Adjustments of Protein Metabolism in Fasting Arctic Charr,Salvelinus alpinus

Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (K_S) in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems) were measured in the same fish. This study is the first to measure both K_S and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills). K_S was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins.     

Determination of relative protein degradation activity at different life stages in rainbow trout (Oncorhynchus mykiss)

Rainbow trout were reared from 5 g to ~ 400 g on a diet formulated to supply the required protein from either fishmeal or plant proteins. The fish were sampled at every weight doubling and liver and muscle samples were obtained. From these tissue samples RNA and protein were isolated and analyzed for the expression of a number of muscle regulatory and protein degradation genes and enzymatic activity for proteins involved in the caspase, calpain, and ubiquitin-proteasome pathways for protein proteolysis. Only MyoD2 showed significant differences in expression between the two diets, while no significant changes over the course of the experiment were determined for MyoD2 or the other muscle factors. For the degradation genes significant changes in expression were determined for calpain1 and calpastatin. Calpastatin also showed a significant increase in expression over the course of the experiment in the muscle of fish fed a fishmeal diet and significant decrease in expression in the liver of fish fed the fishmeal based diet. Differences in proteasome enzyme activity were found between diets in the liver and muscle of fish and for caspase-3 activity in muscle. Significant changes in activity over the course of the experiment were noted for proteasome and calpain activity in the liver and muscle. These findings suggest that diets replacing fishmeal with plant material can have some effects on protein turnover in muscle and that some degradation pathways are differentially regulated during the growth of rainbow trout.     

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.     

Hormonal regulation of protein degradation in skeletal and cardiac muscle

A number of hormones produce either anabolic or catabolic effects on protein degradation in muscle. These effects can account for the changes in muscle proteolysis associated with a variety of physiological and pathological states. Thus the balance of hormones within the organism seems to play an important role in the overall regulation of this process. In the fed state, insulin may be the single most important factor maintaining low rates of proteolysis, whereas the catabolic effects of the glucocorticoid hormones in fasting seem to predominate. The proportions of these hormones may be important not only during starvation, but also in trauma and in diseases associated with their altered production and secretion (e.g., diabetes, Cushing's syndrome). Hyperthyroidism too causes catabolic effects on muscle proteolysis.     

Signal-transduction networks and the regulation of muscle protein degradation

Protein degradation in muscle functions in maintaining normal physiological homeostasis and adapting to new homeostatic states, and is required for muscle wasting or atrophy in various pathological states. The interplay between protein synthesis and degradation to maintain homeostasis is complex and responds to a variety of autocrine and intercellular signals from neuronal inputs, hormones, cytokines, growth factors and other regulatory molecules. The intracellular events that connect extracellular signals to the molecular control of protein degradation are incompletely understood, but likely involve interacting signal-transduction networks rather than isolated pathways. We review some examples of signal-transduction systems that regulate protein degradation, including effectors of proteolysis inducing factor (PIF), insulin and insulin-like growth factor (IGF) and their receptors, and fibroblast growth factor (FGF) and its receptors.     

Intracellular protein degradation: basis of a self-regulating mechanism for the proteolysis of endogenous proteins

The intracellular basal proteolysis system, as distinct from the lysosomal system, is important in sustaining a high flux of proteins required for maintenance, growth and adaptability of cells. Its activity automatically fluctuates with changes in protein synthetic activity, but with a considerably slower response time, since the two processes are only indirectly or passively linked. Since as much as one-third of intracellular proteolysis in mammalian cells is directed as nascent proteins, the consequences are more fully discussed in relation to cell growth state. During rapid growth, cells have to accumulate more than double their original protein mass in order to achieve a 100% increase between divisions. The effects of reducing protein synthesis by inducing quiescence, serum step-down or cycloheximide treatment on intracellular proteolysis are considered, and the possibility that this leads to enhanced degradation of existing proteins has been explored. No substantial evidence was found to support this latter notion. The basal proteolysis system is seen as a constitutive, pervasive and broad-spectrumed collection of hydrolytic enzymes. It destroys proteins randomly, having no means of distinguishing young from old, aberrant from normal. The rate of demise of protein substrates depends on two factors, the ease of access of the hydrolytic enzymes to their peptide bonds, and the length of time that any species of protein remains at risk to this hydrolytic potential. While the former has long been recognized, the importance of the second factor in relation to the ability of proteins to become integrated in the living fabric of the cell is only beginning to be appreciated. The discussion also suggests elaborate regulatory mechanisms akin to those for protein synthesis would be unnecessary for protein degradation, especially if it can now be substantiated that substrate availability determines the turnover rates of proteins by a pervasive and relatively unlimited proteolytic system (Grisolía, 1964).     

USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.     

Effects of insulin-like growth factor-1/binding protein-3 complex on muscle atrophy in rats

Muscle atrophy and wasting is a serious problem that occurs in patients with prolonged debilitating illness, burn injury, spinal injury, as well as with space flight. Current treatment for such atrophy, which often relies on nutritional supplementation and physical therapy, is of limited value in preventing the muscle wasting that occurs. Considerable recent attention has focused on the use of anabolic growth factors such as insulin-like growth factor (IGF-1) in preventing muscle atrophy during limb disuse or with various catabolic conditions. However, potential side effects such as hypoglycemia appear to be limiting factors in the usefulness of IGF-1 for clinical treatment of muscle wasting conditions. The formulation of IGF-1 used in this study (IGF-1/BP3) is already bound to its endogenous-binding protein (BP3) and, as a result, has a greater specificity of action and significantly less hypoglycemic effect. Using a rat model of hind limb suspension (HLS) for 10 days, we induced marked muscle atrophy that was accompanied by enhanced muscle proteolysis and reduced muscle protein content. When HLS rats were treated with IGF-1/BP3 (50 mg/kg, b.i.d.), they retained greater body and muscle mass. Muscle protein degradation was significantly reduced and muscle protein content was preserved. The rate of protein synthesis, although somewhat reduced in HLS muscle, was not significantly elevated by IGF-1/BP3 treatment. Volume density of HLS-treated muscles were increased compared to untreated HLS rats and the actual number of fibers per area of muscle was likewise increased. The results of the current study suggest that IGF-1/BP3 might be useful for inhibiting muscle proteolysis in catabolic conditions and thus preserving muscle protein content and mass.     

Deubiquitinating enzymes in skeletal muscle atrophy—An essential role for USP19

The ubiquitin proteasome system is well recognized to be involved in mediating muscle atrophy in response to diverse catabolic conditions. To date, almost all of the genes that have been implicated are ubiquitin ligases. Although ubiquitination is modulated also by deubiquitinating enzymes, the roles of these enzymes in muscle wasting remains largely unexplored. In this article, the potential roles of deubiquitinating enzymes in regulating muscle size are discussed. This is followed by a review of the roles described for USP19, the deubiquitinating enzyme that has been most studied in muscle wasting. This enzyme is upregulated in muscle in many catabolic conditions and its inactivation leads to protection from muscle loss induced by stimuli that are common in many illnesses causing cachexia. It can regulate both protein synthesis and protein degradation as well as myogenesis, thereby modulating the key processes that control muscle mass. Roles for other deubiquitinating enzymes remain possible and to be explored.     

The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.)

The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass.     

Salmon spawning migration and muscle protein metabolism: the August Krogh principle at work

The August Krogh principle, stating that for any particular question in biology, nature holds an ideal study system, was applied by choosing the anorexic, long-distance migration of salmon as a model to analyze protein degradation and amino acid metabolism. Reexamining an original study done over 20 years ago on migrating sockeye salmon (Oncorhynchus nerka), data on fish migration and starvation are reviewed and a general model is developed on how fish deal with muscle proteolysis. It is shown that lysosomal activation and degradation of muscle protein by lysosomal cathepsins, especially cathepsin D and sometimes cathepsin L, are responsible for the degradation of muscle protein during fish migration, maturation and starvation. This strategy is quite the opposite to mammalian muscle wasting, including starvation, uremia, cancer and others, where the ATP-ubiquitin proteasome in conjunction with ancillary systems, constitutes the overwhelming pathway for protein degradation in muscle. In mammals, the lysosome plays a bit part, if any. In contrast, the proteasome plays at best a subordinate role in muscle degradation in piscine systems. This diverging strategy is put into the context of fish metabolism in general, with its high amino acid turnover, reliance on amino acids as oxidative substrates and flux of amino acids from muscle via the liver into gonads during maturation. Brief focus is placed on structure, function and evolution of the key player in fishes: cathepsin D. The gene structure of piscine cathepsin D is outlined, focusing on the existence of duplicate, paralogous, cathepsin D genes in some species and analyzing the relationship between a female and liver-specific aspartyl protease and fish cathepsin Ds. Evolutionary relationships are developed between different groups of piscine cathepsins, aspartyl proteases and other cathepsins. Finally, based on specific changes in muscle enzymes in fish, including migrating salmon, common strategies of amino acid and carbon flux in fish muscle are pointed out, predicting some metabolic concepts that would make ideal application grounds for the August Krogh principle.     

Downregulation of muscle protein degradation in sepsis by eicosapentaenoic acid (EPA)

Eicosapentaenoic acid (EPA) has been shown to attenuate muscle atrophy in cancer, starvation and hyperthermia by downregulating the increased expression of the ubiquitin-proteasome proteolytic pathway leading to a reduction in protein degradation. In the current study EPA (0.5 g/kg) administered to septic mice completely attenuated the increased protein degradation in skeletal muscle by preventing the increase in both gene expression and protein concentration of the α- and β-subunits of the 20S proteasome, as well as functional activity of the proteasome, as measured by the ‘chymotrypsin-like’ enzyme activity. These results suggest that muscle protein catabolism in sepsis is mediated by the same intracellular signalling pathways as found in other catabolic conditions.     

Intracellular proteolysis in rat cardiac and skeletal muscle cells in culture.

Treatment of adult rats with dexamethasone resulted in an increase in cardiac muscle weight but a decrease in skeletal muscle weight. The different response of skeletal and cardiac muscles to the glucocorticoid was also reflected by a dexamethasone-induced enhancement of myofibrillar protease activity in the gastrocnemius muscle and an inhibition of a similar proteolytic activity in the heart. Newborn rats also exhibit the same, tissue-specific response to the glucocorticoid hormone. Consequently, the difference between cardiac and skeletal muscle responsiveness to conditions of wasting was investigated in culture. Average rates of degradation of intracellular proteins were determined in cultured cells derived from rat skeletal and cardiac muscle by following the release of radioactivity from cells prelabelled with ¹⁴C-phenylalanine. The release of label into the TCA soluble medium as measured during 12 hours of incubation, conformed to a first-order reaction and both cell types were found to degrade intracellular proteins at a similar rate. After 12 hours of incubation in a complete Ham F-10 medium supplemented with serum approximately 18% of total cellular protein was degraded. Incubation in a minimal medium or serum-deprivation enhanced the average rate of proteolysis to a value of 29% degradation at 12 hours indicating that intracellular proteolysis in these cells is responding to nutritional deprivation by increased activity. However, addition of glucose (22.2 nM) or dexamethasone (10^?6M) to the incubation medium failed to affect the rate of net protein degradation. Under no experimental condition could a difference be found between the proteolytic response of skeletal muscle cells to that of cardiac muscle cells and both cell types displayed similar changes in rates of protein degradation under various nutritional and hormonal conditions in culture. Thus, protein sparing in the heart of intact animals under catabolic conditions which enhance protein loss in skeletal muscle can probably not be ascribed to intrinsic differences in the direct response of cellular proteases to the tested hormones and nutrients. Rather, an extracellular factor(s) is apparently required for induction of the differential response of these tissues in the intact animal to protein wasting conditions. Alternatively, cells in culture might have lost the property of differential degradative response which operates in vivo.     

Laboratory evolution of catabolic enzymes and pathways

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.     

Molecular mechanisms of genetic adaptation to xenobiotic compounds.

Microorganisms in the environment can often adapt to use xenobiotic chemicals as novel growth and energy substrates. Specialized enzyme systems and metabolic pathways for the degradation of man-made compounds such as chlorobiphenyls and chlorobenzenes have been found in microorganisms isolated from geographically separated areas of the world. The genetic characterization of an increasing number of aerobic pathways for degradation of (substituted) aromatic compounds in different bacteria has made it possible to compare the similarities in genetic organization and in sequence which exist between genes and proteins of these specialized catabolic routes and more common pathways. These data suggest that discrete modules containing clusters of genes have been combined in different ways in the various catabolic pathways. Sequence information further suggests divergence of catabolic genes coding for specialized enzymes in the degradation of xenobiotic chemicals. An important question will be to find whether these specialized enzymes evolved from more common isozymes only after the introduction of xenobiotic chemicals into the environment. Evidence is presented that a range of genetic mechanisms, such as gene transfer, mutational drift, and genetic recombination and transposition, can accelerate the evolution of catabolic pathways in bacteria. However, there is virtually no information concerning the rates at which these mechanisms are operating in bacteria living in nature and the response of such rates to the presence of potential (xenobiotic) substrates. Quantitative data on the genetic processes in the natural environment and on the effect of environmental parameters on the rate of evolution are needed.     

Proteinases and their inhibitors in cells and tissues

A large body of evidence has been assembled to indicate the substantial importance of proteolytic processes in various physiological functions. It has recently become clear too that endo-acting peptide bond hydrolases provisionally characterized and classified at present as serine, cysteine, aspartic and metallo together with unknown catalytic mechanism proteinases sometimes act in cascades. They are controlled by natural proteinase inhibitors present in cells and body fluids. In the first part of the present monograph the author was concerned to present an overview on the morphological and physiological approach to localization, surveying reaction principles and methods suitable for visualization of proteolytic enzymes and their natural and synthetic inhibitors. In the second part the roles played by proteinases have been summarized from the point of view of cell biology. The selection of earlier and recent data reviewed on the involvement of proteolysis in the behavior of individual cells reveals that enzymes, whether they be exogeneous or intrinsic, can be effective and sensitive modulators of cellular growth and morphology. There exists a close correlation between malignant growth and degradation of cells. It appears likely that as yet unknown or at least so far inadequately characterized factors that influence the survival or the death of cells may turn out to be proteinases. The causal role of extracellular proteolysis in cancer cell metastases, in stopping cancer cell growth and in cytolysis remains for further investigated. Ovulation, fertilization and implantation are basic biological functions in which proteolytic enzymes play a key role. The emergence of new approaches in reproductive biology and a growing factual basis will inevitably necessitate a reevaluation of present knowledge of proteolytic processes involved. The molecular aspects of intracellular protein catabolism have been discussed in terms of the inhibition of lysosomal and/or non-lysosomal protein breakdown. Peptide and protein hormone biosynthesis and inactivation are still at the centre of interest in cell biology, and a number of proteinases have been implicated in both processes. A number of conjectures partly based on the author's own work have been discussed which suggest the possibility of the involvement of proteolysis in exocytosis and endocytosis. The author's optimistic conclusion is that through the common action of biochemists, cell biologists, cytochemists, and pharmacologists the mystery of cellular proteolysis is beginning to be solved.     

Cellular signals activating muscle proteolysis in chronic kidney disease: a two-stage process

Muscle atrophy is a prominent feature of catabolic conditions and in animal models of these conditions there is accelerated muscle proteolysis that is dependent on the ubiquitin-proteasome system. However, ubiquitin system cannot degrade actomyosin or myofibrils even though it rapidly degrades actin or myosin. We identified caspase-3 as the initial and potentially rate-limiting proteolytic step that cleaves actomyosin/myofibrils. In rodent models of catabolic conditions, we find that caspase-3 is activated to cleave muscle proteins and actomyosin to fragments that are rapidly degraded by the ubiquitin system. This initial proteolytic step in muscle can be recognized because it leaves a footprint of a characteristic 14-kDa actin band. Stimulation of caspase-3 activity depends on activation of phosphatidylinositol 3-kinase. When we suppressed this enzyme in muscle cells, protein breakdown increased as did the expression of caspase-3. In addition, there was increased expression of E3-ubiquitin-conjugating enzymes that are involved in muscle proteolysis, atrogin-1/MAFbx and MuRF1. Thus, when phosphatidylinositol 3-kinase activity is low in muscle cells or rat muscle, both caspase-3 and the ubiquitin-proteasome system are stimulated to degrade protein. Additional investigations will be needed to define the cell signaling processes that activate muscle proteolysis in uremia and catabolic conditions.