Some Mechanisms for Hormonal Effects on Substrate Transport

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Some Mechanisms for Hormonal Effects on Substrate Transport

In the first quarter of the 20th century, endocrine physiology began its modern development. As was to be expected, the first steps of the new biological discipline were taken in the realm of the purely descrip- tive. Physiological and pharmacological effects were duly noted and catalogued, at first in the living, intact animal; later under more isolated conditions in vitro.

In the area of hormonal effects on metabolic regulation, studies on mechanism had to wait for the elucidation of the involved enzymatic pathways. The data and concepts in the field of enzymology began to accumulate in exponential fashion during the period 1900-1930, and the outlook derived from them colored our theoretical concepts of the mode of hormone action. This was the era of analysis, the period of enzyme isolation and the study of separated reactions. With few exceptions (notably Hopkins and Peters in England) the prevailing opinion favored the concept that hormones (as well as drugs in general)

Rachmiel Levine

I. Introduction

II. Effects on Sugar Transport III. Amino Acids

IV. Cations

V. Concerning Mechanisms . . VI. Water, Sodium, and A D H VII. Mineralocorticoids

627 628 632 633 634 635 636 637 639 VIII. Effects of Growth Hormone

References

I. I N T R O D U C T I ON

627

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would be found to exert their direct action on a particular enzyme reaction, and that the effects on tissues or the body as a whole would depend upon the modulation of activity of that particular system.

Some workers had from time to time directed attention to the possibility that the plasma membrane may well be of regulatory impor- tance to cell physiology and that drugs and/or hormones may indeed exert their effects via an influence on rates of influx and efflux of critical materials. For many years the work on membrane permeability phenom- ena concerned mainly the flux of electrolytes. In the case of the con- veniently available red blood cell, work on penetration of sugars and other organic substrates was added to the electrolyte investigations.

Here and there attention was drawn to the possibility that transmembrane traffic of substrates may have decisive regulatory effects. However, it was generally thought that the influx of metabolites was regulated primarily by the first irreversible enzymatic reaction undergone by the substrate after it entered the cell. In the case of glucose, the dominant notion was that its rate of phosphorylation by hexokinase determined the rate of cell entry [1,2]. Thus the effect of phlorhizin on the renal reabsorption of glucose or on its rate of intestinal absorption was attributed (without proof) to its supposed inhibition of phosphorylation. The notion of specific carrier molecules which could translocate without transforming was not generally entertained.

The radical change in the situation began in the late forties and early fifties of this century. The years since then have seen a veritable ava- lanche of experimental data on transport across membranes, on carrier systems, and the possible mechanisms by which drugs and hormones may alter rates of penetration.

In this chapter an attempt will be made to provide an outline of the present status of the field. The review does not intend complete cover- age. Rather it is the intention to emphasize the areas which have been firmly established and to indicate the regions of obscurity and doubt.

II. EFFECTS ON SUGAR TRANSPORT

By 1913 insulin had been named but not yet" discovered." In Hoeber's laboratory it was demonstrated that there was great variation among r.b.c. gathered from different species in their rate of uptake of monosaccharides. In the discussion section of a paper from his laboratory, Hoeber speculated that diabetes might be the result of difficulty in membrane permeability of sugar rather than in its "fermentative" fate [3]. Loewi [4,5] thought that he had provided experimental evidence for

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such a view but withdrew his conclusions after failing to confirm his earlier results. Pollak [6] showed that insulin led to a rise in galactose concentration of heart muscle when this sugar was added to the medium bathing the heart. Lundsgaard [7] interpreted his findings on hind limb perfusion with glucose and insulin as consistent with a, permeability change.

Between 1949 and 1955, Levine, Goldstein, and their co-workers [8-12] obtained evidence that insulin increased the rate of membrane transport of a number of monosaccharides which were not phosphory- lated or otherwise transformed under their experimental conditions.

Using eviscerated dogs and rats, they showed that rf-galactose, /-xylose, and rf-arabinose were distributed in a volume equal to total body water in the presence of insulin. In the absence of added hormone the volume of distribution of these sugars was restricted to about 40% of body weight. The effect was not obtained with other monosaccharides such as fructose, /-xylose, or ^/-arabinose. No appreciable fraction of the affected sugars was lost by metabolic transformation. Clearly, then, insulin promoted a process distinguishable from phosphorylation and necessarily preceding it. The free sugars were translocated without being transformed. Certain deductions were made then about the sugar transport or translocation system: (1) It was steriospecific; (2) it did not lead to the intracellular accumulation of the transported sugar; (3) insulin accelerated but did not induce transport de novo, since in the absence of the hormone an increase in concentration of the sugar permitted more transport. The system thus conformed to the type of transport known as "facilitated diffusion" and seemed best explained on the basis of a specific carrier molecule in the cell membrane [13,14].

The work cited above was soon confirmed both in vivo and in vitro (reviewed in [15]). Since these early experiments related only to nonutilizable sugars, it was important to establish the fact that the transport of rf-glucose was similarly affected. Under ordinary conditions, in vivo and in vitro, the intracellular concentration of free glucose is essentially zero, since the activity of the hexokinase system (e.g., in muscle) is relatively so great that the glucose molecule, having entered through the membrane is immediately transformed to G6P. Park and associates, using techniques which either inhibited or overwhelmed the hexokinase system, demonstrated a rise in the free glucose of the intracellular compartment when insulin was added to the medium [16-18].

The work which followed in this and other laboratories established that glucose transport systems were present in all mammalian tissues examined, but that the systems differed from one another in type of transport, in rate, and in sensitivity to insulin. Thus insulin increases

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the transport of glucose across the membranes of skeletal muscle, heart muscle, fibroblasts, and fat cells. The intestinal absorption of glucose and the reabsorption in the kidney show a capacity for active transport against a gradient, but the rates are not influenced by insulin.

The r.b.c. and the cells of the brain also do not react to the hormone [19]. In some tissues, reaction to insulin may be obscured by the rapid destruction of the hormone. Thus a kidney slice in vitro does not increase its sugar uptake even when high amounts of insulin are added to the medium. However, if insulin destruction is inhibited (by oxidizing the GSH of the tissue), it will be reasonably sensitive to the transport activation by insulin [20].

At this point two alternative explanations were possible: (1) The transport systems of insulin-sensitive tissues differed radically from their counterparts in the insulin-insensitive cells; or (2) the transport systems were quite similar in all cells, but insulin could only affect their activity in cells which possessed a "receptor" for insulin. Such a

"receptor" would interact with the hormone, and this interaction would then be transmitted to the transport system itself.

The accumulated evidence to date favors the second alternative, for the following reasons.

1. It was found by Randle [21,22], using muscle tissue in vitro, that anoxia and exposure to uncoupling agents increase the rate of sugar transport. This does not result from a general breakdown of the cell membrane, since the stereospecificity is preserved and the process is reversible after re-exposure to 0² or removal of the uncoupling agents.

It is as if anoxia removed an influence or structure which normally limits the full rate of penetration.

2. More recently it was shown in many laboratories that reasonably mild exposure of isolated fat cells to enzymes which cleave phospho- lipids and proteins activates sugar transport without damaging the transport system itself. The cell system that is being used is the isolated fat cell, and the enzymes which imitate insulin action are the phospholipases and certain proteases [23-27]. Again, this work demonstrated that transport can be activated by perturbations of the membrane without direct damaging effects on the transport system, per se.

3. In addition, it has recently been demonstrated that exposure of a fat cell to trypsin can abolish completely its response to insulin even though the sugar transport system remains functionally intact. During that time the cell is able to transport sugars in normal fashion but is temporarily completely insensitive to insulin. A "rest" of 30-60 minutes repairs the membrane and then insulin can again raise the rate of sugar uptake [28-30].

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4. At age 5 days, the chick embryo heart has a fully developed sugar transport system but one which does not react to insulin. At the 9-day stage, something has been added to the membrane. The basal rate of sugar transport is reduced but now insulin activates the system [31,32].

The assumption which best fits the preceding observations is that the transport system can and does operate independently of hormonal influences.

The transport system is thus a separate molecular aggregate from the

"receptor" with which insulin interacts. The link between the two is unknown. Inhibition of RNA synthesis or of the translational step in protein biosynthesis does not interfere with activation of sugar transport by insulin, even though such procedures do interfere with other insulin effects [33,34].

Additional evidence that insulin action on sugar uptake is exerted by attachment to the outer membrane is provided by the work of Cuatre- cases [35,36] who showed that insulin covalently linked to large particles of sepharose (an inert carbohydrate polymer) affected transport in the same manner as did soluble insulin. The sepharose-insulin particles were, on the average, of the same size as or larger than the isolated fat cells which constituted the test preparation.

The effect of insulin on glucose transport resides wholly in the membrane and is seemingly independent of other cytoplasmic or nuclear events. It can be readily demonstrated in fat cell ghosts and in membrane vesicles [37-39]. The other effects of insulin such as glycogen storage, protein synthesis, and antilipolysis can be inhibited without interfering with the enhancement of transport. While many of the effects of insulin are concerned in some way with modifications of the expression of cAMP activity or concentration, this does not seem to be the case with the membrane transport action [40]. On the other hand, removal of the membrane receptor (by trypsin) abolishes many of the " internal" metabolic effects of the hormone as well as the enhancement of transport [41].

Before we will be able to depict this action of insulin in precise molecular terms, we need to know: (a) the chemical structure and the mode of action of the glucose "carrier" or transport system; (b) the structure and properties of the membrane insulin receptor; and (c) the nature of the influence or signal issuing from the receptor-hormone complex to the transport system.

It should be noted that the data and speculations concerning the mechanism of the insulin effects on transport have been gathered in the main from work on skeletal muscle, the heart, and fat cells, with fragmentary work on the lens and fibroblasts. The brain as a whole or brain tissue in vitro is not sensitive to insulin or its lack. There are,

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however, indications that some hypothalamic neurones do have an insulin-sensitive glucose transport [42] system and that peripheral nerve may be insulin-sensitive [43,44].

The liver cell has a membrane system of such low K^m that glucose transport does not appear limiting [45]. Liver membranes do bind insulin in a seemingly specific manner, i.e., they have insulin receptors, but the effect on transport is, so to speak, unnecessary [46].

Intestinal absorption and renal reabsorption of glucose proceed by means of a modified membrane carrier system which results in transport

" against a concentration gradient." The lumenal portion of the epithelial cell has a bifunctional carrier which combines with the sugar and with N a⁺. When the fully loaded carrier arrives at the inner membrane, the N a⁺ is forcibly unloaded by the "sodium pump," thus releasing the glucose passenger as well. The free glucose level rises in the cell and proceeds " downhill" across the serosal pole of the cell into the circu- lation [47,48]. Insulin does not affect the rates of these epithelial, active transport systems [49]. For reasons not yet understood, glucose absorption in the intestinal tract is said to be faster in the diabetic animal or man [50,51].

Other hormones do affect intestinal absorption of sugar secondarily, as a result of other primary effects. Thus lack of thyroid (probably because of the lower rate of energy production) decreases absorption rates. After adrenalectomy the N a⁺ level of body fluids including those of the g.i. lumen falls. Sugar absorption is decreased, and a normal rate can be restored simply by providing sodium [52].

III. A M I NO ACIDS

The protein anabolic effect of insulin, especially in diabetes, has of course been appreciated for many years. When the insulin action on glucose entry into cells was discovered, it seemed appropriate to test whether the effect of the hormone on protein synthesis was perhaps secondary to an action on the flux of amino acids into cells. Just as in the case of carbohydrates, so also in this instance the first tests were carried out with a nonutilizable substrate. The substance used was aminoisobutyric acid (AIB), and indeed positive results were immediately obtained [53,54]. It was, however, much more difficult to get solid experimental evidence that the transport of the normal /-amino acids was enhanced by insulin. Proof for this was finally put on firm ground by the use of puromycin to inhibit protein synthesis, thus allowing

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amino acid accumulation to take place [55,56]. The effect is not uniform for all the normal amino acids. We are dealing here, of course, with a number of transport systems, and these seem to vary as to their reaction to the presence of insulin [57,58], Some of these systems also are some

how linked to the N a⁺ pump, and are affected by the extra and intra

cellular concentration of this ion [59]. The exact relation between the rate of uptake of amino acids and their incorporation into cell protein is quite obscure. It is, however, known that insulin enhances protein synthesis in the total absence of amino acids in the medium [60,61].

It is postulated that insulin provides a "regulatory" substance (pro

tein?) which serves to enhance the rate of translation at the ribosome level [62,63]. Whether or not this action involves the penetration of a particular amino acid through an intracellular barrier is not known.

Would removal of the membrane receptor for insulin interfere with the enhancement of protein synthesis ? It seems probable that a signal generated in the membrane is necessary for the hormonal effect on protein biosynthesis.

IV. CATIONS

Insulin affects the fluxes of both N a⁺ and K⁺. However, the effect on Κ⁺ is more evident and consistent than that on N a⁺. In muscle, both in vivo and in vitro, insulin stimulates the uptake of K⁺ both in the presence and in the absence of glucose [64]. In the perfused liver, insulin may exert a rapid, very significant effect on K⁺ influx while at the same time no effect is seen on the uptake of glucose [65]. It had long been known that, in the whole animal, insulin administration is followed by a very significant fall in plasma K⁺ and a reduction in the urinary output of K⁺, hence a stimulation of potassium entry into the intra

cellular compartment.

The removal of K⁺ from the medium stimulates uptake of sugars by rat diaphragm [66,67]. This effect is probably due to an inhibition of the N a⁺ pump. Similar results may be obtained by adding ouabain.

The stimulation of N a⁺ transport is less clearly separable from the relation of Na⁴" to transport of some metabolites. Amino acid uptake seems to be linked with that of N a⁺, while glucose flux is related to N a⁺ entry through the g.i. mucosa and possibly in adipose tissue, but not in muscle. Insulin has been shown to stimulate N a⁺ transport in the toad skin [68].

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In muscle and in adipose tissue, the effects of insulin on ion transport are associated with a rise in the membrane potential [69,70]. The presence of glucose in the medium is not necessary for this effect. An effect of insulin on the activity of an isolated Na⁺-K-ATPase has not been reported.

V. C O N C E R N I NG MECHANISMS

At this point we can only speculate, but the picture which emerges may be very helpful. We can imagine that dotting the cell membrane are the various specific transport systems, each type unique in structure and function. While capable of acting independently of one another, they may at times act to assist each other. A hormone or drug (in this case insulin) interacts directly only with its own receptor material, which is distinct from any and all of the transport systems. The interaction causes a perturbation or signal which is propagated and affects the activity of some of the transport systems.

The most recent work makes it very probably that the insulin receptor is a protein. The binding characteristics exhibited by fat cells and by liver cells are sufficiently similar to suggest that the receptors (at least in these two tissues) are very similar or identical [71]. Interposed between the receptor and the "transport" systems are carbohydrate- and lipid-containing structures which may well be the " wires" carrying the signal from the receptor to the " carrier." This is of course highly speculative since we do not know the precise architecture of the molecular assembly of the functional units of the membrane. The only molecule we know in greater detail is insulin itself. But even in this respect we cannot speak with certainty since we do not yet really know whether the hormone attaches to its receptor in the monomeric or the dimeric form. Further knowledge waits for the isolation and character- ization of the receptor protein in order to be able to study the precise locale and manner of binding.

The bulk of available evidence suggests that insulin need not enter the cell to exert its well-known effects on storage and anabolism such as glycogen and fat deposition, protein synthesis, and nucleic acid formation. We must therefore visualize a system of communication to the cell interior in addition to the propagation of a signal in the membrane itself [72,73]. Is a secondary messenger in the form of a small molecule involved here? Since Sutherland's classic work one immediately turns to the cAMP system in this regard. In respect to certain actions

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of insulin, evidence points to a lowering of cAMP levels, either by inhibition of the cyclase or by the activation of the phosphodiesterase.

For other actions no significant cAMP changes can be shown [74]. We thus at present do not know whether the signal to the cell interior is a

"negative" one, i.e., modulation of cAMP downwards, or a "positive"

one, i.e., a messenger molecule other than cAMP specific for insulin.

VI. W A T E R, SODIUM, A N D A DH

The membranes of most cells do not present a barrier to the free movement of water molecules. Water seems to move passively in the direction of an osmotic pull. Some cell surfaces, however, exhibit a barrier to this passive transfer and water is prevented from entering despite the presence of an osmotic gradient. This is the normal situation at the lumenal surfaces of the distal renal tubule, the toad bladder epithelium, frog skin, etc. ADH (vasopressin) is the specific hormone which removes the "barrier" and thus allows the free movement of water [75,76]. In vivo the osmotic gradient is produced by N a⁺ concen

tration differences, but it can be shown that water movement is not dependent uniquely on N a⁺. The separability of H²0 and N a⁺ move

ments is attested by the fact that, in toad bladder, amphotericin Β stimulates N a⁺ transfer but exerts no effect on the movement of water [77].

In the toad bladder, N a⁺ seems to enter the mucosal surface by passive transfer. In the cell, toward the serosal pole an active N a⁺ pump is operating to transport N a⁺ outward. The neurohypophyseal hormones speed up the transfer of N a⁺ across the toad bladder and the frog skin, and most workers feel that they do so by aiding the passive transport at the lumenal, mucosal barrier [78,79]. Some investigators feel that their evidence points to an action of the hormones on the active, intracellularly located, N a⁺ pump [80]. Stimulation of N a⁺ transport by ADH is accompanied by a rise in 0² consumption, and it is reduced by metabolic inhibitors. It is as yet unclear whether the rise in metabolism is itself a primary effect or is due simply to the effect of entry of N a⁺ ions produced by allowing freer passive transfer of N a⁺.

ADH does affect to some extent the transfer of other substances,such as urea, alcohols of low molecular weight, and Ca^{2 +} . The specificity of these effects is unknown. The effects on N a⁺ and H²0 are undoubtedly of primary significance.

Much work has been done trying to link the chemical structures of

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the neurohypophyseal peptides with their cellular effects. The cyclic structure seems essential for hormonal expression. The S-S linkage plays a role in determining degree of activity, but it is possible to show that analogs not containing S may retain a significant degree of activity [81].

The hormones do increase the tissue levels of cAMP, and the cyclic nucleotide mimics the actions of vasopressin in toad bladder, frog skin, and kidney. Since vasopressin must be added to the serosal surface of the toad bladder in order to alter mucosal permeability, it would be reasonable to suppose that a small messenger molecule such as cAMP is the conveyor of the message [82,83].

The changes in metabolic parameters of the cell produced by the hormonal peptides, such as increased 0² consumption, glycogenolysis, phosphofructokinase, and pyruvate kinase activities, can be thought of as secondary to an increase in N a⁺ concentration brought about by the hormone. The increased metabolic activity serves in turn to raise the power of the N a⁺ pump needed to transport N a⁺ outward at the serosal surface.

Nothing definitive is known concerning the nature of the mucosal barrier to the free movement of H²0 and N a⁺. One suggestion is that it may be related to C a^{2 +} bound at the plasma membrane. Under the influence of the hormone, C a^{2 +} would move from the external membrane to the mitochondria. This in turn would remove the barrier to N a⁺ [84,85].

VII. MINERALOCORTICOIDS

The principal adrenal steroid exhibiting effects on the mineral balance is, of course, aldosterone. But it must not be forgotten that many other steroid hormones possess mineralocorticoid activity to varying extents. It is assumed that the cellular mechanism of action of these compounds is uniform.

Aldosterone affects N a⁺ transport in a great variety of epithelial cells-kidney, gut, g.i. glands, skin, bladder, etc. The flux of N a⁺ is visualized as follows: The mucosal membrane of the epithelial cell possesses a saturable carrier for N a⁺ which can account for the "passive" movement of N a⁺ inward. In the cell, probably at or close to the serosal end, an Na⁺-K-ATPase activated by ATP which is generated by the reactions of oxidation extrudes N a⁺ to the intercellular space on the serosal side [86].

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Theoretically a hormone which leads to a greater transcellular flux of N a⁺ could effect this by (a) increasing the " permeaselike" system at the mucosal end of the cell; (b) increasing oxidative rates leading to an increase in ATP generation; and (c) stimulating the N a⁺ pump directly.

In the case of the ADH effect on toad bladder, it seems in accordance with the evidence that it is the mucosal barrier to transport which is affected by the peptide. The action of aldosterone is still a subject of a major controversy. This seems to be due to varying interpretations of laboratory data which depend in a great degree on the assumption of certain actions of drugs used as models or as inhibitors [87,88].

One group of investigators points out that amphotericin-B, which presumably acts directly on the apical (mucosal) membrane of the epithelial cell to make it more permeable to N a⁺, is followed by stimu

lation of cell metabolism and a secondary activation of the N a⁺ pump.

They postulate therefore that aldosterone need only affect the lumenal entry of N a⁺ without having to exert its primary action on the control of cell respiration [87].

On the other hand, it has been pointed out that the effects of adding aldosterone to isolated toad bladder follow a particular time sequence which is best interpreted as a primary effect at the level of nuclear function [89]. Thus there is a 60-90 minute lag period during which aldosterone is accumulated at the nucleus, probably by attachment to a specific protein. It is assumed that the aldosterone-receptor complex induces the production of enzymes of the oxidative pathway. At that time one sees an increased 0² consumption and oxidation of members of the Krebs cycle. ATP would thus be produced which could serve as the energy source for the N a⁺ pump. The activation of the pump in turn would serve to propel N a⁺ to and through the serosal portion of the cell. More N a⁺ would then be "pulled" through the lumenal sur

face into the cell [88].

VIII. EFFECTS OF G R O W TH H O R M O NE

In vivo under physiological conditions, pituitary growth hormone (GH) has a variety of well-established metabolic functions. It promotes general bodily growth and produces a positive Ν balance; it is necessary for adequate mobilization of fat from the adipose tissue; it maintains normal secretory activity and insulin synthetic capacity of the Β cell;

it produces antagonism to the action of insulin, especially in muscle

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[90,91]. In general, GH tends to shift food utilization away from carbohydrate to fat while conserving protein. In most animals the protein synthetic effect in vivo depends upon the simultaneous presence of insulin [92]. The rat seems to be an exception. In this species, GH administration leads to Ν retention even after pancreatectomy [93].

In view of the above, it remains difficult to account for some seemingly paradoxical effect obtainable in vivo and in vitro on sugar uptake. Thus, soon after an injection of GH (most especially in hypophysectionized animals) there is a transitory fall in blood, sugar, and muscle or adipose tissue removed from such animals within 30-60 minutes after such an injection and there is increased sugar transport capacity. This effect is no longer obtained at times longer than 1 hour or after several injections.

Repeated GH administration leads to inhibitory action on transport and resistance to the usual effects of insulin. The inhibitory actions are prevented by treatment of the animals with actinomycin [94,95].

Some of the evidence implies that the transport-promoting activity of a single GH injection depends somehow on insulin, perhaps by inhibi

tion of insulin destruction [96]. However, in adipose tissue the positive action on glucose uptake seems not to be mediated by insulin. Under certain conditions direct in vitro effects have been obtained [97].

The insulin inhibitory effects of more chronic GH administration can be removed by pancreatectomy or alloxan administration [98,99].

The possibility exists that GH which leads to Β cell proliferation in

creases the production of both insulin and another factor which in

hibits insulin action in muscle [99].

There is experimental evidence that GH stimulates the transport of amino acids, both those that are utilizable and others such as AIB [100,101]. GH seems to stimulate the production of a special protein which is seemingly instrumental in the amino acid transport function [102]. The increased amino acid incorporation into muscle protein in general can be experimentally separated from the transport effect, a situation similar to that relating to insulin action on these functions

[103,104].

It is obvious from the preceding recital that the membrane penetra

tion effects of GH are still very much shrouded in obscurity. A specific receptor for GH at the membrane has not been identified. It is possible that these actions of GH are quite nonspecific and resemble more the insulin-mimicking actions of phospholipases, proteases, and certain SH-compounds.

We have no intimate understanding as yet, on a molecular level, of the exact manner by which a hormone affects transmembrane trans

port. In the case of insulin and glucose we are assuming the existence

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of a sugar carrier molecule. In the absence of insulin most of the carrier molecules are prevented from their transport function. Insulin does not interact directly with the carrier, but binds to its specific receptor. We have no knowledge whatever of how this act of binding

"perturbs" the membrane sufficiently to "uncover" carrier sites.

The only hint we have at present is that anoxia, inhibitors of oxidative phosphorylation, phospholipases, and proteases can mimic such an action. We can only hope that further work on the isolation of the receptor and its molecular environment will throw light on the details of membrane perturbation.

ACKNOWLEDGMENTS

The work of the laboratory is supported by grants from the National Science Founda

tion, the American Diabetes Association, and the Pfizer Company.

The chapter was written during tenure of a John Simon Guggenheim Fellowship.

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