Higher Rates Of Protein Degradation Research Articles

Life in the freezer: protein metabolism in Antarctic fish.

Whole-animal, in vivo protein metabolism rates have been reported in temperate and tropical, but not Antarctic fish. Growth in Antarctic species is generally slower than lower latitude species. Protein metabolism data for Antarctic invertebrates show low rates of protein synthesis and unusually high rates of protein degradation. Additionally, in Antarctic fish, increasing evidence suggests a lower frequency of successful folding of nascent proteins and reduced protein stability. This study reports the first whole-animal protein metabolism data for an Antarctic fish. Groups of Antarctic, Harpagifer antarcticus, and temperate, Lipophrys pholis, fish were acclimatized to a range of overlapping water temperatures and food consumption, whole-animal growth and protein metabolism measured. The rates of protein synthesis and growth in Antarctic, but not temperate fish, were relatively insensitive to temperature and were significantly lower in H. antarcticus at 3°C than in L. pholis. Protein degradation was independent of temperature in H. antarcticus and not significantly different to L. pholis at 3°C, while protein synthesis retention efficiency was significantly higher in L. pholis than H. antarcticus at 3°C. These results suggest Antarctic fish degrade a significantly larger proportion of synthesized protein than temperate fish, with fundamental energetic implications for growth at low temperatures.

Primary skeletal muscle cells from chronic kidney disease patients retain hallmarks of cachexia in vitro.

BackgroundSkeletal muscle wasting and dysfunction are common characteristics noted in people who suffer from chronic kidney disease (CKD). The mechanisms by which this occurs are complex, and although progress has been made, the key underpinning mechanisms are not yet fully elucidated. With work to date primarily conducted in nephrectomy‐based animal models, translational capacity to our patient population has been challenging. This could be overcome if rationale developing work could be conducted in human based models with greater translational capacity. This could be achieved using cells derived from patient biopsies, if they retain phenotypic traits noted in vivo.MethodsHere, we performed a systematic characterization of CKD derived muscle cells (CKD; n = 10; age: 54.40 ± 15.53 years; eGFR: 22.25 ± 13.22 ml/min/1.73 m2) in comparison with matched controls (CON; n = 10; age: 58.66 ± 14.74 years; eGFR: 85.81 ± 8.09 ml/min/1.73 m2 ). Harvested human derived muscle cells (HDMCs) were taken through proliferative and differentiation phases and investigated in the context of myogenic progression, inflammation, protein synthesis, and protein breakdown. Follow up investigations exposed HDMC myotubes from each donor type to 0, 0.4, and 100 nM of IGF‐1 in order to investigate any differences in anabolic resistance.ResultsHarvested human derived muscle cells isolated from CKD patients displayed higher rates of protein degradation (P = 0.044) alongside elevated expression of both TRIM63 (2.28‐fold higher, P = 0.054) and fbox32 (6.4‐fold higher, P < 0.001) in comparison with CONs. No differences were noted in rates of protein synthesis under basal conditions (P > 0.05); however, CKD derived cells displayed a significant degree of anabolic resistance in response to IGF‐1 stimulation (both doses) in comparison with matched CONs (0.4 nm: P < 0.001; 100 nM: P < 0.001).ConclusionsIn summary, we report for the first time that HDMCs isolated from people suffering from CKD display key hallmarks of the well documented in vivo phenotype. Not only do these findings provide further mechanistic insight into CKD specific cachexia, but they also demonstrate this is a reliable and suitable model in which to perform targeted experiments to begin to develop novel therapeutic strategies targeting the CKD associated decline in skeletal muscle mass and function.

Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised proteinday-1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate.

Why Age Matters: Inflammation, Cancer and Hormones in the Development of Sarcopenia

In an aging population, the decline in muscle mass and strength in combination with a high prevalence of osteoporosis and cancer leads to a multitude of clinical manifestations. In the recent years, mouse models of wasting in cancer and inflammation, including xenograft, genetic and chemically induced models, allowed to uncover several key mechanisms underlying muscle loss. These include inflammation, hormone alterations and deregulated protein degradation. Inflammation is associated with increased expression of tumor necrosis factor α (TNF-α), nuclear factor κB (NF-κB), and interleukin (IL)-6 and is therefore linked to inflammatory bowel diseases or chronic obstructive pulmonary disease (COPD). Moreover, active NF-κB signaling and IL-6 secretion commonly occurs in malignancies and cancer-induced cachexia. The ubiquitin proteasome-mediated degradation of proteins represents a second pathway underlying sarcopenia and is partially initiated by inflammatory signaling. Consequently, increased levels of the E3 ligases Muscle RING-Finger Protein-1 (MuRF1), Atrogin-1/Muscle Atrophy F-box (MAFbx), and tumor necrosis factor α receptor adaptor protein 6 (TRAF6) are associated with high rates of protein degradation. Furthermore, hormonal alterations, such as the aging-related decline of growth hormone (GH) and insulin-like growth factor 1 (IGF-1), lead to a reduction of muscle mass. Interestingly, experimental targeting of several of those sarcopenia-associated factors in vivo resulted in a rescue of muscle mass and function. While therapeutic options nowadays still need to be evaluated regarding their clinical practicability, IL-6 antibodies, inhibition of cyclooxygenases and inhibitors of myostatin appear promising.

Chemical sympathectomy further increases muscle protein degradation of acutely diabetic rats

The present work investigated the role of the sympathetic nervous system (SNS) in the control of protein degradation in skeletal muscles from rats with streptozotocin (STZ)-induced diabetes. Diabetes (1, 3, and 5 days after STZ) induced a significant increase in the norepinephrine content of soleus and EDL muscles, but it did not affect plasma catecholamine levels. Chemical sympathectomy induced by guanethidine (100 mg/kg body weight, for 1 or 2 days) reduced muscle norepinephrine content to negligible levels (less than 5%), decreased plasma epinephrine concentration, and further increased the high rate of protein degradation in muscles from acutely diabetic rats. The rise in the rate of proteolysis (nmol.mg wet wt(-1).2h(-1)) in soleus from 1-day diabetic sympathectomized rats was associated with increased activities of lysosomal (0.127 +/- 0.008 vs. 0.086 +/- 0.013 in diabetic control) and ubiquitin (Ub)-proteasome-dependent proteolytic pathways (0.154 +/- 0.007 vs. 0.121 +/- 0.006 in diabetic control). Increases in Ca2+-dependent (0.180 +/- 0.007 vs. 0.121 +/- 0.011 in diabetic control) and Ub-proteasome-dependent proteolytic systems (0.092 +/- 0.003 vs. 0.060 +/- 0.002 in diabetic control) were observed in EDL from 1-day diabetic sympathectomized rats. The lower phosphorylation levels of AKT and Foxo3a in EDL muscles from 3-day diabetic rats were further decreased by sympathectomy. The data suggest that the SNS exerts acute inhibitory control of skeletal muscle proteolysis during the early stages of diabetes in rats, probably involving the AKT/Foxo signaling pathway.

Decreased protein catabolism after exercise in subjects with IDDM

We examined whether the increased rates of protein catabolism (proteolysis and leucine oxidation) associated with moderate insulinopenia in subjects with IDDM would be accentuated by prior bicycle exercise (53% VO2max for 82 min). Insulin infusions maintained plasma glucose concentrations on one study day in “tight” control (TC: 6 mmol/l) and on a separate day in “loose” control (LC: 12 mmol/l). Elevations in serum ketone body, plasma NEFA, and whole-blood branched-chain amino acid concentrations on the loose control day during the basal period persisted throughout the post-exercise recovery period. Amino acid kinetics were estimated during a primed, constant infusion of l-[1-13C]leucine from plasma dilution of α-[1-13C]KIC and expired air 13CO2 enrichments. Loose control was associated with increased rates of whole-body leucine oxidation (LC 25±7 vs TC 21±8 μmol · kg−1 · h−1) and protein degradation (LC 127±12 vs TC 118±18 μmol · kg−1 · h−1) (both p<0.05). During the 2-h post exercise recovery period, there were significant decreases in rates of leucine oxidation (LC 21±7, TC 16±7) and protein degradation (LC 112±13, TC 107±11), compared to the basal period (both p<0.05, basal vs recovery). Rates of wholebody protein synthesis were unchanged by prior exercise. In conclusion, moderate insulinopenia is associated with significantly higher rates of protein degradation and leucine oxidation in the basal state. Following exercise, net protein catabolism is diminished due to reduced rates of protein degradation in the presence of maintained rates of protein synthesis. The significantly increased concentrations of fat-derived substrates (ketone bodies, NEFA) may have prevented the predicted increases in protein catabolism which we anticipated would follow acute exercise during periods of relative insulin deficiency.

Decreased protein catabolism after exercise in subjects with IDDM

We examined whether the increased rates of protein catabolism (proteolysis and leucine oxidation) associated with moderate insulinopenia in subjects with IDDM would be accentuated by prior bicycle exercise (53% VO2max for 82 min). Insulin infusions maintained plasma glucose concentrations on one study day in "tight" control (TC: 6 mmol/l) and on a separate day in "loose" control (LC: 12 mmol/l). Elevations in serum ketone body, plasma NEFA, and whole-blood branched-chain amino acid concentrations on the loose control day during the basal period persisted throughout the post-exercise recovery period. Amino acid kinetics were estimated during a primed, constant infusion of L-[1-13C]leucine from plasma dilution of alpha-[1-13C]KIC and expired air 13CO2 enrichments. Loose control was associated with increased rates of whole-body leucine oxidation (LC 25 +/- 7 vs TC 21 +/- 8 mumol.kg-1.h-1) and protein degradation (LC 127 +/- 12 vs TC 118 +/- 18 mumol.kg-1.h-1) (both p < 0.05). During the 2-h post exercise recovery period, there were significant decreases in rates of leucine oxidation (LC 21 +/- 7, TC 16 +/- 7) and protein degradation (LC 112 +/- 13, TC 107 +/- 11), compared to the basal period (both p < 0.05, basal vs recovery). Rates of whole-body protein synthesis were unchanged by prior exercise. In conclusion, moderate insulinopenia is associated with significantly higher rates of protein degradation and leucine oxidation in the basal state. Following exercise, net protein catabolism is diminished due to reduced rates of protein degradation in the presence of maintained rates of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium entry through stretch-inactivated ion channels in mdx myotubes

Recent advances in understanding the molecular basis of human X-linked muscular dystrophies have come from the identification of dystrophin, a cytoskeletal protein associated with the surface membrane. Although there is little or virtually no dystrophin in affected individuals, it is not known how this causes muscle degeneration. One possibility is that the membrane of dystrophic muscle is weakened and becomes leaky to Ca2+. In muscle from mdx mice, an animal model of the human disease, intracellular Ca2+ is elevated and associated with a high rate of protein degradation. The possibility that a lack of dystrophin alters the resting permeability of skeletal muscle to Ca2+ prompted us to compare Ca2(+)-permeable ionic channels in muscle cells from normal and mdx mice. We now show that recordings of single-channel activity from mdx myotubes are dominated by the presence of Ca2(+)-permeable mechano-transducing ion channels. Like similar channels in normal skeletal muscle, they are rarely open at rest, but open when the membrane is stretched by applying suction to the electrode. Other channels in mdx myotubes, however, are often open for extended periods of time at rest and close when suction is applied to the electrode. The results show a novel type of mechano-transducing ion channel in mdx myotubes that could provide a pathway for Ca2+ to leak into the cell.

Mechanisms of Starvation Tolerance in Pearl Millet

The response of pearl millet (Pennisetum glaucum [L.]) seedlings to prolonged starvation was investigated at the biochemical and ultrastructural level. After 2 days of darkness the bulk of the seedling carbohydrate reserves were depleted. After 8 days in the dark the respiratory rate had declined to less than 50% of its initial value and the plants had lost half of their total protein content. Unlike the situation with carbohydrate depletion, protein loss was restricted to specific organs. The secondary leaf and stem (including the apical meristem) showed little or no protein loss during this period. In the primary leaf, seed, and roots, protein loss was substantial. In spite of the high rate of protein degradation in the primary leaf and roots, these organs showed no ultrastructural changes suggestive of tissue, cellular, or subcellular degradation. In addition, ribulose bisphosphate carboxylase was not preferentially degraded during starvation and only a small decline in chlorophyll content was observed after 8 days in the dark. During the period from 8 to 14 days, cell death started at the tip of the primary leaf and gradually spread downward. Both shoot and root meristems remained alive up to 14 days. Consequently, the eventual death of the plant was due to the loss of the carbohydrate-producing regions rather than the meristems. We suggest that these results provide an explanation for the high degree of starvation tolerance exhibited by pearl millet.

Effects on in vivo and in vitro administration of vinblastine on the perfused rat liver—Identification of crinosomes

Livers of nonstarved rats were perfused for up to 4 hr in a recirculating system. Bile production, transaminases, and the lactate/pyruvate ratio remained at normal values. The ultrastructure of the hepatocytes was also well preserved even after the 4-hr perfusion. When vinblastine was given either in vivo or in vitro by addition to the perfusion fluid, it caused a conspicuous expansion of the autophagic-lysosomal compartment. Initially, nascent autophagic vacuoles developed, followed by the appearance of more mature ones and finally an increase in dense bodies was observed. In addition, administration of vinblastine in vivo gave rise to an increased occurrence of a subpopulation of lysosomes laden with VLDL-like particles. The term crinosomes seems appropriate for these lysosomal vesicles, since they apparently evolve by means of fusion between retained secretory granules and preexisting lysosomes (dense bodies). Addition of vinblastine of the perfusion fluid decreased the rate of proteolysis whether four times the serum concentration of amino acids were added or not. However, when vinblastine was given in vivo, proteolysis as measured in the perfusate decreased during the initial 3 hr of VBL treatment, whereas by longer times of pretreatment protein degradation exceeded the control value, constituting an example of catch-up proteolysis. Autophagic vacuoles isolated after short exposure to vinblastine in vivo exhibited high rates of protein degradation when incubated at acid pH. Insufficient proton pumping rather than lack of hydrolytic enzymes seems to be the most plausible explanation for this prompt pH effect.

The effect of insulin and intermittent mechanical stretching on rates of protein synthesis and degradation in isolated rabbit muscle.

Tyrosine balance and protein synthesis were studied during the same incubation in isolated rabbit forelimb muscles. From these measurements, protein degradation was calculated. Isolated muscles were usually in a state of negative amino acid balance, principally as a result of the 75% decrease in protein synthesis. Muscles from rabbits starved for 18 h had lower rates of both protein synthesis and degradation compared with muscles from normally fed rabbits. Intermittent mechanical stretching and the addition of insulin at 100 microunits/ml increased rates of both protein synthesis and degradation. Increases in the rate of protein synthesis were proportionately greater in the muscles from starved animals. In muscles from both fed and starved donors, increases in protein-synthesis rates owing to intermittent stretching and insulin were proportionately greater than the increases in degradation rates. For example, insulin increased the rate of protein synthesis in the muscles from starved donors by 111% and the rate of degradation by 31%. Insulin also increased the rate of protein synthesis when added at a higher concentration (100 munits/ml); at this concentration, however, the rate of protein degradation was not increased. The suppressive effect of insulin on high rates of protein degradation in other skeletal-muscle preparations may reflect a non-physiological action of the hormone.

Protein degradation in cultured skeletal muscle from Duchenne muscular dystrophy patients.

The loss of contractile protein in Duchenne muscular dystrophy could result from low rates of synthesis, abnormally high rates of protein degradation, or a combination of both. We measured overall protein degradation rates in cultured human muscle cells obtained at biopsy from patients with Duchenne dystrophy or various muscle diseases and normal subjects. Measurements were performed on confluent cultures exhibiting no growth and containing a mixed cell population of myoblasts, fibroblasts, and multinucleated myotubes. Using a new double-isotope labeling protocol, we found protein degradation rates in all three groups to be similar (KD = 0.0171-0.0176 hr-1), suggesting no detectable abnormality of overall protein degradation in cells derived from Duchenne dystrophy patients.

Protein degradation in primary monolayer cultures of adult rat hepatocytes. Further evidence for the regulation of protein degradation by amino acids.

Intracellular protein degradation was measured in primary monolayer cultures of adult rat hepatocytes by the loss of radioactivity from proteins pulse-labeled in culture with L-[U-14C]valine. The fractional rate of protein degradation of total protein measured over 18 h was similar to the rate in vivo. The fraction rate of protein breakdown was altered significantly by varying the composition of the chase medium from a balanced salts medium (high rate of protein degradation) to a more complete medium (lower rate of protein degradation). Whereas the vitamin component of the complete medium had some effect, the major portion of the inhibition of protein degradation was due to the essential amino acids in the medium. This inhibition was found to depend on the presence of methonine, phenylalanine, and tryptophan, the latter two of which appear to be primarily responsible. The observed rate of protein breakdown was a function of the level of amino acids in the medium when they were varied in the physiological range for rat plasma. Our results suggest that the regulation of protein degradation by essential amino acids may be physiologically important in maintaining intracellular amino acid pools when the exogenous supply of amino acids is diminished.

Protein metabolism and survival of rat hepatocytes in early culture

Rat hepatocytes, cultured in a serum- and hormone-free medium on a substratum of adsorbed fibronectin, are in negative protein balance throughout the first 24 h of culture. The rate of protein loss at all times equals the difference between the high rate of protein degradation and the low rate of protein synthesis. A continuous decline in the rate of protein degradation gradually reduces the loss of protein, probably the result of medium conditioning as well as progressive culture deterioration. Inhibition of lysosomal protein degradation by amino acids and the protease inhibitor, leupeptin, reduces the protein loss considerably, but does not prevent cell death. The protein balance per se thus does not seem to be the limiting factor for the survival of cultured hepatocytes.

Insulin inhibition of protein degradation in cell monolayers

Protein degradation has been measured in confluent monolayers of eleven lines of contact-inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with [3H]leucine. Insulin, at contrations from 10(-12) M to 10(-6) M, has been added at the beginning of the 4-hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serum-free medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature-sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH1C1, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast-like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum-free medium. Generally higher rates of protein degradation are observed in the contact-inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells.