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Energy Restriction Suppresses Muscle Protein Synthesis, and High Protein Diets Extend Protein Half-Lives Across the Muscle Proteome in Obese Female Zucker Rats

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Energy Restriction Suppresses Muscle Protein Synthesis, and High Protein Diets Extend Protein Half-Lives Across the Muscle Proteome in Obese Female Zucker Rats

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Background Effects of high protein (HP) diets and prolonged energy restriction (ER) on integrated muscle protein kinetics have not been determined. Objective The objective of this study was to measure protein kinetics in response to prolonged ER and HP on muscle protein synthesis (MPS; absolute rates of synthesis) and muscle protein breakdown (MPB; half-lives) for proteins across the muscle proteome. Methods Female 6-wk-old obese Zucker rats (Leprfa+/fa+, n = 48) were randomly assigned to one of four diets for 10 wk: ad libitum-standard protein (AL-SP; 15% kcal from protein), AL-HP (35% kcal from protein), ER-SP, and ER-HP (both fed 60% feed consumed by AL-SP). During week 10, heavy/deuterated water (2H2O) was administered by intraperitoneal injection, and isotopic steady-state was maintained via 2H2O in drinking water. Rats were euthanized after 1 wk, and mixed-MPS as well as fractional replacement rate (FRR), relative concentrations, and half-lives of individual muscle proteins were quantified in the gastrocnemius. Data were analyzed using 2-factor (energy × protein) ANOVAs and 2-tailed t-tests or binomial tests as appropriate. Results Absolute MPS was lower in ER than AL for mixed-MPS (–29.6%; P < 0.001) and MPS of most proteins measured [23/26 myofibrillar, 48/60 cytoplasmic, and 46/60 mitochondrial (P < 0.05)], corresponding with lower gastrocnemius mass in ER compared with AL (–29.4%; P < 0.001). Although mixed-muscle protein half-life was not different between groups, prolonged half-lives were observed for most individual proteins in HP compared with SP in ER and AL (P < 0.001), corresponding with greater gastrocnemius mass in HP than SP (+5.3%; P = 0.043). Conclusions ER decreased absolute bulk MPS and most individual MPS rates compared with AL, and HP prolonged half-lives of most proteins across the proteome. These data suggest that HP, independent of energy intake, may reduce MPB, and reductions in MPS may contribute to lower gastrocnemius mass during ER by reducing protein deposition in obese female Zucker rats.

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... FSR of individual proteins (%/day) was calculated as the fraction of newly synthesized protein present at Pre, Mid, and Post, and the proportion of individual proteins with greater FSR in TEST compared with PLA was also determined. This approach has been implemented previously by our group to examine changes in proteome dynamics (11,25). ...
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Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.
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Caloric restriction (CR) mitigates many detrimental effects of aging and prolongs life span. CR has been suggested to increase mitochondrial biogenesis, thereby attenuating age-related declines in mitochondrial function, a concept that is challenged by recent studies. Here we show that lifelong CR in mice prevents age-related loss of mitochondrial oxidative capacity and efficiency, measured in isolated mitochondria and permeabilized muscle fibers. We find that these beneficial effects of CR occur without increasing mitochondrial abundance. Whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis, which is further supported by lower mitochondrial protein synthesis with CR. We find that CR decreases oxidant emission, increases antioxidant scavenging, and minimizes oxidative damage to DNA and protein. These results demonstrate that CR preserves mitochondrial function by protecting the integrity and function of existing cellular components rather than by increasing mitochondrial biogenesis.
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Calorie restriction (CR), which is thought to be largely dependent on the neuroendocrine system modulated by insulin/insulin-like growth factor-I (IGF-I) and leptin signaling, decreases morbidity and increases lifespan in many organisms. To elucidate whether insulin and leptin sensitivities are indispensable in the metabolic adaptation to CR, we investigated the effects of CR on obese Zucker (fa/fa) rats and lean control (+/+) rats. CR did not fully improve insulin resistance in (fa/fa) rats. Nonetheless, CR induced neuropeptide Y (NPY) expression in the hypothalamic arcuate nucleus and metabolism related gene expression changes in the liver in (fa/fa) rats and (+/+) rats. Up-regulation of NPY augmented plasma corticosterone levels and suppressed pituitary growth hormone (GH) expression, thereby modulating adipocytokine production to induce tissue-specific insulin sensitivity. Thus, central NPY activation via peripheral signaling might play a crucial role in the effects of CR, even in insulin resistant and leptin receptor deficient conditions.
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The mechanism by which insulin controls protein metabolism is not fully understood. Insulin stimulates protein synthesis; it also enhances transport of some amino acids, but the latter action does not appear to be sufficient explanation of the increase in synthesis. The various actions seem to be independent of effects on glucose metabolism. In diabetic muscle there are fewer than normal polysomes, and insulin rapidly enhances attachment of monomers to messenger-RNA. Insulin also increases the effectiveness of cell sap in catalyzing protein synthesis by ribosomal systems. The way in which the hormone may affect either initiation or peptide synthesis is not known. Experiments are reported bearing on whether availability of amino acids could be a mechanism by which effects of insulin are mediated. Activity of liver and muscle soluble fractions declines on fasting and, for the latter tissue, possibly also on a low protein diet. Sap from fasting animals allows a much smaller response of isolated ribosomes to added amino acids. Availability of glutamate in amino acid mixtures may be of special importance. However, insulin can influence the activity of the sap fraction of diaphragm muscle during incubation without the presence of amino acids in the medium. Understanding of what mechanisms are involved will depend on resolution of the critical sap factors.
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Obese Zucker rats exhibit marked hyperphagia when compared to lean littermates but deposit a smaller percentage of total dietary energy as body protein. This study was designed to determine the roles of skeletal muscle protein synthesis, protein degradation, RNA, or DNA in producing the lower muscle mass of obese rats. At 44 days, 3 hindlimb muscles, the extensor digitorum longus (EDL), the gastrocnemius and the plantaris were significantly smaller in the obese animals. At 72 days, the differences in weights of these muscles were more pronounced. Protein synthesis and degradation were determined in the soleus at 44 days of age using an in vitro whole muscle incubation technique. Protein synthesis rate was significantly decreased in the obese animals. These changes were accompanied by reductions in both RNA and DNA levels. Significant changes in nucleic acid levels were observed in both the red and white portions of the gastrocnemius muscle. These changes in the anabolic process of protein accretion appear to be sufficient to account for the reduced muscle mass in the obese Zucker rat.
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Mass isotopomer distribution analysis (MIDA) is a technique for measuring the synthesis of biological polymers. First developed approximately eight years ago, MIDA has been used for measuring the synthesis of lipids, carbohydrates, and proteins. The technique involves quantifying by mass spectrometry the relative abundances of molecular species of a polymer differing only in mass (mass isotopomers), after introduction of a stable isotope-labeled precursor. The mass isotopomer pattern, or distribution, is analyzed according to a combinatorial probability model by comparing measured abundances to theoretical distributions predicted from the binomial or multinomial expansion. For combinatorial probabilities to be applicable, a labeled precursor must therefore combine with itself in the form of two or more repeating subunits. MIDA allows dilution in the monomeric (precursor) and polymeric (product) pools to be determined. Kinetic parameters can then be calculated (e.g., replacement rate of the polymer, fractional contribution from the endogenous biosynthetic pathway, absolute rate of biosynthesis). Several issues remain unresolved, however. We consider here the impact of various deviations from the simple combinatorial probability model of biosynthesis and describe the analytic requirements for successful use of MIDA. A formal mathematical algorithm is presented for generating tables and equations (APPENDIX), on the basis of which effects of various confounding factors are simulated. These include variations in natural isotope abundances, isotopic disequilibrium in the precursor pool, more than one biosynthetic precursor pool, incorrect values for number of subunits present, and concurrent measurement of turnover from exogenously labeled polymers. We describe a strategy for testing whether isotopic inhomogeneity (e.g., an isotopic gradient or separate biosynthetic sites) is present in the precursor pool by comparing higher-mass (multiply labeled) to lower-mass (single- and double-labeled) isotopomer patterns. Also, an algebraic correction is presented for calculating fractional synthesis when an incomplete ion spectrum is monitored, and an approach for assessing the sensitivity of biosynthetic parameters to measurement error is described. The different calculation algorithms published for MIDA are compared; all share a common model, use overlapping solutions to computational problems, and generate identical results. Finally, we discuss the major practical issue for using MIDA at present: quantitative inaccuracy of instruments. The nature and causes of analytic inaccuracy, strategies for evaluating instrument performance, and guidelines for optimizing accuracy and reducing impact on biosynthetic parameters are suggested. Adherence to certain analytic guidelines, particularly attention to concentration effects on mass isotopomer ratios and maximizing enrichments in the isotopomers of interest, reduces error. Improving instrument accuracy for quantification of isotopomer ratios is perhaps the highest priority for this field. In conclusion, MIDA remains the "equation for biosynthesis," but attention to potentially confounding factors and analytic performance is required for optimal application.
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