The hyperglycaemic activity of locust adipokinetic hormone

ABSTRACT. Pure adipokinetic hormone (AKH) extracted from the glandular lobes of locusts induces hyperglycaemia in cockroaches. Larger doses (20–200 pmol) of AKH are required to induce hyperglycaemia in the cockroach than are required to produce hyperlipaemia in locusts (1–20 pmol).     

Studies on the purification of locust adipokinetic hormone

$\text{Locusta}$ adipokinetic hormone (AKH) has been purified from glandular lobes of corpora cardiaca by a series of chromatographic steps that have allowed a 58 to 77% recovery of activity. Methanolic extracts of the glandular lobes were fractionated on Sephadex LH-20 and then on thin-layer plates. The average recovery of AKH activity after separation on Sephadex LH-20 approached 100%, whereas recoveries of between 58 to 77% were obtained after TLC on cellulose plates. In practice, the highest recoveries were obtained with separations of large amounts of the hormone on cellulose plates. In the course of this investigation it was found that AKH activity can be extracted efficiently from aqueous solutions using Florisil. The hormone activity was recovered from the Florisil by eluting with 80% methanol. The purified hormone was found to be homogeneous in composition when subjected to rechromatography on Sephadex LH-20 and to amino acid analysis of the acid hydrolysate. From the amino acid analysis we estimate that one pair of glandular lobes contains on average 188 pmoles of AKH.     

Quantitative studies on the release of locust adipokinetic hormone

Fractionation of methanolic extracts of haemolymph on Sephadex LH-20 made possible the measurement of the titre of adipokinetic hormone in the haemolymph of locusts. Experimentally produced high concentrations of haemolymph carbohydrate caused a delay in the mobilization of lipid during flight, and very low titres of the hormone were present in the haemolymph of locusts injected with trehalose immediately before a 25 min flight. In these locusts flight speed was higher than saline-injected controls. Although delayed lipid mobilization during flight was also seen in locusts injected with sucrose, sucrose is not utilized for flight metabolism and flight speed was not increased by the injection. Tentative estimates of the release rate (c. 1000pg/20min flight) and half life (c. 20 min) of adipokinetic hormone during flight are made. The results described suggest that during flight the rate at which trehalose disappears from the haemolymph does not play a major role in the initiation of the release of adipokinetic hormone.     

Breakdown of locust adipokinetic hormone I by malpighian tubules of Schistocerca gregaria

The degradation of synthetic adipokinetic hormone I (AKHI) was studied using homogenates of the Malpighian tubules (MTs) from Schistocerca gregaria. Three major breakdown products, AKHI-1, AKHI-2 and AKHI-3, were found when the reaction mixture was subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). At pH 7.5 AKHI-1 and AKHI-3 were found in small amounts only, however, at pH 8.0 large amounts could be detected. It was concluded that more than one degradative enzyme must be responsible for the complete breakdown of AKHI. Analysis of the products showed that AKHI-1 contained the AKHI residues 1–6 (pGlu-Leu-Asn-Phe-Thr-Pro), AKHI-2 only residue 8 (Trp) and AKHI-3 residues 8–10 (Trp-Gly-Thr-NH₂). None of the breakdown products exhibited lipid-mobilizing activity when tested at doses of 20 pmol per locust.     

Effect of adipokinetic hormone on the structure and properties of lipophorin in locusts

The reversible association of a low molecular weight hemolymph protein (mol wt 20,000 estimated by SDS-polyacrylamide gel electrophoresis) with lipophorin, following treatment with adipokinetic hormone (AKH), was demonstrated by density gradient ultracentrifugation and by specific precipitation of lipophorin from the hemolymph of resting and AKH-injected locusts. The injection of AKH also stimulated the loading of diacylglycerol from fat body by lipophorin and resulted in a lower density lipophorin ("activated lipophorin"). The activated lipophorin particles (diameter 21.7 +/- 3.0 nm, 15.8 to 33.6 nm) were larger and more heterogeneous in size than those of resting lipophorin (14.5 +/- 1.6 nm, 11.9 to 19.2 nm). A theoretical analysis based on the experimental data (e.g., density gradient profile, electron microscopic observation, and diacylglycerol content) suggests that very large lipophorin particles result from intermolecular fusion of the lipophorin molecules that are activated by AKH. Attempts to demonstrate the effect of AKH on the structure of lipophorin, in vitro, were unsuccessful.     

Synthesis of adipokinetic hormone (AKH-I) in the locust brain

Methanol extracts of vitellogenic female locust brains contain two factors that inhibit protein synthesis in fat body tissue excised from such individuals. One of these factors (BI) elicits lipid mobilization when injected into adult male locusts. The retention times of BI on an RP-18 column and on an RP-4 column are identical to those of synthetic locust adipokinetic hormone (AKH-I) on each of these columns. Half maximal inhibition of protein synthesis in excised adult locust fat bodies is exerted by 0.05 brain extract equivalents of BI, which is equivalent to activity elicited by 1.5 pmol of AKH-I, as previously determined by AKH-radioimmunoassay. Enzymatic hydrolysis of the N-terminal pyroglutamate, followed by amino acid sequence analysis, indicates that the structure of BI is similar to that of the decapeptide AKH-I synthesized in the glandular lobe of the locust corpora cardiaca (CC). Incorporation of [5-³H]tryptophan into BI of locust brains incubated in vitro indicates that the AKH-I present in the brain is synthesized in situ and is not transported from the CC. Similar incorporation of radiolabel into AKH-I is obtained when excised CC are incubated in vitro.     

Adipokinetic hormone causes formation of a low density lipophorin in the house cricket,Acheta domesticus

In the house cricket, Acheta domesticus, injection of adipokinetic hormone induces the formation of a low density lipophorin via uptake of diacyglycerol from the fat body. Cricket low density lipophorin contains apolipophorin-III, which was purified and partially characterized. Cricket apolipophorin-III has a molecular weight of about 18,000, is not glycosylated, and has an isoelectric point of 4.8. Its amino acid composition is more similar to apolipophorin-III from Locusta migratoria than to that from Manduca sexta. The amino terminal sequence of cricket apolipophorin-III shows only limited homology to the amino terminal sequences of apolipophorin-III from L. migratoria and M. sexta.     

Primary structures of locust adipokinetic hormones II

Adipokinetic hormones II from corpora cardiaca of the locusts Schistocerca gregaria and Locusta migratoria, respectively, have been isolated and their primary structures elucidated. Both octapeptides are N-terminally blocked by a 5-oxoproline (pyroglutamate) residue and had to be cleaved by 5-oxoprolyl-peptidase to make them available for the Edman degradation method carried out with a gas-phase sequencer. The C-termini are blocked as both peptides are not cleaved by carboxypeptidase A; the presence of C-terminal amide groups is very likely. AKHII (S. gregaria) Glu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH2 AKHII (L. migratoria) Glu-Leu-Asn-Phe-Ser-Ala-Gly-Trp-NH2.     

Spontaneous aggregation of locust lipophorin during hemolymph collection

Density-gradient ultracentrifugation, gel permeation chromatography, polyacrylamide gel electrophoresis and electron microscopy were used to provide an experimental basis for the proposal that locust lipophorin aggregates spontaneously during the collection of hemolymph through a puncture in the neck or coxal region of the insect.The evidence presented in this paper supports the above proposal and clearly demonstrates that the “puncture” method promotes the aggregation of lipophorin molecules and results in the formation of large aggregates as an artificial product. Thus, the use of this method should be discouraged for the study of lipophorin in locusts.     

Synthesis of a homodimer neurohormone precursor of locust adipokinetic hormone studied by in vitro translation and cDNA cloning

The homodimer neurohormone precursor P1, consisting of 41 residue subunits or A-chains, is synthesized by the glandular neurosecretory cells of the corpora cardiaca (CC) of the locust Schistocerca gregaria. Processing of P1 generates two copies of a 10 amino acid peptide neurohormone (AKH I) and one copy of a homodimer peptide (APRP 1). Here we show that the P1 dimer is formed from two independent A-chain translation products. Translation of CC mRNA in vitro produces a prominent 6.4 kd protein, the synthesis of which can be blocked by oligonucleotides hybridizing to mRNA encoding the A-chain. Northern blot experiments suggest that the 6.4 kd protein is produced by an integral of 500 base mRNA. cDNA cloning reveals a pre-A-chain structure in which a single copy of the A-chain is preceded by a 22 amino acid signal peptide. This evidence indicates that the P1 dimer is synthesized by coupling of very small translational products rather than by folding and processing of a larger protein containing more than one copy of the A-chain.     

Possible involvement of monoamines in the release of adipokinetic hormone in the locust Schistocerca gregaria.

1. The adipokinetic hormone release, which can be induced by anticholinesterases, is reduced by depleting the content of monoamines in the nervous system. 2. The participation of monoamines in the pathway of release of adipokinetic hormone is studied in vivo and in vitro. 3. A possible mechanism for anticholinesterase-induced release of this hormone involving cholinergic and aminergic transmission is postulated.     

Displacement of apolipophorin III from the surface of low density lipophorin by human apolipoprotein A-I

A hybrid low density lipophorin particle (LDLp) was prepared by incubation with human apolipoprotein (apo) A-I in vitro. ApoA-I associated with LDLp in a concentration dependent, saturable manner which was accompanied by dissociation of apolipophorin III (apoLp-III). The apoA-I hybrid LDLp had the same lipid composition, density and morphology as native LDLp indicating that displacement of apoLp-III by apoA-I did not affect its structural properties. The molar ratio of apoLp-I:apoLp-II:apoLp-III was maximally reduced from 1:1:16 to 1:1:2 in native versus hybrid LDLp with the latter particle binding 7 molecules of apoA-I. The inability of apoA-I to displace the remaining 2 apoLp-III supports the concept that these apoLp-III molecules are not equivalent to the other fourteen. Native and hybrid LDLp particles were both metabolized to high density lipophorin in vivo. The displacement reaction represents a novel method for the production of apolipoprotein hybrids of LDLp and the results indicate that apoA-I has an inherently higher affinity for lipid surfaces than apoLp-III.     

A possible role of Schisto FLRFamide in inhibition of adipokinetic hormone release from locust corpora cardiaca

The distribution and actions of FMRFamide-related peptides (FaRPs) in the corpora cardiaca of the locust Locusta migratoria were studied. Antisera to FMRFamide and SchistoFLRFamide (PDVDHVFLRFamide) label neuronal processes that impinge on glandular cells in the glandular lobe of the corpora cardiaca known to produce adipokinetic hormones. Electron microscopic immunocytochemistry revealed that these FaRP-containing processes form synaptoid contacts with the glandular cells. Approximately 12% of the axon profiles present in the glandular part of the corpus cardiacum contained SchistoFLRFamide-immunoreactive material. Retrograde tracing of the axons in the nervus corporis cardiaci II with Lucifer yellow revealed 25–30 labelled neuronal cell bodies in each lateral part of the protocerebrum. About five of these in each hemisphere reacted with the SchistoFLRFamide-antiserum. Double-labelling immunocytochemistry showed that the FaRP-containing processes in the glandular lobe of the corpora cardiaca are distinct from neuronal processes, reacting with an antiserum to the neuropeptide locustatachykinin. The effect of the decapeptide SchistoFLRFamide and the tetrapeptide FMRFamide on the release of adipokinetic hormone I (AKH I) from the cells in the glandular part of the corpus cardiacum was studied in vitro. Neither the deca- nor the tetrapeptide had any effect on the spontaneous release of AKH I. Release of AKH I induced by the phosphodiesterase inhibitor IBMX, however, was reduced significantly by both peptides. These results point to an involvement of FaRPs as inhibitory modulators in the regulation of the release of adipokinetic hormone from the glandular cells.     

Preliminary characterization of glycogen phosphorylase activating hormone and adipokinetic hormone from Manduca sexta corpora cardiaca

ABSTRACT. An attempt was made to separate glycogen phosphorylase activating hormone (GPAH) and adipokinetic hormone (AKH) from the corpora cardiaca (CC) of the moth Manduca sexta (Lepidoptera: Sphingidae) by separating extracts of CC on various chromotographic media, but it was not possible to conclude whether GPAH and AKH are activities of one or of two different peptides. Both activities elute together from glass beads, from Sephadex G-25 and from Sephadex LH-20 columns. In the separation experiments with glass beads and G-25 the activities eluted as a single peak, but using LH-20 we found two peaks exhibiting both activities. The major peak eluted at 1.25 × V_t, which is very similar to locust AKH, while the smaller second peak eluted at O.74 × V _t. Cross injections of CC extracts from M. sexta into Locusta migratoria and CC extracts from L. migratoria into M. sexta suggest that GPAH and the AKH from M. sexta are not identical with the decapeptide AKH from locusts.     

Binding of lipophorin to the fat body of the migratory locust

The interaction of locust high density lipophorin (HDLp) with pieces of fat body tissue was studied at 33°C using a radiolabelled ligand binding assay. Under the assay conditions, binding of tritium-labelled HDLp ([³H]HDLp) was demonstrated to correlate linearly with tissue concentration up to ∼ 7 mg of fat body protein per ml of incubation medium. The [³H]HDLp binding that was displaceable by a 20-fold excess of unlabelled HDLp (which is an approximation of the specific binding) reached equilibrium after ∼ 2 h, whereas low levels of non-displaceable binding increased linearly during this time interval. Analysis of the concentration dependent total binding of [³H]HDLp revealed the presence of a specific binding site with an equilibrium dissociation constant of K_d = 3.1 (±0.5) × 10⁻⁷ M and a maximal binding capacity of 9.8 (±0.5) ng μg⁻¹ tissue protein. Competition experiments demonstrated that the affinity of unlabelled HDLp for the binding site is similar to the affinity of [³H]HDLp. Unlabelled low density lipophorin (LDLp), however, was shown to have an approx. 20-fold lower affinity for the binding site.     

Interaction of lipophorin with the plasma membrane of locust flight muscles

Binding of high-density lipophorin (HDLp) to a plasma membrane preparation of locust flight muscle tissue was studied using a radiolabelled ligand binding assay and ligand blotting techniques. Analysis at 33 degrees C of the concentration-dependent total binding of tritium-labelled HDLp ([3H]HDLp) to the membrane preparation revealed the presence of a single specific binding site with an equilibrium dissociation constant of Kd = 9 (+/- 2) X 10(-7) M and a maximal binding capacity of 84 (+/- 10) ng X (micrograms protein)-1. Unlabelled HDLp as well as unlabelled low-density lipophorin (LDLp) competed with [3H]HDLp for binding to the identified binding site. In addition, ligand blotting demonstrated that both HDLp and LDLp bind specifically to a 30-kDa protein in the plasma membrane preparation, suggesting the involvement of this protein in the binding of lipophorins to the isolated membranes. A possible relationship between the identified binding of lipophorins and the observed co-purification of lipophorin lipase activity with the plasma membranes is discussed.     

Further characterization of low density mononuclear cells: FACS-assisted analysis of human MLR stimulators

Previously, we reported that all of the potent stimulators of the allogeneic mixed leukocyte reaction (MLR) are contained in a heterogeneous low density fraction of human peripheral blood mononuclear (PBM) cells. We have further characterized human MLR stimulators by staining them with highly specific monoclonal antibodies, and then analyzing and separating them with the fluorescence-activated cell sorter. These studies revealed two populations of low density cells with potent allogeneic stimulatory activity. One population is a monocyte subset that reacts with anti-OKM1, MO.2, and expresses C3b as well as Fc-IgG receptors. The second population exhibits even greater stimulatory capacity and does not express any of these monocyte markers. Moreover, these cells are not phagocytic and do not react with alpha-naphthyl esterase. They comprise approximately 10% of the low density fraction or 0.5% of PBM. These cells are most likely lymphoid dendritic cells, described in various species as potent MLR stimulators. In contrast to the allogeneic MLR, only the low density cell type exhibiting dendritic cell characteristics induced a potent autologous MLR.