D. Umadevi et al. IRJP 2012, 3 (2) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407 Research Article HYPERLIPAEMIC AND HYPERGLYCAEMIC EFFECTS OF A METABOLIC PEPTIDE HORMONE FROM THE NEURONAL TISSUES OF THE MANGO LEAF WEBBER ORTHAGA EXVINACEA D. Umadevi*, M. Gokuldas and A.P. Ajaykumar Department of Zoology, Division of Insect Biochemistry and Physiology, University of Calicut, Kerala- 673635, India Article Received on: 06/01/12 Revised on: 09/02/12 Approved for publication: 18/02/12 *Email: [email protected] ABSTRACT The peptide hormone present in the brain-retrocerebral complexes of the mango leaf webber Orthaga exvinacea (Pyralidae: Lepidoptera) belonging to the adipokinetic hormone/red pigment-concentrating hormone family have different functions. The activity of the hormone extract tested both in vivo (heterologous bioassays against the polyphagous plant bug Iphita limbata) and in vitro clearly indicated that they are involved in lipid (adipokinetic hormones) and carbohydrate (hyperglycaemic hormones) release by activating fat body lipase and glycogen phosphorylase respectively. Injection of hormone extract (5 µl) containing one gland pair equivalent (gpe) elicited significant hyperlipaemic (up to 15%, P<0.001) and hyperglycaemic effects (up to 18%, P<0.05), whereas in the control, injection of 5 µl of insect saline did not evoke any such effects. The brain-retrocerebral complex extract showed significant effect on fat body lipid mobilization (up to 17%, P<0.05) and fat body sugar release (up to 18%, P<0.001). HPLC separation of the peptides followed by analysis of the fractions for activities confirmed that the peptide hormone extracted has a pivotal role in the mobilization of these metabolites. Key words: Adipokinetic hormone, hyperlipaemia, hyperglycaemia, Orthaga exvinacea, Iphita limbata, HPLC. INTRODUCTION employing HPLC analysis. Furthermore, the role of this Neuropeptides make up the largest and the most diverse class hormone in carbohydrate and lipid metabolism was also of signaling molecules used in nervous system confirmed by fraction testing studies. communication. The importance of insects as biochemical MATERIALS AND METHODS models is supported by the fact that many discoveries in Experimental insect insects are applicable to vertebrate systems as well. Because Larvae of O. exvinacea were collected from their natural arthropod nervous systems contain a manageable number of habitat, mango trees, and were transferred to plastic basins neurons, many of which exhibit consistent morphological and and reared in the insectary by feeding mango leaves. The physiological properties between animals, these organisms colony was maintained at 27+ 2˚C and 70%-80% RH. Sixth provide excellent model systems for investigating instar larvae were separated from the colony and used for neuromodulation in well-defined networks. The intermediary experiments. The insects of both sexes were used for metabolism in insects is regulated by small neuropeptides of hormone extraction and bioassay experiments. the adipokinetic hormone/red pigment concentrating hormone Adults of the plant bug, Iphita limbata were collected from (AKH/RPCH) family1 present in the neuronal tissues (brain- University campus and maintained in the insectary on a diet retrocerebral complexes). They are structurally similar to the of germinating seeds of green gram. Mature adults were used red pigment-concentrating hormone (RPCH) of crustaceans2. for the experiments. Common characteristics of the known AKHs are that the Preparation of brain-retrocerebral extract and Synthetic peptides have a length of 8-10 amino acids, are amino Lom-AKH terminally blocked by a pyroglutamate residue and end in a The brain retrocerebral complexes were removed with the carboxy terminal amide. They contain at least two aromatic help of fine forceps under a stereozoom binocular amino acids with tryptophan in position 4. The AKHs exert a microscope. The tissues were immediately put in to ice cold wide range of effects- it acts on the fat body to mobilize 80% methanol and stored at -4˚C until extraction. They were stored lipids and carbohydrates, activate glycogen sonicated for 1 min on ice using ultrasonicator. The extract phosphorylase, accumulate cAMP3,4 and inhibit the synthesis was centrifuged at 4˚C and 10000 rpm for 10 min, the of proteins5,6, lipids7, and RNA8. AKHs exhibit similarity supernatant was collected in to an eppendorf tube and with vertebrate hormone at functional level. AKHs resemble vacuum dried. The dried supernatant was stored at -4˚C until glucagon9, a peptide hormone from the α-pancreatic islets used for analysis. The synthetic peptide Lom-AKH cells in vertebrates and the vertebrate catecholamine (GenScript Corp; USA) was dissolved in 80% methanol adrenalin10. Another vertebrate candidate whose function can (HPLC grade). Required concentrations of peptide solutions be compared with AKHs is vertebrate adiponectin, a hormone were prepared from this stock solution. discovered recently from vertebrate adipose tissue11 which Preparation of standard solutions increases the oxidation of fat and thereby reduces the The lipid standard solution was prepared by dissolving 500 intracellular triglyceride content of the liver and muscle and mg of chromatographically pure glycerol trioleate in 50 ml of increases the cellular sensitivity to insulin. chloroform. A working standard solution was prepared by The present investigation was carried out to study the effect diluting 1 ml of stock solution to 10 ml with chloroform so as of brain-retrocerebral complex extract of Orthaga exvinacea to get 1 µg lipid/1 µl. Appropriate volumes of this solution on lipid and carbohydrate metabolism using various in vivo were taken for the quantitation of lipids in the experimental (heterologous bioassay in Iphita limbata) and in vitro samples. experiments and to separate the peptide hormone by Page 271 D. Umadevi et al. IRJP 2012, 3 (2) A standard stock solution for carbohydrate estimation was from all vials to determine the amount of lipids and sugars prepared by dissolving 100 mg trehalose in 10 ml distilled using the methods already described above and using water. From this, different solutions having sugar ranging standard values and the changes in lipid and sugar titres were from 10 µg-200 µg were prepared by diluting with distilled measured colorimetrically with appropriate standards. water. HPLC analysis Detection of biological activity in vivo The dried extract made from the retrocerebral complexes The dried brain retrocerebral extract prepared was dissolved from O. exvinacea was resuspended in 20 ml of 80% in insect saline (the buffer used for bioassays and fat body methanol (HPLC grade). The extract was filtered using a incubation contained NaCl, 130 mM; KCl, 5 mM; Na2HPO4, sample filtration unit (Millipore, U.S.A) with pore size 0.45 1.9 mM and K2HPO4, 1.7 mM, the pH was adjusted to 7.5) to µm. A sample of 20 µl of the brain-retrocerebral extract was get a final concentration of one gland pair equivalent (gpe) directly injected into the instrument by a Hamilton micro- per 5 µl. A sample of haemolymph (2 µl) was collected syringe. HPLC separations were carried out using Shimadzu directly from the cut end of antenna in to the precalibrated system (SPD M 10AVP, LC 10 ATVP, LC-10 ATVP) with a capillary tube. The sample was transferred in to the bottom of reversed phase column (C18) 250 mm long, 4.6 mm i.d. The a small test tube. An aliquot of the extract (5 µl) was then separation was done in a binary gradient from 43% to 53% injected in to the acceptor plant bug, Iphita limbata, through solvent B in 20 min with a flow rate of 1 ml/min. the intersegmental membrane between thoracic and first Trifluoroacetic acid (TFA) 0.01% in water (HPLC grade) was abdominal segment. The needle was kept in position for a used as solvent A, solvent B was 60% acetonitrile in solvent while to allow mixing of the material with haemolymph and A. All the solvents were filtered through 0.45 µm pore size to avoid loss through oozing haemolymph droplet. After 60 Millipore filter. The eluants were monitored at 210 nm using min, haemolymph samples were collected (2 µl) directly a photo diode array (PDA) detector. One minute fractions from the cut end of the other antenna in to another capillary starting from 1 min up to 20 min were collected manually, tube and were then transferred in to the bottom of another test dried by vacuum concentrator, and were used for testing their tube. Haemolymph samples taken before injections were biological activities. Similarity of retention time of any taken as controls and 60 min after injection as experimentals. materials of the extract of O. exvinacea with that of already Similar experiments were carried out using 5 µl of insect reported peptide, the synthetic locust AKH was tested by saline instead of extract. Haemolymph samples collected in overlaying this profile with that obtained for Lom- AKH in a various experiments were used to measure lipid and similar HPLC run. carbohydrate concentrations. The total concentration of lipids Hyperlipaemic and hyperglycaemic activities of fractions in the haemolymph samples were measured by separated on HPLC phosphovanillin method12 and the total sugar concentration of One minute fractions of the extract of retrocerebral the haemolymph were measured by anthrone method13. For complexes of O. exvinacea fractionated on HPLC, were the quantitation of haemolymph lipids, concentrated collected manually.