The hormonal control of protein turnover

CLINICALNUl-RITION (1990) 9: 115-126 , Longman Group UK Lrd 1990

THE ARVID WRETLIND

LECTURE

0261-5614/90iuoo9-o115’810.00

1989

The Hormonal Control of Protein Turnover D. J. Millward Nutrition NW1 2PE, UK

INTRODUCTION reviewing hormonal control of protein turnover I want to explore several themes. The first relates to our perception of the system we are trying to research. If we are to understand whole body protein metabolism, its regulation and hormonal control and the nature of dietary needs for protein and amino-acids, we need to have a model of the system and its behaviour. I want to introduce and elaborate the model which we are developing [ 11. After this, I want to explore the specific role of insulin, but in doing that I need to address the problem we face in building a coherent story from animal studies to explain regulation in man, when there appear to be species differences in responses to individual hormones. There are puzzling aspects of the response to insulin in man compared with what we would expect from the established wisdom deriving from animal data, and these must be reconciled before the specific role of insulin in the endocrinological orchestra can be resolved. This will be the ultimate focus of my paper, the question of how to explain the marked nutritional influence of dietary protein, on protein metabolism and growth which we have termed the anabolic drive [2]. I will show that insulin can be viewed as having a pivotal role in mediating the homeostatic and homeorhetic influence of dietary amino-acids.

1. Modelling man’s need for dietary protein and amino acids The model which seems to be implied by most current discussions of protein requirements is shown in Figure la. Dietary amino-acids are needed to replace the obligatory nitrogen losses and to provide for growth and any other net protein needs, and this dietary provision is always inefficient. The current adult mean requirement of 0.6g/kg [3] implies an efficiency of only 60:, in replacing the obligatory nitrogen losses of 0.34 g/kg. The key question is whether this inefficiency is fixed for an individual. If it is, then the requirement is fixed. If it is not, then the requirement is variable. In fact this question cannot currently be answered unequivocally. How-

Medicine, St. Pancras

ever, as described elsewhere [4] there is a considerable degree of disparity between the apparent requirements as judged by the various published short term balance studies. The most plausible explanation of this is a variable efficiency in response to extrinsic factors which are not yet suthciently understood, and consequently not always controlled for in balance studies. In fact, there has been little discussion in the literature of this apparent inefficiency. Munro [5] has described it as the ‘non-linear response’, explaining that, for a high quality protein, the organism utilises it with perfect efficiency to provide for obligatory needs until equilibrium is approached, at which point excretion increases to match any further intake. Thus the cuwature which causes the non linearity is assumed to be an indication of the organism’s less than perfect ability to sense equilibrium and adjust immediately. This is an inadequate explanation, since, as pointed out elsewhere [1, 4, 61, reports indicate linear responses throughout the range: others show quite concave curves, with poor utilisation at low intakes and better utilisation near equilibrium. This variability in responses is also observed in studies examining the requirement for high quality protein as well as studies examining the relative utilisation (i.e. the biological value) of different proteins. Indeed, because of the lack of reproducibility of balance studies it is extremely difficult to demonstrate consistent differences in human adults between N balance responses to proteins as disparate in their indispensable amino-acid content as egg and wheat gluten [4]. We can only conclude that it is due to factors which have not yet been identified. After reviewing the large volume of balance data and considering what is known about amino acid and protein metabolism, Millward and Rivers [1] came to the conclusion that the real problem is that the model within which we formulate requirements is simply inadequate. Having decided this it was a relatively simple task to develop a new one (Fig. 1b). The model attempts to explain the inefficiency of utilisation of dietary protein by identifying two factors which result in oxidative losses of amino-acids. The first factor is the recognition that the organism does not tolerate high concentrations of most of the indispensable amino-acids. If they are not deposited as protein 115

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Protein gain growth

transient

I-G-I

obligatory

(diurnal cycling)

I-G-l

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THE ANABOLIC c

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inoreaaed loaaea on feeding: creatinine urinary

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‘non-linear reaponae’

and faecal nitrogen

losses

regulatory

loaaea

Fig. 1

Models for protein requirements. la) Model imnlied in most current discussions of orotein reauirements. id) New model proposed by Millward and River;[l, 2,5]. -

after a meal they are oxidised by high capacity highly regulated oxidative pathways (Lr, regulatory losses). It is most likely that both the capacity and activity of these pathways are sensitive to acute and chronic dietary protein and possibly other influences. The second factor which influences these losses is the periodicity of food intake i.e. the diurnal pattern of feeding and fasting. The argument here is that body protein is lost in the post-absorptive (PA) state and that the extent of these losses is likely to be a variable, also influenced by the protein intake. The model also includes an additional concept, that of the anabolic drive. This is the idea that indispensable amino-acids are needed to exert a regulatory influence on the organism and this is of central importance to my story.

able with the diet [l]. What is most important is whether PA losses are variable with the diet and the two possible situations are shown in Figure 2. If there is a constant low PA loss, with no adaptation to the dietary protein intake, then there should be a fixed low level of fed-state gain with fed state oxidation

nitrogen balance feeding

(a)

gain ---“~_-“~_“*._yn fasting

loss

A

-ve -overall balance

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Model validation The model implies that requirements must be viewed in terms of meeting intrinsic needs (G,, L, and L,) and needs determined by extrinsic influences on diurnal cycling and L,. Thus the efficiency of utilisation of protein or an individual amino-acid is determined by L, which varies in a complex way in response to acute and chronic dietary influences and according to the periodicity of the intake. We have recently started to explore this latter aspect. The inclusion of diurnal cycling in the model arises from the simple fact that, with few exceptions (patients on TPN), there is a diurnal pattern of eating and fasting so we have to deposit protein in the fed state to match post-absorptive losses. The key question is how oxidative losses are regulated. There is no doubt that losses increase in the fed state and that these losses are vari-

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Fig. 2 Alternative models of diurnal cycling on varying protein intakes. (a) Constant low post-absorptive losses: As intake changes balance is adjusted by changes in fed-state oxidation, allowing a fixed fed-state gain to balance post-absorptive losses, and constant amplitude of diurnal cycling. Adjustment to new protein intake should be immediate. (b) Variable post-absorptive losses: As intake changes balance is adjusted by changes in both fed and post-absorptive oxidation, requiring a variable fed-state gain to balance postabsorptive losses, and varying amplitude of diurnal cycling. Adjustment to a lower level of protein intake is not immediate, since the need for gain is determined by prior post-absorptive loss.

CLINICAL

adjusting immediately to eliminate all surplus intake. The amplitude of diurnal cycling should be constant and adjustment to new protein intake should be immediate. However, from what we know [l], we can predict that L, is likely to increase in both fed and PA state as dietary protein is increased; there are implications flowing from this if it is true. If there is an increased postabsorptive loss as protein intakes are increased, and if such dietary conditioning persists for any appreciable time when the diet is changed, then the pattern of response would be quite different. At low intakes there will be insufficient fed state gain to balance PA losses so there will be negative balance. As intakes increase, there will be increasing fed state gains to balance increased PA losses. This means that fed state deposition needed for balance will be determined by prior post-absorptive losses, in turn conditioned by previous intakes. Thus, after a period on a high intake a switch to a low intake should result in marked negative balance with insufficient fed state gain for balance whilst the high PA losses persist. In other words adjustment to a new level of protein intake should take some time. In an attempt to differentiate between these two models we have made measurements in individuals fed increasing protein diets. After two weeks on diets containing either low (0.35g/kg, i.e. equal to the ONL), current safe intakes (0.75 g/kg), or twice the safe level (,1.5 g/kg), we have studied the amplitude of diurnal cycling over a 48 h period during alternate 12 h periods of feeding and fasting, measuring 12 h urinary nitrogen losses, corrected for changes in the body urea pool, and whole body protein turnover in the fed and fasting state with a combination of stable isotope methodologies [7]. These preliminary results (Fig. 3) show that increasing the intake from the safe allowance of 0.75 g protein/ kg per day to twice that level increased PA losses and post-prandial (PP) gains whilst approximate overall balance was maintained. This was accompanied by an increase in the rate of whole body protein turnover. On the low intake there was insufficient PP gain to balance the PA loss, with a negative overall balance. When the diet was switched from a high to a low intake, the high rate of PA and PP loss persisted so that there was insufficient gain to balance the loss, with a very marked negative balance. These data do suggest that our basic assumptions are accurate and that the central feature of our model is correct, that the requirement at any time is conditioned by previous intakes. What we still have to determine of course is how long this conditioning persists after the diet is changed. In many ways of course the model has to be correct, since it is built on the long established phenomenon of labile protein reserves. In this new model, labile protein reserves

are seen

as a manifestation

of the kinetic

and

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N-balance mgN/kg per 12 hrs

Nitrogen turnover 0.82 (gN/kg per d)

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protein intake (mgN/kg per d) Fig. 3 Diurnal cycling in adults fed varying intakes of protein. 12 h nitrogen balances in normal adults measured over 48 h at the end of 2 weeks on the diets. Protein turnover calculated from “CO, excretion during a constant infusion of [W-l] leucine and the rate of nitrogen excretion. The amplitude of diurnal cycling increased with increased protein intakes as did protein turnover and the persistance of the elevated post-absorptive losses as predicted by model b in Figure 2 with insufficient fed-state gain resulted in a very marked negative balance [7].

adaptive response to varying protein intakes. However the key question is how these acute and chronic changes in protein balance are achieved and this brings us back to the anabolic drive.

2. Hormonal control of protein turnover: The anabolic drive The concept of the anabolic drive is a simple one. Nutrients are assumed to serve a dual role, acting as substrates for protein deposition and metabolism, and exerting an important regulatory function stimulating anabolic processes. As described elsewhere [6], these regulatory functions include homeostatic influences i.e. the acute regulation associated with immediate responses to food intake and other environmental influences, homeorhetic influences i.e. influences which sustain an important non-steady state biological response such as growth, and those influences which relate to functional regulation. In this paper I want to deal with the homeostatic and homeorhetic components of the anabolic drive since, as discussed elsewhere, we have as yet little information on the nature of functional regulation. There is no doubt that dietary amino-acids are the most important dietary stimulus of the anabolic drive [2,8,9]. It is obvious to all that growth in terms of protein deposition cannot occur without dietary protein. However as described elsewhere [9], it is also the case that (a) protein deposition cannot occur without an

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intact system of hormonal regulatory responses, (b) that these regulatory responses are so powerful that in the absence of sullicient dietary energy, body fat will be mobilised to provide the energy, and (c) that high concentrations of dietary protein will elicit muscle and bone growth even when the organism is in negative energy balance. These facts are easily demonstrated. Hypophysectomised rats will not grow without growth hormone treatment, but when treated with growth hormone and not allowed to increase food intake, will increase muscle mass and decrease body fat [Q]. Normal rats fed restricted intakes of diets of increasing protein concentrations (i.e. maintaining constant protein intakes) continue to deposit muscle even with 50% food restriction (again resulting in loss of body fat and falling body weights) [Q]. These data demonstrate the power of the anabolic drive of protein. The fact that indispensable amino-acids (IAA) play a central role in these phenomena would be predicted from the large literature on the influence of protein quality on growth, from specific investigations of the influence of selected IAA depletions on growth retardation [lo], as well as studies showing effects of IAA as opposed to dispensable amino acids on nitrogen balance and IGF-I levels during refeeding [l I]. The problem posed by any regulatory anabolic mechanism requiring substantial intakes of IAAs is that most IAAs cannot be tolerated by the organism and are rapidly oxidised if concentrations rise. This is evident when the regulation of indispensable amino-acid levels in body tissues is examined. It is the case that whilst very high levels of some amino-acids are tolerated (glutamine may be more than 20 m Molar), most indispensable amino-acids are kept at very low concentrations. This happens because the oxidative enzymes do not allow the concentrations to rise [l]. For the branchedchain amino-acids, for example, although they are the most abundant in protein, the branched-chain a-oxo dehydrogenase, the rate limiting enzyme in their oxidation, will not allow their concentration to rise, and they are oxidized rapidly after feeding unless incorporated into protein. Judging by the responses to high levels of the BCAs and the aromatic amino-acids in children with inborn errors of metabolism, these amino-acids can be judged to be relatively toxic compared with the non essential amino-acids. Any regulatory influence is therefore only likely to be transient, since dietary IAAs are rapidly removed either by net protein synthesis or oxidation. Clearly, as discussed elsewhere, the extent to which amino-acids can serve this dual role as substrates and as a regulatory influence, without promoting excessive oxidation, will determine both the efficiency of protein utilisation and the overall protein requirement. The magnitude of the intakes required to exert this regulatory role remains to be determined.

3. Insulin as a mediator of homeostatic regulation As to the nature of the regulatory influence of dietary amino-acids, most would expect insulin to be particularly important in homeostatic regulation given its ability to respond immediately to each meal and to promote protein deposition in muscle (augmenting the deposition of hepatic protein which occurs after every meal [ 131). However, our understanding of hormonal control is based primarily on animal studies and it is important to examine the relevance of these studies to man before we proceed any further.

(a) Studies in the rat: insulin secretion My own interest in insulin originated from observations that Peter Garlick and I made during studies of meal feeding in rats. Having demonstrated increases in muscle protein synthesis in response to feeding [ 121, we were trying to define e mechanisms involved. We were struck by the fact I at, whilst insulin secretion was stimulated by a protein-containing meal, the response to a protein-free meal was almost completely blunted 1131. We now know that insulin, more than any other anabolic hormone, is able to respond acutely and sensitively to nutrient intake. Its secretion reflects a complex interaction between the content of glucose and amino-acids in the diet and the prevailing state of the organism [ 141. The importance of amino-acids as a stimulus for insulin secretion has been confirmed in our recent studies of the responses of rats to various low-protein diets fed ad libitum. Plasma insulin levels reflected protein intake and not non-protein energy [ 151. In addition to this, in these experiments, insulin levels reflected T, status and we have confirmed this statistical relationship in other studies of insulin secretion in response to refeeding fasted rats [6]. In these fasted rats, plasma T3 levels were low. However not only was the increase in plasma insulin in response to refeeding dependent on the plasma TS level, but pre-treatment with thyroid hormones of these fasted rats and increasing their TS levels, increased the insulin response to refeeding. This influence of T, on the insulin response to feeding is consistent with the studies of Okajima and Ui [ 171. Recent studies show that in addition to thyroidal influences on insulin secretion, beta-cells of the pancreas are also sensitive to insulin-like growth factor 1 (IGF1) and this influences insulin secretion [18J. The importance of these thyroidal and IGF-1 influences on insulin secretion means that, to the extent that these hormones are a reflection of the more chronic nutritional state, insulin secretion is also sensitive to chronic as well as acute changes in food intake. This aspect is

CLINICAL NUTRITION

important in relation to insulin’s homeorhetic role discussed below. Insulin a&on. The marked increase in insulin levels in response to a protein meal [ 131 suggested to us that the accompanying stimulation of muscle protein synthesis might be mediated by insulin in line with then current studies by Jefferson and Morgan [ 191. We were generally unimpressed by arguments that amino-acids, and particularly the branched-chain amino-acids (BCAS), might have direct influences on muscle protein synthesis because of our observations on the way in which their intracellular concentrations changed. Thus, whilst we observed small increases in the BCA concentrations in muscle with feeding, there were much more marked changes with fasting [13] and in a variety of other catabolic states [20] when protein synthesis was inhibited. Clearly some other factor was responsible for this inhibition and we thought that insulin was the most likely factor judging from our observation that, in the diabetic rat, muscle protein synthesis was severely inhibited [20]. We showed subsequently that amino-acids and glucose could not stimulate this reduced muscle protein synthesis in the diabetic rat, whereas insulin induced rapid restoration [21]. Furthermore we showed in refeeding experiments that within 20min of food pots being put in the cages of fasted rats muscle protein synthesis was stimulated (Fig. 4), a response which was highly correlated with the increase in insulin, (at least at the lower end of the physiological range of insulin levels (Fig. 5) and which was partially inhibited by antiinsulin treatment [22]. It is therefore clear to us that, in the rat, insulin is an essential factor for muscle protein synthesis, most likely activating the initiation of translation, with initiation factor eIF, as a likely target [23], possibly through suppressing the action of an inhibitory protein that phosphorylates eIF* [24] which accumulates in the absence of insulin [25]. The ability of insulin to exert this stimulatory effect on protein synthesis is dependent, of

synthesis rate (%/d) *r

protein synthesis (g/gRNA/d) 20

1 * 15

i

*

I

Fig. 5 Correlation between skeletal muscle protein synthesis and insulin in refed fasted rats [ 161.

course, on the absence of counter-regulatory factors which induce insulin insensitivity (Fig. 6). Glucocorticoids are the best described example [21], with the high levels in starvation accounting for a major part of the catabolic response and the rapid fall of corticosterone on refeeding fasted animals appears to be necessary for the maximum insulin stimulation of protein synthesis [22]. Another factor is the inflammatory response which is associated with marked insulin insensitivity. Indeed, as we have shown [26], the inhibition of muscle protein synthesis can be accompanied by a hyperinsulinemia due to a direct prostaglandin-dependent cytokine-mediated stimulation of insulin secretion [27]. However, according to our studies [28], insulin action on muscle protein synthesis is not prostaglandindependant as suggested by others [29,30]. The major problems in relating this experience of insulin action to man start when we ask questions about insulin and proteolysis. This is a difficult question since, in the rat, it is more difficult to study this in the in vivo experiments which we have focused on. For many years it has been thought that insulin plays a major role in inhibiting muscle protein degradation (e.g. [31]), and this has certainly been repeatedly observed in incubated or in perfused muscle. However, the physiological relevance of most of the incubated muscles studies can be questioned, since in rabbit muscles (which have a lower glucocorticoids endotoxinslcytokines

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Fig. 4 Increases in muscle protein synthesis on refeeding fasting rats [22].

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cyclooxygenase inhibitors(fenbufen) Fig. 6 Factors which influence the action of insulin on skeletal muscle protein synthesis.

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metabolic rate, are less likely to become anoxic and are consequently better maintained than rodent muscles) incubated under tension the addition of physiological levels of insulin has been shown to have parallel stimulatory effects on muscle protein synthesis and proteolysis [29,30]. It is interesting in the light of these studies to note that in a recent study from Peter Garlick’s laboratory [32] it was observed that, in post-absorptive rats, insulin alone stimulated muscle protein synthesis, albeit requiring higher concentrations when administered with amino-acids. It seems to me that, in order for insulin to act on protein synthesis alone, it is highly unlikely that it could be inhibiting proteolysis at the same time, since the free amino-acid pool would vanish under these circumstances. I would interpret these results as not only confirming the anabolic influence of insulin on protein synthesis but also confirming that, in rat skeletal muscle in vivo, insulin does not inhibit proteolysis. Indeed, a stimulation of proteolysis is one possible interpretation of this data. We have reported other data which suggests that insulin might stimulate proteolysis in rat muscle in vivo. We examined [33] rates of muscle growth, protein synthesis and degradation and plasma insulin, free Ts and protein and non-protein energy intakes in rats growing at different rates on various intakes of dietary energy and protein. With enough measurements of these variables it is possible not only to determine their interrelationships, but also, by means of partial correlation analysis, to dissect these inter-relationships in considerable detail. When this was done, a scheme emerged in which insulin stimulates muscle proteolysis. (b) Studies in man and other species My own initiation into the regulation of protein metabolism in human muscle were studies we carried out some years ago with colleagues in London (Rennie, Edwards and Halliday) and in St Louis in the US (Mathews) looking at the response of protein synthesis in human muscle to feeding and fasting with stable isotopes and needle biopsy [34]. We showed that the low rate of protein synthesis in fasting was stimulated by feeding. These early pioneering studies have been subsequently confirmed with further [“C-l] leucinebiopsy studies from Halliday’s laboratory in both normal subjects and patients with diseased muscle [35], and extended by studies involving the forearm model showing that feeding stimulates muscle protein synthesis with little influence on protein degradation [36]. These studies are quite comparable to the responses observed in the rat. The question we have to ask, however, is whether they are achieved by the same mechanisms and, in particular, what is the role of insulin? In fact there are

DIETARY INFLUENCES r value

\ protein 0.457 C-peptide

Fig. 7

= -17.3 + O.Ol(Kcals) + O.OSl(gCHO) + O.O49(gPROT)

Dietary factors which influence insulin secretion [36].

obvious differences which can be identified at the outset. Insulin secretion in man is much less dependent on dietary amino-acids than in the rat, to the extent that this particular component of the complex series of influences on insulin secretion is often ignored. In fact dietary protein does play an important role. The studies of Hoogwerf et al. [37] on the dietary factors which influence secretion, as assessed by urinary C-peptide excretion, showed that protein intake was an important determinant. Indeed, on a weight basis, protein appears to be a more powerful stimulus than carbohydrate (Fig. 7). These studies mean that insulin may well mediate some of the influence of dietary amino-acids on muscle protein synthesis. The major problem arises when we look at the responses of muscle protein synthesis to insulin administration. In a small unpublished series of leucine biopsy studies from Halliday’s laboratory, Nair showed that the administration of insulin to insulin-withdrawn diabetics had no stimulatory influence on muscle protein synthesis, a response confirmed in subsequent studies by Pacy and Halliday with both [“C-l] leucine-biopsy [38] and forearm studies [39]. This is not to say that insulin has no effect, since close arterial infusion of insulin has certainly been shown significantly to improve protein balance across the forearm [40], but the mechanism appears to involve an inhibition of proteolysis, a different response to that observed in the rat. Pacy has also observed this type of response with the forearm model in insulin treated diabetics [39], the improvement in balance appearing to involve a lowering of degradation and possibly protein synthesis (see Fig. 8). However, the most clear indication of what is going on has been reported in studies on growing lambs. By means of arterio-venous monitoring of leucine kinetics, Oddy et al. [41] showed clearly that insulin treatment induces a lowering of both proteolysis and protein synthesis, changes which were significant in the fasted lambs (Fig. 9). The most likely explanation of these effects in man and in the lamb is that there are obvious species differences in the relative sensitivity of muscle protein synthesis and degradation to regulatory stimuli. If in man insulin acts primarily to inhibit proteolysis

CLINICAL

nmol phe/min/lOOml

leucine disposal and release in muscle 250r

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and if, in contrast to the rat, amino-acids have a dominant role in regulating protein synthesis independently of insulin, we would expect insulin treatment to inhibit proteolysis, which would lower amino-acids and inhibit protein synthesis. In fact, we now have good evidence that this interpretation is probably correct. Bennett, working in Rennie’s laboratory, has shown with both biopsy [42] and arterio-veneous-kinetic studies across the leg [personal communication], that amino-acids do stimulate muscle protein synthesis in man directly, with no effect on proteolysis. Clearly, if the role of insulin in the response to feeding is to be understood, then insulin must be given with sticient amino-acids to maintain intracellular levels. Bennett has done this [personal communication] and, as shown in Figure 10, a stimulation of protein synthesis and inhibition of proteolysis is observed. These species differences between rat and man relating to the influence of insulin and amino-acids on muscle protein turnover are summarised in the schemes in Figure 11. In the rat (Fig. 1 la), dietary amino-acids synthesie

or degradation

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Fig. 10 Influence of insulin and amino-acids on skeletal muscle protein synthesis and degradation in human type 1 diabetics [Bennett and Rennie, personal communication].

Fig. 8 Influence of insulin on skeletal muscle protein synthesis in insulin treated type 1 diabetics [38].

Protein

syn deg -basal-

.

increase plasma insulin which stimulates muscle protein synthesis as the primary response, with a parallel stimulatory influence on proteolysis, if any at all. Any influence of amino-acids such as glutamine [43] appear to need the presence of insulin in order to be effective. In contrast, in man (Fig. llb), insulin alone acts primarily to inhibit proteolysis, with amino-acids having the dominant role in regulating protein synthesis independently of insulin. Thus, insulin treatment inhibits proteolysis, lowering amino-acids and inhibiting protein synthesis. The response to feeding would appear therefore to involve events similar to those in Figure 1 lc, with the stimulation of protein synthesis only possible when dietary amino acids are sufficient to increase levels in muscle, at which time insulin can stimulate protein synthesis as well as inhibit proteolysis. Unanswered questions still remain of course, for example, why proteolysis is obviously inhibited by insulin and amino acids in the diabetic but with no obvious inhibition occurs in response to feeding (361. It would appear therefore that insulin exerts an important homeostatic influence in man, interacting with amino-acids to regulate the gains and losses of protein in muscle during feeding and fasting, although the detailed mechanisms may differ between man and the rat. It should be borne in mind that these homeostatic influences of the anabolic drive also include protein deposition and mobilisation from liver where it is clear that amino-acids exert the major influence [44, 451. However there is considerable evidence, again from animal studies, that it also exerts a powerful homeorhetic influence which is an equally important component of the anabolic drive.

I”.“,,”

Fig. 9 Influence of insulin on skeletal muscle protein synthesis and degradation in fed and fasted growing lambs 1W.

4. Insulin as mediator of homeorhetic influences In Figure 12, both homeostatic and homeorhetic

com-

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(6) HUMAN SKELETAL MUSCLE response to insulin alone

(A) RAT SKELETAL MUSCLE response to insulin and amino acids

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l

AMINO

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(Cl HUMAN SKELETAL MUSCLE response to insulin and amino acids Schemes showina influences of insulin and amino-acids on muscle protein turnover. (a) Effect of insulin and amino-acids in the rat. (b) Effect of insulin alone on human muscle. (c) Effect of insulin and amino-acids on human muscle. Fig. 11

ponents of the anabolic drive are represented as a combination of direct regulatory influences of dietary protein on tissues together with indirect hormonal influences of insulin together with growth hormone, IGF-1 and T,: see [9]. This is not to ignore the large number of other hormones known to exert both positive and negative influences on growth, but rather to focus on those hormones known both to mediate anabolic effects and to be nutritionally sensitive. Distinction is made in Figure 12 between nutritional influences exerted centrally, acting on growth hormone and (through TSH) T4 production, or peripherally, influencing the GH mediated synthesis of IGF-1, T4Ta conversion and insulin production. As already indic-

~GHRH Central

Dietary protein GI

I

peripheral regulation

ANABOLIC Fig. 12

bRiVE

Hormonal components of the anabolic drive.

ated, the sensitivity of insulin to amino-acid intake allows it to mediate its homeostatic role acting with amino-acids. However, it is the indirect influence of insulin mediated through its involvement in the peripheral metabolism of growth hormone and thyroid hormone metabolism and action which constitutes its homeorhetic role. Insulin’s regulation of T3 and IGF-I metabolism and action The apparent statistical dependence of T, on insulin concentration on rats fed low-protein diets [33] is explainable by the fact that, in many tissues, the T4 deiodenase is an insulin-dependent hormone [46, 471. Thus, the adaptation in thyroid hormone metabolism in starvation, which results in low T3 levels [16], reflects, at least in part, the low insulin levels which occur in response to the absence of food intake. As would be expected from this, Tj levels fall markedly in experimental diabetes [48]. The influence of insulin on IGF-1 production is less well understood, but indirect evidence suggests it to be very important. Thus, diabetes is associated with low levels of IGF-1 [49] which persists with growth hormone treatment [50]. Although the influence of dietary protein on plasma IGF-1 concentrations is often stressed [51], with evidence for specific influences of dietary IDAs [IO, Ill, such relationships could well reflect the influence of insulin on IGF-1 production.

CLINICAL NUTRITION

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rats fed protein deficient diets (Yahya and Millward, unpublished).

Fig. 14 IGF-1 in plasma, muscle and bone (tibia1 growth plate), of protein deficient rats (Yahya and Millward, unpublished).

Given these relationships between insulin, TS and IGF1, it is not surprising to observe that, in rats fed low protein diets, the fall in plasma insulin is accompanied by falls in both Ta and IGF-1, correlations which are most marked in the low insulin range (Fig. 13). Clearly, a major problem in attempting to evaluate the anabolic drive in this way concerns the relevance of changes in plasma concentration of hormones to net effects on their various targets. Changes in receptor number and sensitivity, associated with both stimulatory and inhibitory influences from other factors, mean that measurements of plasma hormone concentrations provide, at best, a crude qualitative guide to their actual physiological role in growth. For both TS and IGF-1, the problem can be particularly difficult. Given the intracellular location of both TS receptors and the T4 deiodenase, TS can be considered to exert its action, to some extent in an autocrine manner, and TS concentrations at the receptor could be quite different from plasma levels. Plasma IGF-1 almost certainly reflects liver-produced hormone, and since extra-hepatic target tissues such as muscle and bone can produce their own IGF in paracrine or autocrine fashion, the relevance of changes in plasma IGF-1 is increasingly under scrutiny. There is a further problem posed by binding proteins. In the case of TS in the protein-deficient rat, concentrations of free TS change in the opposite direction to total T, [52], due to increases in binding proteins. In the case of IGF-1, a large mwt IGF-1 binding protein in plasma (IGF-1 BP3) possibly increases the half life of IGF-1 [53]. In addition, IGF-l-receptor interaction appears to be influenced (inhibited) by a low mwt. binding protein (IGF-1 BPl), possibly produced by target tissues [54], and under regulation by insulin which inhibits it production [ 551. These problems mean that, in contrast to insulin,

where we can be reasonably certain that changes in circulating hormone levels usually have biological significance, the acute changes in these other hormones are more difficult to interpret. Certainly, changes in plasma IGF-1 in response to dietary variation may overestimate the actual nutritional sensitivity of IGF-1 at the target receptors, and this is shown by the concentrations of IGF-1 in plasma muscle and bone of protein deficient rats (Fig. 14). The marked falls in plasma hormone levels were accompanied by very little change in tissue levels, even though muscle and bone growth was markedly depressed. Our recent studies on changes in tibia1 length growth and IGF-1 concentrations in protein deficiency tend to support this (Fig. 15). Notwithstanding these uncertainties, we can suggest a scheme based on our own and other studies [9] and some speculation. In this scheme (Fig. 16), insulin mediates homeostatic regulation by controlling the reversible deposition of protein (shown here for skeletal muscle but also including liver). However, homeorhetic regulation of growth involves events in the myofibre, satellite cells and fibroblasts of muscle and in the proliferating chondrocytes of the growth plate of bone. This requires a sustained nutrient input in which insulin levels are elevated for long enough to increase levels of IGF-1, T, and the other growth factors which together mediate such responses. Clearly, the scheme is not intended to be comprehensive. IGF-l’s role in muscle connective tissue growth has yet to be confirmed, but seems to me most likely, especially since satellite cell proliferation is most sensitive to IGFs rather than insulin [56]. The interactions between insulin and IGF-1 action via modulation of the IGFlBP1[55] are still somewhat tentative, with the role of TS in satellite cell and muscle connective tissue cell not yet confirmed but based on no more than its similar known role in chondrocyte maturation [57]. However,

124

THE

HORMONAL

CONTROL

OF PROTEIN

TURNOVER

o,s!ibial growth (mm/d)

0.,&y

growth plate IGF-1 (U/gm)

t-3

days of diet

days of diet Fig. 15 Chances in tibia1 length growth

and IGF-1

concentration

(Yahya and Millward,

unpublished).

ACKNOWLEDGEMENTS fibroblast

The ideas and arguments expressed in this paper have de-

Fig. 16 Scheme summarising putative homeostatic and homeorhetic influences of insulin on skeletal muscle and bone.

the important feature of the scheme is the concept that growth hormone-IGF-1 and T, levels represent an integrated response to food intake with sufficient torque in their regulation that several meals need to be missed before malnutrition is sensed and growth is shut down.

veloped over several years during discussion with many colleagues including John Waterlow, Peter Garlick, George Grimble, Bhanu Odedra, John Brown, Geoff Laurent, Peter Bates, Margaret Jepson, Zainal Yahya and Gill Price. I am particularly grateful to Dave Halliday, Paul Pacy, Mike Rennie and Willie Bennet, who have given me the benefit not only of their own ideas but who have also shared with me their recent unpublished experimental data. I am grateful to the Medical Research Council, The British Diabetic Association, The Muscular Dystrophy Group of Great Britain, the Wellcome Trust, the Nestle Nutrition Programme and the Leverhulme trust for past and present Financial support. The development of my ideas relating to models for protein requirements resulted from a collaboration with John Rivers, to whom I dedicate this paper.

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CONCLUSION

[31

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Submission date: 23 November

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1990

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.“,