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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Treatment of low density lipophorin with lipoprotein lipase: diacylglycerol content has no effect on dissociation of apolipophorin III from low-density lipophorin.

The mechanism of the conversion of low-density lipophorin (LDLp) to high-density lipophorin (HDLp) in long-distance flight insects was investigated using a lipoprotein lipase from a bacterium, Alcaligenes sp. Diacylglycerol of LDLp was steadily hydrolyzed in vitro by the lipase, resulting in a 90% loss of diacylglycerol from LDLp during incubation. The "lipase-treated LDLp" thus obtained still contained associated apolipophorin-III (apoLp-III). These data suggest that the dissociation of apoLp-III is independent of the depletion of diacylglycerol from LDLp, and that the decrease in particle diameter caused by the depletion of diacylglycerol does not force the dissociation of apoLp-III from the lipophorin particle. Some physico-chemical properties of the lipase-treated LDLp were measured.     

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.     

Binding of locust high-density lipophorin to fat body proteins monitored by an enzyme-linked immunosorbant assay

Binding of locust high-density lipophorin (HDLp) to fat body proteins coated on immunoassay plates was studied using the ELISA method and ligand blotting techniques. HDLp binding proved to be correlated with the amount of fat body protein coated. From the concentration-dependent total HDLp binding an equilibrium dissociation constant could be calculated (Kd = 1.6 x 10(-8) M). Heparin inhibits the HDLp binding, indicating that positively charged groups are involved in the HDLp-fat body interaction. These groups were shown to be arginyl residues, as the arginine-specific treatment of HDLp by 1,2-cyclohexanedione resulted in a approximately 50% decrease in the binding ability of HDLp. HDLp binding is also affected by the pH. A decrease from pH 7.5 to pH 6.5 increases the binding affinity by approximately 250%. A monoclonal antibody specific for apolipophorin-II (apoLp-II) hampered the HDLp binding significantly, whereas a monoclonal anti-apoLp-I had no effect. Locust fat body HDLp binding proteins are highly specific for locust HDLp.     

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm,Bombyx mori

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24 h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.     

Further characterization of natural killer cells induced by Kunjin virus

The natural killer (NK) cell induced one to two days after Kunjin virus infection of BALB/c mice is cytotoxic for a wide range of syngeneic, allogeneic and xenogeneic cell lines. It is also weakly cytotoxic for some non-malignant cells including mouse fibroblasts, macrophages and thymocytes, but not lymph node cells. Levels of lysis of non-tumour target cells are dependent on their genotype. Furthermore, malignant cell lines may become resistant following transplantation in vivo then susceptible again after culture in vitro. The virus-induced NK cell is elicited as readily in athymic (nude) as in normal mice. X-irradiation inhibits its development if administered prior to infection. It is labile on culture at 37 degrees. The cell carries Fc receptors but its NK activity is not antibody-dependent.     

Further characterization of the silkworm diapause hormone A

One of two diapause hormones (DH-A) was studied. DH-A was stable to acids, bases (except to 1·0 N NaOH), acylation agents and periodate oxidation. The hormonal activity was quickly lost by trypsin as well as by non-specific proteolytic enzymes but slowly or hardly at all by α-chymotrypsin and carboxypeptidase A. The hormone contains 14 kinds of amino acids and 2 kinds of amino sugars. The amino sugars appear not to be essential for the hormonal activity.     

Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein

Insect plasma lipid transfer particle (LTP) catalyzes vectorial net transfer of diacylglycerol (DAG) from Manduca sexta larval high density lipophorin (HDLp-L) to human low density lipoprotein (LDL) producing an LDL of lower density and lipophorin subspecies of higher density. At equilibrium, a stable DAG-depleted very high density lipophorin species (density = 1.25 g/ml) is formed. Electrophoretic analysis of the substrate and product lipoproteins showed that apoprotein exchange or transfer between human LDL and lipophorin did not occur during the lipid transfer reaction. Facilitated net transfer of cholesteryl ester, free cholesterol, and phospholipid occurred to a much lower extent than DAG net transfer, indicating that under these conditions, LDL serves as a sink for lipophorin-associated DAG. This reaction, therefore, provides a method whereby the mass of lipid associated with human LDL can be modified in vitro without alteration of its apoprotein component. The DAG content of LDL increased in a linear manner with respect to LTP concentration and time during the initial phase of the reaction, demonstrating the utility of this system as a quantitative assay method for LTP-mediated net DAG transfer. When [3H]DAG-labeled LDL was prepared and employed in transfer experiments with unlabeled lipophorin, labeled DAG was recovered in the HDLp-L fraction. The amount of labeled DAG recovered in the HDLp-L fraction was dependent on the ratio of LDL to HDLp-L in the reaction. Thus, in this system, LTP-mediated DAG redistribution is bidirectional, suggesting that the final equilibrium distribution of lipid may be dictated by the properties of potential donor/acceptor lipoproteins rather than by an inherent particle substrate specificity of LTP.