Lutz Auerswald

Mode of action of neuropeptides from the adipokinetic hormone family

Neuropeptides of the adipokinetic hormone (AKH) family regulate inter alia mobilisation of various substrates from stores in the fat body of insects during episodes of flight. How is this achieved? In insects which exclusively oxidise carbohydrates for flight (cockroaches), or which oxidise carbohydrates in conjunction with lipids (locusts) or proline (a number of beetles), the endogenous AKHs bind to a G(q)-protein-coupled receptor, activate a phospholipase C and the resulting inositol trisphosphate releases Ca(2+) from internal stores. In addition, influx of extracellular Ca(2+) is increased and, via a kinase cascade, glycogen phosphorylase is activated, glucose-1-phosphate produced, and transformed to trehalose, which is released into the haemolymph. In locusts, additionally, adenylate cyclase is activated and cyclic AMP is synthesised. In insects which use lipids for sustained flight (locust, tobacco hornworm moth) or proline for flight (certain beetles), adenylate cyclase is activated after the AKHs bind to their respective G(s)-protein-coupled receptor. The resulting cyclic AMP, together with the messengers intra- and extracellular Ca(2+), activate a triacylglycerol lipase, which results in the production of 1,2 diacylglycerols (in locusts, moths) or (hypothetically) free fatty acids (fruit beetle).

Beetles' choice--proline for energy output: control by AKHs.

Many beetle species use proline and carbohydrates in a varying ratio to power flight. The degree of contribution of either fuel varies widely between species. In contrast, dung beetle species investigated, thus far, do not have any carbohydrate reserves and rely completely on proline to power energy-costly activities such as flight and, probably, walking and ball-rolling. While the fruit beetle, Pachnoda sinuata, uses proline and carbohydrates equally during flight, proline is solely oxidised during endothermic pre-flight warm-up, as well as during flight after prolonged starvation. Thus, proline seems to be the essential fuel for activity in beetles, even in flightless ones and in those that use proline in combination with carbohydrates; the latter can be completely substituted by proline in certain circumstances. It is apparent from the rapid decline of energy substrates in flight muscles and haemolymph after the onset of flight that mobilisation of stored fuels of the fat body is necessary for prolonged flight periods. This task is performed by AKH-type neuropeptides. In beetles, like in other insects, these peptides mobilise glycogen via activation of glycogen phosphorylase. They also stimulate proline synthesis from alanine and acetyl-CoA in the fat body. Acetyl-CoA is derived from the beta-oxidation of fatty acids and we propose that the neuropeptides activate triacylglycerol lipase.

The fate of proline in the African fruit beetle Pachnoda sinuata

Abstract Metabolic pathways of proline consumption in working flight muscles and its resynthesis were investigated in the African fruit beetle, Pachnoda sinuata . Mitochondria isolated from flight muscles oxidise proline, pyruvate and α-glycerophosphate, but not palmitoyl-carnitine. At low proline concentrations, the respiration rate during co-oxidation of proline and pyruvate is additive, while at high proline concentrations it is equal to the respiration rates of proline oxidation. Flight muscles have high activities of alanine aminotransferase and NAD + -dependent malic enzyme which are involved in proline metabolism. Glycogen phosphorylase and glyceraldehyde-3-phosphate dehydrogenase (carbohydrate breakdown) also display high activities, whilst 3-hydroxyacyl-CoA dehydrogenase (fatty acid oxidation) showed low activity. During the oxidation of proline, mitochondria isolated from flight muscles produce equimolar amounts of alanine. The rates of oxygen consumption by the mitochondria during this process lead to the conclusion that proline is partially oxidised. This is confirmed by the incorporation of radiolabel from pre-injected [U- 14 C] proline into alanine during a flight experiment with P. sinuata . Proline is resynthesised, in vitro, from alanine and acetyl-CoA in the fat body. High activities of enzymes catalysing such pathways (alanine aminotransferase, 3-hydroxyacyl-CoA dehydrogenase and NADP + -dependent malic enzyme) were found. The in vitro production of proline from alanine is equimolar suggesting that resynthesis of one proline molecule is accomplished from one alanine molecule and one acetyl-CoA molecule. One source of the acetyl-CoA for the in vitro synthesis of proline is the lipid stores of the fat body. Proline synthesis by fat body tissue is controlled by feedback. Alanine aminotransferase is competitively inhibited by high proline concentrations.

Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MS(n) (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.

Substrate usage and its regulation during flight and swimming in the backswimmer, Notonecta glauca

Abstract.  The metabolites that are generally used by insects during exercise are present in quite different concentrations in the haemolymph of the backswimmer Notonecta glauca L. Lipids are most abundant (between 10 and 20 mg/mL), whereas carbohydrates (2–3 mg/mL) and proline (approximately 1 mg/mL) are at very low concentrations. Injection of an extract of conspecific corpora cardiaca causes pronounced hyperlipaemia in the backswimmer. A neuropeptide with the same effect was isolated from the corpora cardiaca in a single high‐performance liquid chromatography (HPLC) step; the primary sequence was deduced from mass spectrometric measurements (matrix‐assisted laser desorption/ionization‐time of flight and electrospray quadrupol time‐of‐flight mass spectrometry) of whole corpora cardiaca, and the mass was confirmed in the HPLC fraction that had adipokinetic activity. The biologically active octapeptide has the sequence pGlu‐Val‐Asn‐Phe‐Ser‐Pro‐Ser‐Trp amide, which was characterized previously from the corpora cardiaca of the Emperor dragonfly, Anax imperator, and denoted Anaim‐adipokinetic hormone (AKH). The synthetic Anaim‐AKH peptide causes lipid mobilization when injected at a dose of 1 pmol into N. glauca. When other synthetic AKH members that occur in Hemiptera are injected into N. glauca at the same dose, the hyperlipaemic responses are significantly lower than after injection of Anaim‐AKH. Because only lipids increase upon activity, such as continuous swimming for 1 h or during a 1‐h rest period after a 3‐min flight episode in the laboratory, it is assumed that Anaim‐AKH serves as a true adipokinetic hormone in the backswimmer during bouts of natural swimming and flight.

Effects of metabolic neuropeptides from insect corpora cardiaca on proline metabolism of the African fruit beetle, Pachnoda sinuata.

The effect of neuropeptides from the corpora cardiaca of the fruit beetle Pachnoda sinuata on proline metabolism has been investigated in vivo. Conspecific injections of a crude extract from corpora cardiaca cause an increase of the concentration of proline in the haemolymph by nearly 20% and a decrease of the concentration of alanine, the precursor in proline synthesis, by about 64% when compared with a water-injected group. Purification of an extract of corpora cardiaca on reversed-phase liquid chromatography revealed two distinct UV absorbance and fluorescence peaks that cause hyperprolinaemia in the fruit beetle. The major peak is the previously identified octapeptide Mem-CC; the second peak is also a peptide, but its primary sequence remains, as yet, unidentified. Synthetic Mem-CC elicited time- and dose-dependent increases/decreases of the concentrations of proline and alanine in the haemolymph respectively. Furthermore, the receptor for this peptide seems to be specific in P. sinuata: only peptides of the large family of adipokinetic hormones with an Asp, Asn or Gly residue at position 7 could elicit biological activity, whereas those with a Trp, Ser or Val residue at this position did not have any activity.

Metabolic changes in the African fruit beetle, Pachnoda sinuata, during starvation

Specimens of the fruit beetle Pachnoda sinuata were starved for up to 30 days. The weight of the beetles declined consistently throughout the starvation period. Concentrations of carbohydrates and alanine in flight muscles, fat body and haemolymph decreased rapidly after onset of starvation, while the concentration of proline remained high. Whereas the lipid concentrations in the haemolymph did not change significantly upon starvation, the lipid content in flight muscles and fat body decreased significantly.Beetles that had been starved for 14 days responded to injection of Mem-CC, the endogenous neuropeptide from its corpora cardiaca, with hyperprolinaemia and a decrease in the alanine level, but no such effect was monitored after prolonged starvation of 28 days. Regardless of the period of starvation, Mem-CC injection could not cause hypertrehalosaemia or hyperlipaemia, although carbohydrates were increased in fed beetles after injection.Flight ability of beetles that had been starved for 15 or 30 days was apparently not impaired. During such periods, beetles used proline exclusively as fuel for flight as evidenced by the increase in the level of alanine in the haemolymph and decrease of the level of proline; the concentrations of carbohydrates and lipids remained unchanged.Activities of malic enzyme and alanine aminotransferase (enzymes involved in transamination in proline metabolism), glyceraldehyde-3-phosphate dehydrogenase (enzyme of glycolysis), 3-hydroxyacyl-CoA dehydrogenase (enzyme of beta-oxidation of fatty acids) and of malate dehydrogenase (enzyme of Krebs cycle) were measured in fat body and flight muscles. In flight muscle tissue the maximum activity of NAD(+)-dependent malic enzyme increased, while that of glyceraldehyde-3-phosphate dehydrogenase decreased during starvation, and malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and alanine aminotransferase were unchanged. In fat body tissue, activities of NADP(+)-dependent malic enzyme and 3-hydroxyacyl-CoA dehydrogenase increased during food deprivation and activities of glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and alanine aminotransferase remained unchanged.

Insect neuropeptides regulating substrate mobilisation

Insect flight muscles perform their work completely aerobically, and working flight muscles are known to be the most metabolically active tissue in nature with respect to oxygen uptake. Various substrates can be oxidised and utilised as fuels for flight. Insects such as Diptera and Hymenoptera power their flight muscles by the breakdown of carbohydrates, whereas lipids are the predominant fuel for the contracting flight muscles of Lepidoptera and Orthoptera during long-distance flight. The amino acid proline can also be used as a substrate for flight, especially in tsetse flies and beetles (Colorado potato beetle, blister beetles, certain dung beetles). Neuropeptides from the corpus cardiacum are well-known to be responsible for carbohydrate and lipid mobilisation from the fat body. In this short overview, we show that peptides belonging to the large adipokinetic hormone/red pigment-concentrating hormone family are also thought to be the chemical messengers for initiating proline homeostasis. The peptides...

The role of calcium in the activation of glycogen phosphorylase in the fat body of the fruit beetle, Pachnoda sinuata, by hypertrehalosaemic hormone

The role of calcium in the mediation of the hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in vitro and in vivo in the cetoniid beetle Pachnoda sinuata. The presence of Mem-CC increases the influx of extracellular 45Ca(2+) into the fat body as well as the efflux of 45Ca(2+) from pre-loaded fat body into the incubation medium. Extracellular calcium is essential to exert maximal activation of the fat body glycogen phosphorylase by saturating doses of Mem-CC (0.3 nM). This effect of extracellular Ca(2+) is dose-dependent: maximal activation of glycogen phosphorylase by Mem-CC is achieved at calcium concentrations of approximately 1.2 mM and the ED(50) was calculated to be 0.6 mM. Both, thimerosal and thapsigargin caused a stimulation of carbohydrate metabolism in the fat body, suggesting that a release of calcium from the endoplasmic reticulum is involved in this process. However, neither entry of extracellular calcium nor the release from the endoplasmic reticulum are sufficient alone for a full activation of the phosphorylase. The results of the present study suggest that calcium from extracellular as well as from intracellular sources is part of the second messenger system for the transduction of the hypertrehalosaemic signal of Mem-CC in the fat body of P. sinuata.

Flight substrates and their regulation by a member of the AKH/RPCH family of neuropeptides in Cerambycidae

The pattern of metabolic changes during tethered flight with lift-generation was investigated in two South African species of long-horned beetles (family: Cerambycidae), namely Phryneta spinator and Ceroplesis thunbergi. Energy substrates were measured in haemolymph and flight muscles at rest, after a flight period of 1 min at an ambient temperature of 25-29 degrees C, and 1 h thereafter. Flight diminished the levels of proline and carbohydrates in the haemolymph and proline and glycogen in the flight muscles of both species, and caused an increase in the levels of alanine in both compartments. The concentration of lipids in the haemolymph, however, was not changed upon flight in either species. The resting period of 1 h following a 1 min flight episode, was sufficient to reverse the metabolic situation in haemolymph and flight muscles to pre-flight levels in both species. Purification of an extract of the corpora cardiaca from the two beetle species on RP-HPLC, resulted in the isolation and subsequently in the identification (by mass spectrometry, Edman degradation and RP-HPLC) of an octapeptide of the AKH/RPCH family, denoted Pea-CAH-I (pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trpamide), present in each species. It was demonstrated that low doses of Pea-CAH-I elicited increases in the concentration of proline, as well as carbohydrates, in the haemolymph of both species. The levels of lipids, however, remained unchanged upon injection of this peptide. It is concluded that, upon stimulation by flight, the peptide Pea-CAH-I is released from the corpus cardiacum of a cerambycid beetle and is responsible for the regulation of the major flight substrates, proline and carbohydrates, of these beetles.