CHAPTER 22
NITROGEN:
METABOLISM AND EXCRETION
The Malpighian tubules and rectum of insects form a physiological complex that serves as the functional kidney at the organismal level. A primary urine is formed in the Malpighian tubules, followed by movement to the rectum where selective resorption of ions and water can take place. These secretion and resorption processes are responsible for maintaining osmotic balance between the intracellular and extracellular compartments.
In addition to maintaining osmotic balances, most organisms must also metabolize and eliminate the ammonia nitrogen that is formed by routine amino acid metabolism within cells. The normal dietary intake of protein by most animals is considerably different from ordinary protein needs. Typical diets provide amino acids in excess of the amounts required for synthesis of new protein, and often in quite different compositions. Cells are nearly always in the processes of protein turnover. This may not be an intuitively clear situation. We most often read about the nutritional importance of amino acids and proteins with respect to satisfying needs for essential amino acids. The point to be taken, however, is that dietary supplies of amino acids are rarely in optimal balance for protein synthesis within individual cells. Considerable levels of interconversions and other metabolic alterations of dietary amino acids follow from these imbalances between dietary supplies and instantaneous amino acid needs.
As in regulation of salt balance, it might be thought that insects regulate amino acid balance at the level of the alimentary tract, then just excrete unnecessary amino acids. There are cases where this sort of regulation does occur, but for the most part amino acids are transported across the midgut epithelium into the hemolymph, and thence to various body cells where diverse metabolic fates ensue. The protein amino acids have a backbone of two carbon atoms and at least one nitrogen atom. Amino acids are distinguished by an R group, where R stands for some side-chain that gives the individual amino acid its identity and particular chemical properties. Once the alpha-amino group is removed from the amino acid, by a metabolic process known as deamination, a carbon skeleton remains. The carbon skeleton can undergo a number of metabolic fates.
Possibilities include reamination, thereby becoming an amino acid again, and, alternatively, entering pathways of oxidative metabolism, mainly at various points in the glycolytic and Kreb's pathways. By entering these metabolic pathways, a potentially important energy source is not lost to the animal. Depending on the energy state of a cell, carbon skeletons from amino acids can be taken into a reverse of glycolysis called gluconeogenesis in which glucose can be made form non-carbohydrate precursors. The ultimate fates of the individual carbon skeletons from amino acids is often the domain of a nutrition course given over to "nutritional metabolism". Most of you will probably be taking such a course next spring.
We want to focus our attention on the alpha-amino group that is removed from amino acids prior to carbon metabolism. All amino acids have an alpha-amino group; some amino acids feature an amino group in the side chain as well as the alpha-amino group in the main skeleton.
Most of the protein amino acids undergo transamination reactions. The first figure illustrates a typical reaction. Here the amino acid alanine reacts with the Kreb's cycle intermediate alpha-keto-gluterate to yield the transamination product glutamate plus pyruvate. The central point is that the amino acid donates its amino group to something else. It does not just drop the nitrogen from the skeleton. The donation makes the remaining carbon skeleton available for other metabolic fates. All but two of the major amino acids are subject to transamination reactions. The next metabolic step, also shown in the figure, is the removal of the amino group, known as a deamination reaction. This step regenerates the alpha-keto-gluterate and releases free ammonia.
Just recently, in 1937, it was first suggested that these two events are elegantly combined within cells. The next figure shows the most general expression, which has been given the term transdeamination. First, in the cytosol the amino group is transferred from the amino acid to alpha-keto-gluterate, yielding a carbon skeleton and glutemate. As shown, this reaction most often occurs in cytosol, although there are transaminases within mitochondria. The glutamate then crosses the mitochrondrial membrane where the release of the ammonia group attends regeneration of alpha-keto-gluterate and the reduction of NAD+ to yield NADH + H+. Please note that we have another example of the biological significance of the vitamin niacin, again playing its essential role in electron transfer. This biochemistry has been most well studied, as usual, in various laboratory mammals. But it has also been worked out in a few of our own sorts of lab animals, namely, in the cockroach Periplaneta americana and the fruitfly Drosophila melanogaster. It has also been shown in the flight muscles of several other insects. The main point here is to appreciate the metabolic source of ammonia in insects.
A fundamental precept in nitrogen metabolism is that nitrogen is toxic to animal cells. For example, in an atmosphere of 500 ppm of ammonia humans die within 60 minutes. Your basic, off-the-shelf laboratory rat dies within 16 hours in an atmosphere containing 1000 ppm of ammonia. Ammonia tolerance is less well known in insects, but in the blowfly Lucilia cuprina the amount of ammonia in the body fluids is only slightly higher than the range of body fluid ammonia found in many mammals.
Why is ammonia toxic, at one level of exposure or another, to so many organisms, including fungi, plants and animals? We consider the two leading hypotheses. One mechanism of toxicity appears to be general pH effects. Some enzyme systems release ammonia in the unprotonated form, NH3. At physiological pH within most cells, about 7.0 to 7.4, most ammonia takes on a proton to become NH4+. This has the effect of removing hydrogen ions from the cytosol, and creates a local increase in pH, which can in turn negatively effect enzyme activity and membrane function. Please note this line of reasoning will probably not hold as a general mechanism when we realize that in the most common reaction, the glutamate dehydrogenase system we just looked at, the proton comes from the reactants and there is no local pH change. Hence, general pH effects probably account for some but not all of ammonia toxicity.
The main effects of ammonia are probably their direct action on membrane biology. Ammonia salts can inhibit the active transport of sodium and of chloride, and can inhibit water resorption in several tissues. Oddly enough, ammonia does not seem to effect ion transport in nervous tissue. Drawing on our treatment of salt and water balance, it is clear that inhibition of transport in excretory tissues could have very profound effects on physiological function.
In addition to general membrane effects, there is one specific effect. NH4+ directly inhibits the formation of ATP by the electron transport chain within mitochondrial membranes. We can understand this in terms of the driving force behind oxidative phosphorylation in mitochondria and chloroplastal membranes. The electron transport chains are found within the membranes of these organelles. A hydrogen ion gradient is maintained across these membranes; the hydrogen ion gradient is thought to drive formation of ATP. It is thought that ammonia moves across these membranes, which abolishes the hydrogen ion gradient necessary for ATP formation. This has yet to be definitively proven, but it will most likely turn out to be the most direct mechanism of ammonia toxicity in cells. The specific mechanisms differ among toxic compounds, but oxidative phosphorylation is interrupted by a number of poisons, including carbon monoxide and cyanide as well as ammonia.
We have, then, some idea of how ammonia arises is general metabolism within cells and why it is poisonous to animals. Our next concern will be how ammonia is detoxified so that it is not poisonous while it is in an insect body. There are two major pathways, and several minor ones that we will not worry about. The first pathway is the formation of urea, a very important pathway in mammals. This is a relatively minor pathway in insects. The second pathway is formation of uric acid, which is very common in birds, reptiles and insects. In point of history, the uric acid pathway was first worked out using avian liver slices.
A brief outline of urea formation is provided in the next figure. Ammonia is linked to carbon dioxide and a phosphate at the expense of energy in the form of ATP. This new nitrogen compound, carbamoyl phosphate, joins with the non-protein amino acid ornithine to form citruline. The citruline combines with another amino acid, aspartate to form the larger molecule arginosuccinate. This large compound breaks down into the Kreb's cycle intermediate fumerate and the amino acid arginine. Splitting arginine releases urea and regenerates ornithine. Here are the key points to remember.
We want to look at this information again in the next figure to show that part of this cycle occurs in mitochondria and part of it occurs in cytosol. As with all the pathways presented in this course, you are urged to resist the temptation to memorize the steps. Instead, please note that ammonia enters the cycle in the mitochondria and that urea exits the cycle in the cytoplasm. This simply reiterates a common theme of recognizing the important pathways, knowing where they are located within cells, and knowing what chemicals are taken into the pathway and what chemicals are released.
The second major pathway for detoxification of ammonia is formation of uric acid. The next figure shows, again, that glutamate is transdeaminated to yield a free ammonia molecule. In this case the ammonia is joined to a second molecule of glutamate, thereby producing the common protein amino acid glutamine. The glutamine effluxes from mitochondria to cytosol, wherein a complex pathway leads to xanthine, which is oxidized to uric acid by xanthine oxidase, the terminal enzyme in biosynthesis of uric acid from glutamine. There are four nitrogen atoms in the ring structures of uric acid. These all derive from various amino acids: glycine contributes N-7; aspartate donates N-1 and the last two nitrogens, N-3 and N-9, derive from the amide nitrogen of two gluatamine molecules. The amount of energy that is invested in biosynthesis of uric acid is five ATPs per uric acid molecule. There are two parallels with the urea cycle.
Pulling together, we have looked at the major sources of ammonia, at why it is toxic to cellular functions and how it is metabolized into non-toxic forms. Our next point is to consider excretion of nitrogen. The most general statement is that urea and uric acid diffuse out of cells into circulating hemolymph. Uric acid and urea are secreted into lumens of Malpighian tubules, possibly by active transport processes, and follow the now familiar anatomical route to excretion. Let us consider environmental correlates with respect to nitrogen metabolism.
Fishes and many aquatic insect larvae live in a situation where water tends to enter a body and needs be secondarily removed to maintain optimal osmotic balances. These animals generally excrete ammonia per se without further metabolism. Such animals are called ammonotelic. The vast majority of nitrogen in the excreta of several aquatic larvae is in the form of ammonia. Following release of ammonia within mitochondria, ammonia diffuses out of cells, and into hemolymph. The ammonia probably enters Malpighian tubules with the water that is constantly removed from hemolymph. Since it takes considerable amounts of energy to metabolize ammonia to uric acid or to urea, an advantage in terms of energy economy attends direct elimination of ammonia. There are, however, some general requirements for the ammonotelic strategy. First, direct elimination of ammonia requires simultaneous elimination of substantial amounts of water, so ammonotelic animals are mostly aquatic or amphibious. Second, these insects have relatively low metabolic rates, and hence, low rates of ammonia production. Finally, ammonotelic insects probably have a higher tolerance of tissue ammonia than do many terrestrial insects. Higher ammonia tolerance is certainly true of fishes.
Most terrestrial insects are what we call uricotelic. The vast majority of their excreted nitrogen is in the form of uric acid. Uric acid can diffuse from cells into hemolymph, whence it is taken up into Malpighian tubules, most likely by active transport, although some textbooks say it is by passive transport. The uric acid moves toward the alimentary canal along with the flow of water. Depending on the histological structure of Malpighian tubules, the proximal portion of the tubules may be involved in the resorption of some water and salts. When the water is resorbed, either in proximal portion of Malpighian tubules or in rectum, the uric acid precipitates out of solution and the uric acid crystals can be seen in hindgut or rectum. Since uric acid crystals fall out of solution in water, the water can be resorbed and the ammonia metabolite excreted with minimal loss of water. Here, then, is the main environmental correlate with uricotelism: in those insects (and other animals) where water conservation is important, the toxic ammonia can be ultimately excreted in a form that does not require loss of body water.
A strategy called storage excretion occurs in some insects. Since uric acid is a harmless repository of ammonia, and it can be stored in the body. Periplaneta and Apis have specialized fat body cells called urate cells. Uric acid accumulates in these cells, often to the point that it can be seen under dissecting microscope. Uric acid also accumulates in non-specialized fat body cells of other insects, for example the mosquito Culex. Finally, uric acid may be involved in the coloration of some insects. The white wings of Pieris butterflies is due to uric acid storage in the wing scales.
Urea makes up about 10% of the excreted nitrogen in larval mosquitoes, and is also found in the excreta of other insects. It is much more solubile than uric acid and more water is required to eliminate urea in feces. We use the term ureotelic for animals in which urea makes up a large proportion of the excreted nitrogen. In general, insects can not be called ureotelic because urea makes up a relatively small proportion of their excreted nitrogen. Nonetheless, the pathway for urea synthesis is present in many insects.
In general terms, most insects can be easily classified as ammonotelic or uricotelic. Chapman tells us that most insects do excrete minor proportions of their nitrogen as other than the main excretory forms, but for the most part, they will fit into the general scheme. On the other hand, a good number of insects have adapted strategies that are outside the main line. For example, the pyrrhocorid Dysdercus excrete most of their nitrogen as allantoin. Allantoin is a single metabolic step beyond uric acid.
Still other insects excrete a good bit of amino acids without further modification, after first absorbing them. In tsetse, arginine and histidine from the blood of hosts are absorbed and excreted without metabolism. This may be quite adaptive because these compounds are relatively high in nitrogen. Arginine, for example, has a total of four nitrogens per molecule. These nitrogens would require considerable amounts of energy to metabolize along the normal pathways. The required energy is greater than the amount of energy that could be derived from the carbon skeletons.