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Some basic aspects of protein-energy interrelationships

V.R. YOUNG*

* Laboratory of Human Nutrition, School of Science, Massachusetts Institute of Technology, Cambridge, MA 02142-1308, U.S.A.

1. Introduction
2. Energy dependency of protein and amino acid metabolism
3. Summary and conclusions
References

1. Introduction

In subsequent papers in this volume the nutritional and health aspects of the interactions between protein and energy metabolism, and factors which affect these interactions, will be discussed in considerable detail. The purpose of this short, introductory paper is to review, in brief, qualitative aspects and the quantitative importance of some of the underlying cellular and metabolic processes that are responsible for joining energy and protein metabolism into an intimate partnership. For further detail, the reader is encouraged to consult a recent comprehensive review of energy metabolism, with particular reference to its interactions with protein metabolism and other factors (KINNEY and TUCKER, 1992).

2. Energy dependency of protein and amino acid metabolism

2.1. Qualitative aspects
2.2. Quantitative aspects
2.3. Correlations between energy and protein metabolism

2.1. Qualitative aspects

Initially, it is worthwhile to consider, briefly, those major processes that require energy in the form of high-energy phosphate bonds. They are intimately involved in the utilization of nitrogen and amino acids, in the formation of polypeptides and their further assembly within or outside the cell and, ultimately, in the degradation of proteins and the catabolism of their constituent amino acids (YOUNG et al., 1992a). Table 1 is a list of some of the major processes that are known to be dependent on a source of ATP or GTP (YOUNG et al., 1991).

In addition to the well-established role of these high-energy intermediates in the initiation and elongation of polypeptide synthesis, during the past few years, scientists have exposed and clarified the mechanisms responsible for the regulation of cellular processes, such as protein folding and aggregation (BECKMANN et al., 1990), intracellular traffic of newly synthesized proteins (LINGAPPA, 1989), and transport of proteins across membranes (RAPOPORT, 1990; ROTHMAN and ORCI, 1992), including the import of proteins into mitochondria (BAKER and Schatz, 1991) (more than 95% of the mitochondrial proteins are encoded by nuclear genes), and the transduction of signals from outside of cells to their appropriate intracellular sites (e.g., BERRIDGE, 1987; BERRIDGE and MITCHELL, 1988). Most of these involve the participation of energy, in the form of ATP or GTP or their derivatives (cAMP; cGMP), through a series of complex protein-protein interactions and second messenger cascades.

Table 1. Some energy-dependent processes associated with protein turnover and amino acid homeostasis¹

¹. From Young et al (1991).

One such system involves a bewildering array of proteins, such as the heterotrimeric G proteins, that are turned on when bound to GTP and turned off by hydrolyzing GTP to GDP (BOURNE et al., 1991), and monomeric GTP-binding proteins (SIMON et al., 1991). These proteins are involved in controlling a diverse set of essential cellular functions, including growth, differentiation, intracellular vesicle transport and secretion, to name just a few (HALL, 1990). In essence, however, it is these various processes, including transcription of DNA, followed by splicing of pre-mRNA (COMPANY et al., 1991; SCHWER and GUTHRIE, 1991) that account in a major way, for the molecular and cellular basis of the interactions between protein, nitrogen and energy metabolism and their nutritional significance. Examples are the peptide-chain initiation (GUPTA, 1987), the attachment of an amino acid to its cognate tRNA followed by peptide-chain elongation (MOLDAVE, 1985), together with the energy required for protein degradation, via various ATP-dependent and GTP-dependent mechanisms (e.g., RECHSTEINER, 1987; PLOMP et al., 1987).

2.2. Quantitative aspects

While the processes mentioned above, as well as others, are energy-dependent, it remains uncertain as to how much food energy is required to drive most of them in vivo. This has been pointed out by WATERLOW and MILLWARD (1990). Hence, although we now appreciate better the molecular and cellular events and processes that underlie the interactions between protein and energy metabolism, there remains a considerable challenge to develop the necessary techniques and approaches for quantifying the energy required by these processes in vivo. Furthermore, not only the quantity of total protein formed and degraded is of importance, but also the qualitative nature of the protein turnover should be considered in the comprehensive assessment of the energetics of protein metabolism. For example, it is to be expected that in disease the pattern of gene expression will differ from that in health.

This may well mean that the processing of proteins in various intra- and extra-cellular compartments will also differ, with varying energy requirements involved in their aggregation and trafficking. For example, does the synthesis of acute phase proteins and their appearance in the circulation require the same amount of energy per mole of protein, than an equivalent rate of albumin synthesis? The answer is likely to be no. Hence, it will be necessary to learn more about the pattern of protein turnover, as well as the rate of total protein turnover, before we understand fully the quantitative significance of protein metabolism in relation to the energy needs and expenditure of the organism.

Considering that the body makes more than 100000 different proteins, this is truly a difficult task. In any case, it is now abundantly clear that the energy costs of protein turnover exceed the utilization of the ATP and GTP used for activation of amino acids and for the formation of the peptide bonds during the elongation phase of mRNA translation. Hence, earlier estimates of the energy costs of protein turnover based principally on these processes alone (MILLWARD et al., 1976, WATERLOW et al., 1978) will underestimate the functional significance of protein turnover in whole body energetics, as has also been emphasized by HAWKINS (1991).

There is still a good deal of uncertainty concerning the energy cost of protein degradation which, in the adult, would essentially amount to the equivalent of the daily rate of protein synthesis. The complex process of degradation is now beginning to be worked out. It is known that this proceeds via a series of lysosomal and non-lysosomal mechanisms and that inhibitors of energy metabolism can arrest protein degradation (Goldberg and ST. JOHN, 1976; BALLARD, 1977). For example, it appears that at least 14% of ATP utilization in reticulocytes is involved in intracellular protein degradation (SIEMS et al., 1984). Several mechanisms can account for this energy expenditure, including (1) ubiquitin conjugation, (2) a non-lysosomal protease which apparently requires two ATPs for the cleavage of each peptide bond (GOLDBERG et al, 1987; MENON et al., 1987), and (3) the ATP requirement for entry of proteins into lysosomes and to drive the proton pumps involved in maintaining a pH gradient (DEAN and BARRETT, 1976). Clearly, energy is utilized during protein turnover, but exactly how much is still unknown.

Another point to be made in reference to the energetics of protein turnover is that we need to understand the quantitative and functional aspects of the link between protein turnover and ion pumps, particularly the Na⁺, K⁺ - ATPase complex. A major function of this pump is to convert the chemical energy from the hydrolysis of ATP into a gradient for Na⁺ and K⁺; these gradients are used as free energy sources for a number of processes such as (1) formation of the membrane potential, (2) cell volume regulation, and (3) transport of glucose and amino acids into cells against concentration gradients (SKOU, 1990). The contribution of Na⁺, K⁺ -ATPase activity to cellular and organ energy metabolism has been reviewed recently by PARK et al. (1992). They conclude that it accounts for 9-45, 16-51 and 17-61% of the oxygen utilization in the skeletal musculature, liver and gastrointestinal tract, respectively, under a variety of physiological and nutritional conditions. I find a recent study by QIAN et al. (1991) of interest in this regard, since they showed that the A system amino acid transporter and the mRNA for the µ_-1 subunit of the Na⁺, K⁺ -ATPase are coordinately controlled by the regulatory gene Rl. Furthermore, as already implied above, the Na⁺ electrochemical gradient, established by the Na⁺, K⁺ -ATPase, across the cell membrane serves as the driving force for the A system of amino acid transport. From these various observations it would seem reasonable to suggest that the energy needs of ion pumping must be taken into account in any quantitative consideration of the energy cost of protein and amino turnover. The problem, of course, is the proportionate extent to which the energetics of ion movement ought to be assigned as a primary cost of protein turnover.

Even if it were possible to better quantify the needs for high-energy phosphate bonds for the various processes discussed above, the next problem to be resolved is the dietary energy required to meet the ATP (or GTP) requirements. Thus, FLATT (1992) has shown that the dietary energy required may be higher than the commonly assumed value of approximately 19 kcal (80 kJ) per mole ATP (e.g., BLAXTER, 1971). Certainly the problem is more complex than usually appreciated, but has to be dealt with in any attempt to define the quantitative impact of changes in energy intake and status on protein and amino acid metabolism and vice versa.

2.3. Correlations between energy and protein metabolism

While the energy costs of the various processes referred to above are not known, it is possible to gain an idea about their total impact by exploring relationships between in vivo rates of protein synthesis and of energy expenditure. Thus, as shown in Table 2, rates of protein synthesis, when expressed per unit body weight, are high in the smaller mammalian species when compared to rates of larger mammals. When expressed to the three-quarter power of body weight (kg^0.75), there is a relative constancy among the various species listed here, although the rate is considerably higher in avian as compared with mammalian species.

Table 2. Some estimates of protein synthesis in adults of various mammalian and avian species¹

1. Partial summary from Reeds & Harris (1981) and Muramatsu (1990). With addition of data from White et al (1988) for the wallaby.

This summary indicates that protein synthesis is correlated with resting energy metabolism, since the 0.75 power of body weight (in kg; metabolic body size) is also the weight function that approximately equalizes body energy expenditure across the different mammalian species (KLEIBER, 1947; BLAXTER, 1989). Therefore, it can be seen in Table 3, which is based largely on WATERLOW (1984), that the change in the intensity of body protein turnover parallels that for differences in their metabolic rates (kJ); expressed as kJ metabolic rate per g protein turnover, the values shown in Table 3 range from 11-23, with a mean of about 15.

It is of possible interest to point out, from Table 3, that the lower rate of protein turnover in marsupials is also associated with a lower metabolic rate (WHITE et al, 1988), such that the basal energy expenditure per unit of protein synthesis then approximates that for eutherian mammals. Similarly, the higher rate of protein turnover in chickens is paralleled by a higher basal or resting metabolic rate; it can be estimated (Table 3) that approximately 4 kcal or 16 kJ resting energy expenditure are associated with each g of protein synthesis in this species. This is roughly comparable to that for mammals. Therefore, from these various pieces of data it appears that, on average, about 4-5 kcal or 15-20 kJ of basal energy expenditure are 'connected' with each g protein synthesis.

Table 3. Whole body protein turnover in relation to resting metabolic rate (MR) in adults of mammalian & avian species¹

1. From Waterlow (1984), with addition of data for birds from Muramatsu (1990) and White et al (1988) for the wallaby.

Assuming that about 3 kJ (0.7 kcal) are expended minimally in the formation of peptide bonds per g protein synthesis, this implies that approximately 20% of basal metabolism is due to the process of polypeptide chain elongation. However, as noted above, this represents an underestimate of the total energy cost of protein synthesis and turnover because of the significant but uncertain quantitative estimates of the energy needs of the various processes responsible for the synthesis and positioning of proteins within their functional regions of the body and their subsequent breakdown.

Another approach taken for estimating the relationship between protein turnover and energy expenditure is offered by the study of WELLE and NAIR (1990) who examined the variation of leucine flux and its relationship with metabolic rate in a population of 26 adult men and 21 women. These investigators observed a high correlation between leucine flux and resting metabolic rate (RMR). From their regression analysis it was concluded that the contribution of protein turnover to RMR was about 20% in an average subject. This is comparable to the value given above, and as derived from the interspecies comparison.

A recent paper by SOARES et al (1991) is relevant here. These investigators found, in groups of Indian men, that the slope of the regression of protein synthesis rate (g/d) on BMR (kJ/d) was approximately 9 kJ/g (2.1 kcal/g) or about twice the value derived from the stoichiometry of peptide bond synthesis. This again suggests that the energetics of protein turnover may account for about 40% of the BMR.

Finally, it might be worth mentioning that the relationships discussed above between total body protein turnover and energy metabolism also appear to hold for the turnover of tRNA and rRNA. This is demonstrated in the novel studies of SCHÖCH et al (1990) who measured, in urine, modified RNA catabolites as an index of the turnover of individual classes of RNA. They applied, therefore, a concept which had been earlier proposed for measurement of muscle protein turnover in vivo, based on estimates of the urinary excretion of the derived amino acid, N-methylhistidine (YOUNG and MUNRO, 1978). tRNA and rRNA turnover among the various, but limited, number of mammalian species examined by SCHÖCH et al. (1990) was found to be related to the 0.78 and 0.69 power of body weight, respectively, or to a similar body weight function relating protein turnover among these different species (e.g., Table 2). The intriguing and challenging question emerging from this and similar work is the causal, or mechanistic, link between the energy flux and the turnover of these macromolecules.

SCHÖCH et al. (1990) hypothesize that the irreversible denaturation of macromolecules is basically a function of the energy flux per unit body mass, and that there is a compensatory resynthesis of these macromolecules in order to maintain a steady state. As we learn more about: (a) the mechanisms of protein degradation, which might well be promoted by oxidative damage or modification of specific amino acid residues (e.g., STADTMAN, 1988; DEAN, 1987), and also due, in part, to formation of protein adducts, coming from oxidant byproducts arising from normal metabolism, and (b) the macromolecular and functional link between protein degradation and protein synthesis, we will be in a better position to explain more completely the metabolic basis for the whole body energy-protein relationships just discussed.

3. Summary and conclusions

Energy and protein (amino acid) metabolism interact at various levels of biological complexity, as indicated above. It is not surprising, therefore, that changes in energy intake will give rise to a complex pattern of responses in amino acid and protein metabolism, depending in part upon overall nutritional background and host conditions involved. A number of the molecular and cellular processes that are involved in the interaction were summarized, and while this new knowledge has not yet helped us to better quantify the in vivo interrelationships between energy and protein metabolism, it has expanded our appreciation of the functional significance of dietary energy and protein interactions. It may also help stimulate, here at this workshop or when we return to our laboratories, new thinking about how to define and understand the mechanisms involved.

Molecular and cellular studies have shown, for example, that redox potential can affect the overall thiol-disulfide status of the cell, which in turn can influence the redox state of individual proteins (CAPPEL and GILBERT, 1988; ZIEGLER, 1985) and so determine the activity of metabolic processes. A recent example of this, from ABATE et al. (1990), concerns the DNA binding of the Fos-Jun heterodimers, which functions as an intermediary transcriptional regulator in signal transduction. This is modulated by reduction-oxidation (redox) of a single conserved cysteine residue in the DNA-binding domains of the two proteins, so implicating a possible redox mechanism in the regulation of transcriptional activity mediated by AP-1 binding factors. It appears that not only are ATP formation and availability significant factors in relation to the dynamic state of protein (and energy) metabolism, but also the pattern of fuel utilization (YOUNG et al., 1992b), through an influence on the redox state of the cell could be quite important in determining the quantitative relationships between protein turnover and body energy metabolism. As ZIEGLER (1985) points out, there is not yet sufficient evidence for a major role of changes in the redox state of cell proteins that modulate metabolic pathways in response to physiological stimuli. However this is a hypothesis that would be well worth exploring.

Similarly, it is to be expected that the level, and possibly source, of protein intake and status of protein nutriture would influence energy metabolism (e g., SAMONDS and HEGSTED, 1978; COYER et al., 1987; AUSMAN et al., 1989), mediated, in part, via effects on these various processes and promoted by alterations in the functioning and activity of the endocrine system (e.g., MILLWARD, 1990; YOUNG and MARCHINI, 1990; LONG and LOWRY, 1990). This emphasizes why it is worthwhile for us to explore, in further detail during this workshop, the metabolic interactions between energy and nitrogen and their nutritional implications. It is also still intriguing to clarify further the molecular and cellular features of the pathophysiology of the various forms of protein-energy malnutrition, with the extremes being marasmus and kwashiorkor. In doing so it is hoped that this basic knowledge would encourage development of more effective approaches for preventing significant tissue and organ protein depletion under conditions of stress, and for replenishing tissue and organ proteins in affected individuals and populations.

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