We had a brief, straight forward look at the major pathways of oxidative metabolism, namely glycolysis, ß-oxidation, Kreb's cycle and the electron transport chain. We should be familiar with where these energy-yielding pathways are located within cells, and what principle substrates are taken into these pathways. Let us expand our information just a bit, by introducing the idea of metabolic scope. Rates of metabolism can be measured by oxygen uptake rates. When oxygen uptake is measured at rest and during rigorous activity the range between these minimal and maximal metabolic rates is called the metabolic scope. The point to be made is that insect flight muscle tissue can be characterized as having the largest metabolic scope in the animal kingdom. Here are some examples:

Metabolic rates during flight are about 4 to 6 times higher than metabolic rates at rest in hummingbirds. The actual values are about 10 to 14 ml oxygen /gram body weight/ hour at rest and 40 to 85 ml oxygen /gram body weight/ hour at rest. By comparison, metabolic rates during flight in some insects can be 200 times the resting rates. Some butterflies increase their metabolic rates from 0.5 to 100 ml oxygen /gram body weight/ hour when they begin flying. These data just make the point that differences between resting and flight metabolic rates - that is, metabolic scopes - are much larger in insects than they are in even the most rapidly metabolizing vertebrates - hovering birds. Such large metabolic scopes pose the problem of supplying fuel to the flight muscles such that the hemolymph supply responds to changes in metabolic demand. We can imagine some odd things.

1) soon after initiation of flight the hemolymph reserves of fuels are used up. If more fuel is not mobilized from the fat body and released into the hemolymph in a timely way, the insect may effectively "run out of gasoline". Who knows what would happen after that.

2) at another extreme, after flight activity has ended, if fuels were continuously released into the hemolymph at rates sufficient to support the necessarily high rate of energy metabolism involved in flight, the circulatory system would soon become so clogged up as to effectively block hemolymph movement.

The release and transport of metabolic fuels must, perforce be regulated in relationship with metabolic demands. The topic of this discussion is the hormonal, or endocrine, control of hemolymph fuel levels.

We first recall that the circulatory system of insects is an open system. Hemolymph is aspirated into the heart, or dorsal vessel in the abdominal segments and pumped forward, through the thorax and on into the head. Hemolymph flows out of this single circulatory tube into the open hemocoel and percolates back through the body tissues to the abdomen for recirculation. Hemolymph circulation is driven by contractions of muscles connected to the dorsal vessel and by volume changes in the abdomen and thorax. This circulation is responsible for the transport of fuels and for transporting various hormones and other nutrients throughout the organism.

One of the most thoroughly studied cases of endocrine regulation of fuel transport in the hemolymph is the role of adipokinetic hormone (AKH) in the desert locust. As the name suggests, this is a lipid mobilizing hormone.

Early on in the study of flight energetics it was shown that after locusts fly for more than a few minutes there is an increase in the lipid content of the hemolymph. The increased lipid is mainly in the form of diacylglycerol (DG) as a transport form of metabolic lipid. A key point is this increase in lipid concentration occurs even though there are considerable increases in the rate of lipid metabolism in flight muscles and in the rate of lipid uptake from circulating hemolymph. We understand this to mean that even though lipids are being rapidly removed from the hemolymph by transport into flight muscles there is a net increase in hemolymph lipid concentration. It appeared from these observations that the hemolymph concentration of lipid is regulated. The regulatory mechanism was guessed to be hormonal regulation.

Recall that the first observations showed that hemolymph lipid concentrations increase after some minutes of flight in the locust. The first endocrinology experiments were to surgically remove several tissues from locusts, then create aqueous extracts of the tissues. These extracts were individually injected into non-flying locusts. Extracts of most tissues did not induce an increase in hemolymph lipid levels. When extracts of an endocrine organ, the corpus cardiacum (CC), were injected into resting locusts, an increase in hemolymph lipid concentration followed. The increases occurred in a time course similar to the elevation in lipid that was regularly seen as a result of sustained flight activity. Furthermore, when the CC extracts were applied to isolated fat body tissue in vitro, DG was released into the incubation medium. These observations provide the basis of a classical endocrine system: an endocrine gland releases a factor into the circulatory system. The factor is carried by way of the circulation to some target organ distant from the endocrine gland. A physiological effect follows.

As the history of this inquiry went, people moved from these organismal observations to trying to characterize the endocrine factor. The active principle was found to be stable to boiling for an hour or so; stability to boiling is a property of small molecules. The activity of CC extracts was destroyed by treatment with proteolytic enzymes; this is interpreted to suggest the factor is a peptide or protein. Combined with the heat stability, it was thought the factor was a small peptide. It was also found that hemolymph from resting locusts contained no traces of the factor, while hemolymph from locusts that had been flying for the last 90 minutes or so had small, but detectable quantities of the factor. The factor began to look very much like a real hormone.

In 1976 an English group succeeded in purification of the hormone, then went on to determine the chemical structure. It turned out to be a peptide hormone composed of 10 amino acids. This was the first complete identification of an insect peptide hormone, then named adipokinetic hormone (AKH).

I used to say in this lecture, the only one done so far, but that is no longer even close to true. Several groups worldwide now work on peptide hormones in insects. Usually people try to isolate the factor, then determine amino acid composition, then get amino acid sequences. Two things have helped speed this business along. First is development of very beautiful columns that can be used to separate peptides on HPLC. The second thing is development of peptide sequenators than can work with as little as a few picograms of material. Between the HPLC for isolation of peptides and the sequence technology, the endocrinology of insect peptide hormones is developing into a wonderfully productive area of insect biochemistry and physiology.

Back to AKH. The next step was to confirm the structure of the peptide. The generally accepted approach to confirmation is to synthesize the peptide, and then show that the biologically activity of the synthetic product is identical to the activity of the naturally occurring hormone. At this juncture, we saw a biological phenomenon, namely the increase in hemolymph lipid titres after some time in flight activity, and some of the experiements supporting the idea that hemolymph lipid concentration is under endocrine control.

Before going on, we need to review the anatomy of the main endocrine organs in insects. The important landmarks include the brain with optic lobe, the pars intercerebralis (PI) and two sets of neurosecretory cells - median neurosecretory cells and lateral neurosecretory cells. Neurosecretory cells resemble typical nerve cells or neurons, with elongate axons, but they are characterized by microscopic evidence of secretion. The secretion is granular with characteristic staining properties in histological preparations. The visible granules are probably only large carrier molecules with a much smaller hormone molecule attached. Histological studies have allowed construction of the following series of events: peptide hormones are produced in the cell body, then attached to a large carrier protein and moved down the axon process for export at the terminus of the neurosectretory axon. When the hormone is released it is separated from the carrier molecule and enters the circulating hemolymph. This is a common mechanism to release hormones from the brain.

Let's consider a cross-section of the CC from the locust. The CC are paired endocrine glands that often form part of the aorta. The CC contain nerve endings from neurosecretory cells of the brain. Part of the CC store and release hormones from these neurosecretory cells from the brain. In this storage and release function, the CC are properly called neurohemal organs. Association with the aorta is thought to facilitate broadcast of hormones because the hormones can be directly secreted into circulation.

The CC also contain intrinsic secretory cells. Unlike the neurohemal portion of the CC, these cells produce and release their own hormones. Sometimes the neurohemal and intrinsic parts of the CC are intermingled, but in the locust they occur as separate lobes. Because these lobes could be surgically separated, it was possible to show that most, and probably all, of the AKH of the locust CC was present in the intrinsic rather than neurohemal lobes. We conclude that AKH is produced in the CC rather than in the brain. This idea was tested by removing the intrinsic lobes from locusts, then allowing them to fly. The surgery prevented the normal rise in hemolymph lipid that accompanies flight. Also, cutting the nervous connections between the brain and the CC similarly blocked the normal rise in hemolymph lipids. This suggests that nervous stimulation from the brain triggers the release of AKH by the CC.

We might imagine a control system as follows: prior to a lengthy flight a locust has a standing hemolymph reserve of trehalose and DG associated with lipophoren. During the first few minutes of flight, the carbohydrate is transported into the flight muscles and oxidized. This is followed by a shift to lipid oxidation. Sometime during this shift the brain sends a nerve impulse to the CC, resulting in the release of AKH which finally circulates to target cells in the fat body. The fat body cells are triggered to mobilize storage triacylglycerol and release DG to lipophorin in the hemolymph.

This is a reasonable enough system, but there is evidence that AKH may also regulate the balance of carbohydrate and lipid oxidation in flight muscles. The first line of evidence is that the release of the hormone from the CC appears to be at least partly regulated by the trehalose concentration in the hemolymph. Injection of trehalose into locusts to create above-normal sugar levels inhibits AKH release and lipid mobilization in flown locusts. The second point is that AKH seems to act on the flight muscles directly to influence oxidation of lipid over carbohydrate.

So, we modify our picture to think that a drop in hemolymph sugar is sensed in the brain, followed by a release of AKH from the CC which acts on the fat body to mobilize and release lipid and acts on the flight muscle to stimulate a shift in substrate utilization.

Now, just as we think we are beginning to get a grip on things, we introduce a third role for AKH. In resting locusts the hemolymph lipids are associated with lipophorins, known as high density lipophorin (HDLp). HDLp is also involved in transport of lipids from the site of absorption in the alimentary canal to the fat body and in the transport of sterols and lipoidal hormones. Aside from lipophorins, there is another hemolymph protein first called C2 by one group and finally named apolipophorin-III (apo-Lp-III) by another group. It is called apo-Lp-III because the major lipophorin, HDLp, is composed of two subunits called lipophoren-I and lipophorin-II. Without further concern for nomenclature, here is how the system works.

Following injection of CC extracts, or of synthetic hormone, or after a period of flight, additional DG is found in the hemolymph associated with a new lipophorin, low density lipophorin (LDLp); at the same time the amount of apo-Lp-III in the hemolymph is depleted. It is thought that during lipid mobilization some apo-Lp-III combines with HDLp to produce LDLp. This form has greater lipid carrying capacity than HDLp. Here is a bit of data on the carrying capacity of the various forms of Lp.

Summarizing, we imagine that AKH has three physiological roles: mobilization of fat body lipid reserves, a shift in flight muscle oxidative balance to favor use of lipids and a change in lipophorin pattern to allow increased lipid carrying capacity.

While most of what we know about AKH comes from work on locusts, there is also evidence for similar hormones in other insect species. The tobacco hornworm is another model system for these studies and also the beetle Tenebrio was reported to have an AKH response to CC extracts. The migratory monarch butterfly, which relies on lipid reserves for its long flights, shows elevated hemolymph DG upon injection of CC extracts.

On the other hand, as we have said before, insect physiologists are of necessity comparative physiologists. This is because insects as a group have evolved many different physiological and biochemical adaptations to similar situations. As comparative physiologists we must take care not to generalize too broadly. For example, injection of CC extracts from either the cockroach Periplaneta americana or from Locusta into cockroaches causes a decrease in hemolymph DG. Conversely, injection of cockroach extracts into locusts results in an AKH effect that matches the locust AKH effect. At this point I would speculate that most insects have an AKH, but details of the broader physiology remain rather fuzzy.

As a last point in this discussion, one of the criticisms that frequently arises in hormone research is the assessment of the physiological relevance of hormones. For example, a high dose of AKH from locusts can cause a hyperglycemic effect in cockroaches. A hyperglycemic effect is an increase in hemolymph sugar concentrations. These sorts of non-specific effects of high hormone dosages are called pharmacological as opposed to physiological effects. The question, then, is what information do we need to critically assess physiological activity of a hormone?

The question is quite important because many people are able to isolate peptide factors in a fairly straightforward way. The problem is, the isolators have no idea relative to the biological meaning of their factors. The isolators usually fnd some collaborators to help, and newly isolated factors are routinely tested by bioassay. It is best if you can send your factors to several people. Somebody might test the adipokinetic hormone activity of your factor; somebody else might test the diuretic hormone activity in a completely different system. My colleague, David Petzel, once tested the diuretic hormone activity of a cockroach factor - using mosquito Malpighian tubules. He got a positive response alright, but was his response a physiological activity of a hormone?

Let us consider these points:

Technical problems can be counted upon to make such complete information impossible to obtain except in rare cases. Let us turn again to AKH, where research on the locust system is most complete. We attempt a critical assessment. AKH has been purified and its chemical structure determined:

The structure has been verified by peptide synthesis and recording the biological activity of the synthetic peptide. Surgical experiments showed the hormone is present in the intrinsic portion of the CC. Also, a specific target tissue, the fat body, responds to the hormone in a dose-dependent manner. The hormone has been found in the circulating hemolymph under appropriate physiological conditions - that is, it appears in the hemolymph of locusts after about 90 minutes of flight, but not in resting locusts. The hormone appears to be released in response to a nerve impulse from the brain as a result of changing hemolymph trehalose concentrations. This last point is still a little bit weak; nevertheless, the available information on AKH permits judgement that this is, indeed, a physiological endocrine system.