 24 h recovery). Compared with our earlier work showing that a single exposure to cold significantly affects mitochondrial parameters, here, repeated exposure to cold triggered an acclimatory response, resulting in unchanged mitochondrial bioenergetics. Immediately after cold exposure, we observed an increase in the overall pool of ATP and a decrease in typical antioxidant enzyme activity. We also observed decreased activity of uncoupling protein 4 in muscle mitochondria. After 24 h of recovery, we observed an increase in expression of antioxidant enzymes in muscles and the fat body and a significant increase in the expression of UCP4 and HSP70 in the latter. This indicates that processes related to energy conversion and disturbance under cold stress may trigger different protective mechanisms in these tissues, and that these mechanisms must be activated to restore insect homeostasis. The mitochondrial parameters and enzymatic assays suggest that mitochondria are not affected during FTR but oxidative stress markers are decreased, and a 24 h recovery period allows for the restoration of redox and energy homeostasis, especially in the fat body. This confirms the crucial role of the fat body in intermediary metabolism and energy management in insects and in the response to repeated thermal stress.

Anticancer activity of glycoalkaloids from Solanum plants: A review.

Cancer is still one of the main causes of death worldwide. For this reason, new compounds that have chemotherapeutic potential have been identified. One such group of substances is Solanaceae glycoalkaloids (GAs). They are natural compounds produced by plants widely used in traditional medicine for healing many disorders. Among others, GAs exhibit significant antitumor properties, for example, a strong inhibitory effect on cancer cell growth. This activity can result in the induction of tumor cell apoptosis, which can occur via different molecular pathways. The molecular mechanisms of the action of GAs are the subject of intensive research, as improved understanding could lead to the development of new cancer therapies. The genetic basis for the formation of neoplasms are mutations in protooncogenes, suppressors, and apoptosis-controlling and repair genes; therefore, substances with antineoplastic properties may affect the levels of their expression or the levels of their expression products. Therapeutic compounds can be applied separately or in combination with other drugs to increase the efficiency of cancer therapy; they can act on the cell through various mechanisms at different stages of carcinogenesis, inducing the process of apoptosis, blocking cell proliferation and migration, and inhibiting angiogenesis. This review summarizes the newest studies on the anticancer properties of solanine (SN), chaconine (CH), solasonine (SS), solamargine (SM), tomatine (TT) and their extracts from Solanum plants.

Mitochondria as a target and central hub of energy division during cold stress in insects.

Temperature stress is one of the crucial factors determining geographical distribution of insect species. Most of them are active in moderate temperatures, however some are capable of surviving in extremely high as well as low temperatures, including freezing. The tolerance of cold stress is a result of various adaptation strategies, among others the mitochondria are an important player. They supply cells with the most prominent energy carrier-ATP, needed for their life processes, but also take part in many other processes like growth, aging, protection against stress injuries or cell death. Under cold stress, the mitochondria activity changes in various manner, partially to minimize the damages caused by the cold stress, partially because of the decline in mitochondrial homeostasis by chill injuries. In the response to low temperature, modifications in mitochondrial gene expression, mtDNA amount or phosphorylation efficiency can be observed. So far study also showed an increase or decrease in mitochondria number, their shape and mitochondrial membrane permeability. Some of the changes are a trigger for apoptosis induced via mitochondrial pathway, that protects the whole organism against chill injuries occurring on the cellular level. In many cases, the observed modifications are not unequivocal and depend strongly on many factors including cold acclimation, duration and severity of cold stress or environmental conditions. In the presented article, we summarize the current knowledge about insect response to cold stress focusing on the role of mitochondria in that process considering differences in results obtained in different experimental conditions, as well as depending on insect species. These differentiated observations clearly indicate that it is still much to explore.

Characterization of Gromphadorhina coquereliana hemolymph under cold stress.

Low temperatures in nature occur together with desiccation conditions, causing changes in metabolic pathways and cellular dehydration, affecting hemolymph volume, water content and ion homeostasis. Although some research has been conducted on the effect of low temperature on Gromphadorhina coquereliana, showing that it can survive exposures to cold or even freezing, no one has studied the effect of cold on the hemolymph volume and the immune response of this cockroach. Here, we investigated the effect of low temperature (4 °C) on the abovementioned parameters, hemocyte morphology and total number. Cold stress affected hemocytes and the immune response, but not hemolymph volume. After stress, the number of circulating hemocytes decreased by 44.7%, but the ratio of apoptotic cells did not differ significantly between stressed and control individuals: 8.06% and 7.18%, respectively. The number of phagocyting hemocytes decreased by 16.66%, the hemocyte morphology drastically changed, and the F-actin cytoskeleton differed substantially in cold-stressed insects compared to control insects. Moreover, the surface area of the cells increased from 393.69 µm2 in the control to 458.38 µm2 in cold-treated animals. Together, our results show the links between cold stress and the cellular immune response, which probably results in the survival capability of this species.

Thermal stress causes DNA damage and mortality in a tropical insect.

Cold tolerance is considered an important factor determining the geographic distribution of insects. We have previously shown that despite its tropical origin, the cockroach Gromphadorinha coquereliana is capable of surviving exposures to cold. However, the freezing tolerance of this species had not yet been examined. Low temperature is known to alter membrane integrity in insects, but whether chilling or freezing compromises DNA integrity remains a matter of speculation. In the present study, we subjected the G. coquereliana adults to freezing to determine their supercooling point (SCP) and evaluated whether the cockroaches were capable of surviving partial and complete freezing. Next, we conducted single cell gel electrophoresis (SCGE) assays to determine whether heat, cold and freezing altered hemocyte DNA integrity. The SCP of this species was high and around -4.76°C, which is within the typical range of freezing-tolerant species. Most cockroaches survived to 1 day after partial ice formation (20% mortality), but died progressively in the next few days after cold stress (70% mortality after 4 days). One day after complete freezing, most insects died (70% mortality), and after 4 days, 90% of them had succumbed. The SCGE assays showed substantial levels of DNA damage in hemocytes. When cockroaches were heat-stressed, the level of DNA damage was similar to that observed in the freezing treatment, though all heat-stressed insects survived. The present study shows that G. coquereliana can be considered as moderately freeze-tolerant, and that extreme low temperature stress can affect DNA integrity, suggesting that this cockroach may possess an efficient DNA repair system.

The The TOB/SAM complex composition in mitochondria of Dictyostelium discoideum during progression from unicellularity to multicellularity.

Despite its complex life cycle including unicellular and multicellular stages, the slime mold Dictyostelium discoideum, a well-known model in biomedical research, has not been used as a model organism in studies on mitochondrial import, including its significance in cellular processes. Moreover, data concerning mitochondrial protein import machinery in D. discoideum mitochondria is limited and nothing is known about the impact of that machinery on slime mold life cycle. Here, we focused on the TOB/SAM (topogenesis of the mitochondrial outer membrane ß-barrel proteins/sorting and assembly machinery) complex. This complex is localized in the mitochondrial outer membrane and is indispensable for the formation of metabolite exchange and protein import pathways in the membrane, and substantially contributes to the regulation of mitochondrial morphology and distribution. Furthermore, the available data suggests that the TOB/SAM complex variants differ between mitochondria of multicellular and unicellular eukaryotes. Therefore, we decided to determine these variants of the TOB/SAM in mitochondria of D. discoideum progressing from single cells to early multicellular stages, when the cells stream together to form a multicellular organism. The results revealed two complex variants of the TOB/SAM complex of about 160 and 600 kDa molecular weight, present in mitochondria of D. discoideum cells at the studied stages. The discussed complex variants resemble the ones that have been already detected for the yeast Saccharomyces cerevisiae, fungus Neurospora crassa and human cells, and one of investigated variants differentiates unicellular and initial multicellular stages of the D. discoideum life cycle.

Sulfakinin Signalling Influences Fatty Acid Levels and Composition in Tenebrio Molitor Beetle.

BACKGROUND: Sulfakinins are arthropod neuropeptides that are structurally and functionally similar to vertebrate gastrin-cholecystokinin. Sulfakinins with sulfated tyrosine (sSK) or nonsulfated tyrosine (nSK) in the C-terminated heptapeptide XY(SO3H)GHMRFamide display different biological functions, including myotropic activity, inhibition of food intake, stimulation of digestive enzymes and regulation of carbohydrate and lipid content. OBJECTIVE: To reveal the mechanisms by which sulfakinin signalling modulates lipid homeostasis, we analysed the changes in the level and composition of fatty acids and organic compounds in the fat body and haemolymph of Tenebrio molitor larvae after nSK and sSK treatment. METHODS: Fatty acids in fat body and haemolymph of insects were analysed using Gas Chromatography - Mass Spectrometry (GC-MS). RESULTS: The direction of the changes observed for major fatty acids, 18:1 and 18:2, and the less abundant fatty acids, 16:0, 18:0, 16:1 and 14:0, was the same for unsaturated (UFAs) and saturated (SFAs) fatty acids, and elevated after nSK application in both analysed tissues. However, the action of sSK in fat body tissue evoked distinct effects and induced either significant decreases in individual fatty acids or UFAs and SFAs. Administration of nSK and sSK significantly increased the level of total organic compounds in the haemolymph, contrary to the effect of sSK in fat body, where the level of total organic compounds decreased, although changes differ between individual chemicals. CONCLUSION: Sulfakinins are engaged in the precise modulation of fatty acid levels and composition, but their action depends on the presence of sulfate group on the tyrosyl residue of the peptide what determines the different roles of these peptides in insect physiology.

Beetles as Model Organisms in Physiological, Biomedical and Environmental Studies - A Review.

Model organisms are often used in biological, medical and environmental research. Among insects, Drosophila melanogaster, Galleria mellonella, Apis mellifera, Bombyx mori, Periplaneta americana, and Locusta migratoria are often used. However, new model organisms still appear. In recent years, an increasing number of insect species has been suggested as model organisms in life sciences research due to their worldwide distribution and environmental significance, the possibility of extrapolating research studies to vertebrates and the relatively low cost of rearing. Beetles are the largest insect order, with their representative - Tribolium castaneum - being the first species with a completely sequenced genome, and seem to be emerging as new potential candidates for model organisms in various studies. Apart from T. castaneum, additional species representing various Coleoptera families, such as Nicrophorus vespilloides, Leptinotarsa decemlineata, Coccinella septempunctata, Poecilus cupreus, Tenebrio molitor and many others, have been used. They are increasingly often included in two major research aspects: biomedical and environmental studies. Biomedical studies focus mainly on unraveling mechanisms of basic life processes, such as feeding, neurotransmission or activity of the immune system, as well as on elucidating the mechanism of different diseases (neurodegenerative, cardiovascular, metabolic, or immunological) using beetles as models. Furthermore, pharmacological bioassays for testing novel biologically active substances in beetles have also been developed. It should be emphasized that beetles are a source of compounds with potential antimicrobial and anticancer activity. Environmental-based studies focus mainly on the development and testing of new potential pesticides of both chemical and natural origin. Additionally, beetles are used as food or for their valuable supplements. Different beetle families are also used as bioindicators. Another important research area using beetles as models is behavioral ecology studies, for instance, parental care. In this paper, we review the current knowledge regarding beetles as model organisms and their practical application in various fields of life science.

Insect Peptides - Perspectives in Human Diseases Treatment.

BACKGROUND: Insects are the largest and the most widely distributed group of animals in the world. Their diversity is a source of incredible variety of different mechanisms of life processes regulation. There are many agents that regulate immunology, reproduction, growth and development or metabolism. Hence, it seems that insects may be a source of numerous substances useful in human diseases treatment. Especially important in the regulation of insect physiology are peptides, like neuropeptides, peptide hormones or antimicrobial peptides. There are two main aspects where they can be helpful, 1) Peptides isolated from insects may become potential drugs in therapy of different diseases, 2) A lot of insect peptide hormones show structural or functional homology to mammalian peptide hormones and the comparative studies may give a new look on human disorders. In our review we focused on three group of insect derived peptides: 1) immune-active peptides, 2) peptide hormones and 3) peptides present in venoms. CONCLUSION: In our review we try to show the considerable potential of insect peptides in searching for new solutions for mammalian diseases treatment. We summarise the knowledge about properties of insect peptides against different virulent agents, anti-inflammatory or anti-nociceptive properties as well as compare insect and mammalian/vertebrate peptide endocrine system to indicate usefulness of knowledge about insect peptide hormones in drug design. The field of possible using of insect delivered peptide to therapy of various human diseases is still not sufficiently explored. Undoubtedly, more attention should be paid to insects due to searching new drugs.

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.

NONSULFATED SULFAKININ CHANGES METABOLIC PARAMETERS OF INSECT FAT BODY MITOCHONDRIA.

We investigated the effect of neuropeptide, the nonsulfated sulfakinin (SK) Zopat-SK-1 (pETSDDYGHLRFa) on the mitochondrial oxidative metabolism in the Zophobas atratus larval fat body. Mitochondria were isolated from beetle fat bodies 2 and 24 h after hormone injection. The administration of 20 pmol of Zopat-SK-1 to feeding larvae led to decreased mitochondrial oxidative activities in larval fat body. Diminished activities of citrate synthase and the cytochrome pathway, that is, nonphosphorylating and phosphorylating respiration during succinate oxidation, were observed. However, the effect of Zopat-SK-1 was more pronounced in fat body of insects after 24 h since hormone application. In hormone-treated larval fat bodies, mitochondrial respiration was decreased at the level of respiratory chain and the TCA cycle as well as at the level of mitochondrial biogenesis, as indicated by decreased activities of mitochondrial marker enzymes in fat body homogenates. The inhibition of succinate oxidation may indicate the role of Zopat-SK-1 in the regulation of mitochondrial complex II activity. Moreover, decreased respiratory chain activity was accompanied by the reduced activity of mitochondrial energy-dissipating pathway, uncoupling protein 4. The observed decrease in mitochondrial oxidative metabolism may reflect the Zopat-SK-1-induced reduction in the metabolic rate of larval fat body linked to actual energetic demands of animal.

Uncoupling proteins of invertebrates: A review.

Uncoupling proteins (UCPs) mediate inducible proton conductance in the mitochondrial inner membrane. Herein, we summarize our knowledge regarding UCPs in invertebrates. Since 2001, the presence of UCPs has been demonstrated in nematodes, mollusks, amphioxi, and insects. We discuss the following important issues concerning invertebrate UCPs: their evolutionary relationships, molecular and functional properties, and physiological impact. Evolutionary analysis indicates that the branch of vertebrate and invertebrate UCP4-5 diverged early in the evolutionary process prior to the divergence of the animal groups. Several proposed physiological roles of invertebrate UCPs are energy control, metabolic balance, and preventive action against oxidative stress. © 2016 IUBMB Life, 68(9):691-699, 2016.

Activation of Mitochondrial Uncoupling Protein 4 and ATP-Sensitive Potassium Channel Cumulatively Decreases Superoxide Production in Insect Mitochondria.

It has been evidenced that mitochondrial uncoupling protein 4 (UCP4) and ATP-regulated potassium channel (mKATP channel) of insect Gromphadorhina coqereliana mitochondria decrease superoxide anion production. We elucidated whether the two energy-dissipating systems work together on a modulation of superoxide level in cockroach mitochondria. Our data show that the simultaneous activation of UCP4 by palmitic acid and mKATP channel by pinacidil revealed a cumulative effect on weakening mitochondrial superoxide formation. The inhibition of UCP4 by GTP (and/or ATP) and mKATP channel by ATP elevated superoxide production. These results suggest a functional cooperation of both energy-dissipating systems in protection against oxidative stress in insects.

The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.

Cold induced changes in lipid, protein and carbohydrate levels in the tropical insect Gromphadorhina coquereliana.

Insects cope with thermal stressors using mechanisms such as rapid cold hardening and acclimation. These mechanisms have been studied in temperate insects, but little is known about their use by tropical insects in response to cold stress. Here, we investigated whether cold stress (1×8 h and 3×8 h at 4°C) triggers a metabolic response in the Madagascar cockroach Gromphadorhina coquereliana. We examined the effects of cold on the levels of selected metabolites in the fat body tissue of G. coquereliana. After cold exposure, we found that the quantity of total protein increased significantly in the insect fat body, whereas glycogen decreased slightly. Using antibodies, we observed upregulation of AQP-like proteins and changes in the HSP70 levels in the fat body of G. coquereliana when exposed to cold. We also examined the content and nature of the free sugars in the G. coquereliana hemolymph and discovered an increase in the levels of polyols and glucose in response to cold stress. These results suggest an important role of the fat body tissue of tropical insects upon cold exposure.

New metabolic activity of the nonsulfated sulfakinin Zopat-SK-1 in the insect fat body.

Insect sulfakinins are multifunctional neuropeptides homologous to vertebrate gastrin/cholecystokin (CCK) neuropeptide hormones. We investigated the action of the nonsulfated sulfakinin Zopat-SK-1 (pETSDDYGHLRFa) on the levels of chosen metabolites in the Zophobas atratus beetle fat body. Samples of fat body were collected 2h and 24h after hormone injection. The administration of 20pmol of Zopat-SK-1 to feeding larvae significantly increased concentrations of lipids and proteins and decreased the content of glycogen in fat body tissue in the 24h experimental group. In contrast, the only increase in total lipid concentration in prepupal fat bodies was observed 24h after Zopat-SK-1 treatment. Simultaneously, changes in the quality and quantity of free sugars in the hemolymph were measured. In larval hemolymph, a marked increase in free sugar concentration and a decrease in glucose content were observed 24h and 2h after Zopat-SK-1 application, respectively. No changes in the prepupal stage were observed. For the first time we show potent metabolic activity of sulfakinin in the fat body tissue of an insect. Our findings imply a physiological function of the nonsulfated form of sulfakinin in energy storage and release processes in fat body tissue of larvae and prepupae was indicated. We suggest a role for sulfakinin signaling in the regulation of energy metabolism in insect tissues.

Adipokinetic hormone induces changes in the fat body lipid composition of the beetle Zophobas atratus.

In insects, neuropeptide adipokinetic hormone (AKH) released from the corpora cardiaca mobilizes lipids and carbohydrates in the fat body. We examined the developmental differences in the action of Tenmo-AKH, a bioanalogue belonging to the adipokinetic/hypertrahelosemic family (AKH/HrTH), on the lipid composition of larval and pupal fat bodies in the beetle Zophobas atratus. Tenmo-AKH was administered to the beetle larvae and pupae either as a single dose or as two doses of 20 pmol during a 24h interval. Extracts of fat bodies were used to analyse the lipid composition by gas chromatography (GC) combined with mass spectrometry (GC-MS). Control extracts were analyzed using the same method. Fatty acids (FA) and fatty acid methyl esters (FAME) were the most abundant compounds in the fat bodies from both developmental stages. We observed significant differences in their concentrations following hormonal treatment. Tenmo-AKH also induced a distinct increase in larval sterols, fatty alcohols and benzoic acid.

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect.

In the present study, we describe the existence of mitochondrial ATP-dependent K(+) channel (mitoKATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoKATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoKATP in insect mitochondria. MitoKATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.