Measure Synthesis Rates Research Articles

ER Unfolded Protein Response in Liver In Vivo Is Characterized by Reduced, Not Increased, De Novo Lipogenesis and Cholesterol Synthesis Rates with Uptake of Fatty Acids from Adipose Tissue: Integrated Gene Expression, Translation Rates and Metabolic Fluxes.

The unfolded protein response in the endoplasmic reticulum (UPRER) is involved in a number of metabolic diseases. Here, we characterize UPRER-induced metabolic changes in mouse livers in vivo through metabolic labeling and mass spectrometric analysis of lipid and proteome-wide fluxes. We induced UPRER by tunicamycin administration and measured synthesis rates of proteins, fatty acids and cholesterol, as well as RNA-seq. Contrary to reports in isolated cells, hepatic de novo lipogenesis and cholesterogenesis were markedly reduced, as were mRNA levels and synthesis rates of lipogenic proteins. H&E staining showed enrichment with lipid droplets while electron microscopy revealed ER morphological changes. Interestingly, the pre-labeling of adipose tissue prior to UPRER induction resulted in the redistribution of labeled fatty acids from adipose tissue to the liver, with replacement by unlabeled glycerol in the liver acylglycerides, indicating that the liver uptake was of free fatty acids, not whole glycerolipids. The redistribution of adipose fatty acids to the liver was not explicable by altered plasma insulin, increased fatty acid levels (lipolysis) or by reduced food intake. Synthesis of most liver proteins was suppressed under UPRER conditions, with the exception of BiP, other chaperones, protein disulfide isomerases, and proteins of ribosomal biogenesis. Protein synthesis rates generally, but not always, paralleled changes in mRNA. In summary, this combined approach, linking static changes with fluxes, revealed an integrated reduction of lipid and cholesterol synthesis pathways, from gene expression to translation and metabolic flux rates, under UPRER conditions. The reduced lipogenesis does not parallel human fatty liver disease. This approach provides powerful tools to characterize metabolic processes underlying hepatic UPRER in vivo.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Accounting for non‐steady state conditions during long‐term deuterium oxide‐based measurement of protein turnover

IntroductionMost isotope calculations to measure synthesis rates require steady state conditions. In skeletal muscle, steady state refers to a period of time in which there is neither loss or gain of protein. Deuterium oxide (D2O) is a stable isotopic tracer that can measure protein synthesis over long periods of time. This is advantageous when measuring changes in protein synthesis in response to long‐term interventions such as exercise training or pharmacologic interventions. However, prolonging the period of measurement increases the likelihood of non‐steady state conditions. The purpose of this study was to demonstrate that non‐steady state conditions such as growth can change interpretation of tracer data and validate a model that accounts for non‐steady state conditions when assessing muscle protein synthesis. We hypothesized that use of the appropriate model can account for non‐steady state conditions when measuring muscle protein synthesis.MethodsTo demonstrate the potential implications of non‐steady state conditions, we used D2O to measure skeletal muscle protein synthesis over two weeks in three month old male Sprague Dawley rats with access to running wheels during a period of growth. Then we established a model to account for non‐steady state conditions and validated the model in cultured C2C12 myoblasts and myotubes during periods of cell growth and atrophy. Finally, we applied this model in vivo by measuring protein synthesis in four month old male CB6F1 mice with steady weight or weight loss over a month.ResultsDuring a period of growth, active rats gained less body mass than their sedentary counterparts. There was a significant (p<0.05) positive relation between changes in body size and fractional synthesis rates (FSR) (R2 = 0.50, 0.475, 0.50 for myofibrillar, cytosolic, and mitochondrial FSR, respectively) that obscured any exercise‐related increases in protein synthesis. In vitro studies validated the model parameters that rely on a change in protein mass. We grouped mice based on weight maintenance versus weight loss during the period of measurement. We used our modeling parameters to estimate changes in protein synthesis during the steady and non‐steady conditions.ConclusionsNon‐steady state conditions occur with greater likelihood in long‐ versus short‐term protein synthesis studies and should be accounted for. We showed that a period of growth could account for ~50% of all variation in changes in protein synthesis in all subfractions of skeletal muscle, and this growth effect could mask more subtle effects that may be important for hypothesis testing. We have developed a model to account for non‐steady state conditions over long‐term interventions. We applied this model to a one month intervention to account for body mass changes in response to dietary restriction. By applying this model to future long‐term interventions, we can more accurately discern the effects of interventions on protein synthesis in non steady‐state conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling the contribution of individual proteins to mixed skeletal muscle protein synthetic rates over increasing periods of label incorporation.

Advances in stable isotope approaches, primarily the use of deuterium oxide ((2)H2O), allow for long-term measurements of protein synthesis, as well as the contribution of individual proteins to tissue measured protein synthesis rates. Here, we determined the influence of individual protein synthetic rates, individual protein content, and time of isotopic labeling on the measured synthesis rate of skeletal muscle proteins. To this end, we developed a mathematical model, applied the model to an established data set collected in vivo, and, to experimentally test the impact of different isotopic labeling periods, used (2)H2O to measure protein synthesis in cultured myotubes over periods of 2, 4, and 7 days. We first demonstrated the influence of both relative protein content and individual protein synthesis rates on measured synthesis rates over time. When expanded to include 286 individual proteins, the model closely approximated protein synthetic rates measured in vivo. The model revealed a 29% difference in measured synthesis rates from the slowest period of measurement (20 min) to the longest period of measurement (6 wk). In support of these findings, culturing of C2C12 myotubes with isotopic labeling periods of 2, 4, or 7 days revealed up to a doubling of the measured synthesis rate in the shorter labeling period compared with the longer period of labeling. From our model, we conclude that a 4-wk period of labeling is ideal for considering all proteins in a mixed-tissue fraction, while minimizing the slowing effect of fully turned-over proteins. In addition, we advocate that careful consideration must be paid to the period of isotopic labeling when comparing mixed protein synthetic rates between studies.

Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria

Traditional proteomics provides static assessment of protein content, but not synthetic rates. Recently, proteome dynamics with heavy water ((2)H2O) was introduced, where (2)H labels amino acids that are incorporated into proteins, and the synthesis rate of individual proteins is calculated using mass isotopomer distribution analysis. We refine this approach with a novel algorithm and rigorous selection criteria that improve the accuracy and precision of the calculation of synthesis rates and use it to measure protein kinetics in spatially distinct cardiac mitochondrial subpopulations. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated from adult rats, which were given (2)H2O in the drinking water for up to 60 days. Plasma (2)H2O and myocardial (2)H-enrichment of amino acids were stable throughout the experimental protocol. Multiple tryptic peptides were identified from 28 proteins in both SSM and IFM and showed a time-dependent increase in heavy mass isotopomers that was consistent within a given protein. Mitochondrial protein synthesis was relatively slow (average half-life of 30 days, 2.4% per day). Although the synthesis rates for individual proteins were correlated between IFM and SSM (R(2) = 0.84; P < 0.0001), values in IFM were 15% less than SSM (P < 0.001). In conclusion, administration of (2)H2O results in stable enrichment of the cardiac precursor amino acid pool, with the use of refined analytical and computational methods coupled with cell fractionation one can measure synthesis rates for cardiac proteins in subcellular compartments in vivo, and protein synthesis is slower in mitochondria located among the myofibrils than in the subsarcolemmal region.

Fractional synthesis rate of creatine from arginine in healthy adult men

Creatine is synthesized from the amino acids arginine, glycine and methionine. We performed a study using 15N2‐guanidino arginine, to elucidate synthesis of creatine from dietary arginine in adult men. A primed, intermittent infusion of labeled 15N2‐guanidino arginine was given enterally to 5 healthy men fed a standardized milkshake diet. Blood was sampled at baseline and at plateau, between 1.5 and 3 hours. Enrichment of M+2 isotopomers of arginine, guanidino acetate (GAA), creatine and creatinine was measured by LC‐tandem MS. Plasma enrichment of the arginine tracer was 6.1 APE (Stdev ±1.05%), giving a flux estimate of 93 ìmol/kg/hr. M+2 isotopomers of creatine and GAA from the arginine tracer were detected in all subjects. There was no measurable M+2 creatinine. Enrichment of creatine was 0.55 APE (±0.1%) and GAA 4.92 APE (±0.8%). The fractional synthesis rate of GAA from arginine was 81% and 9% for creatine from arginine, these results are in keeping with synthesis of GAA being the rate limiting step in the creatine synthesis pathway. We have demonstrated synthesis of creatine from labeled dietary arginine in healthy adult humans; this method will allow in vivo studies in humans to directly measure synthesis rates of creatine.Supported by a grant from the CIHR.

Measuring serotonin synthesis: from conventional methods to PET tracers and their (pre)clinical implications

The serotonergic system of the brain is complex, with an extensive innervation pattern covering all brain regions and endowed with at least 15 different receptors (each with their particular distribution patterns), specific reuptake mechanisms and synthetic processes. Many aspects of the functioning of the serotonergic system are still unclear, partially because of the difficulty of measuring physiological processes in the living brain. In this review we give an overview of the conventional methods of measuring serotonin synthesis and methods using positron emission tomography (PET) tracers, more specifically with respect to serotonergic function in affective disorders. Conventional methods are invasive and do not directly measure synthesis rates. Although they may give insight into turnover rates, a more direct measurement may be preferred. PET is a noninvasive technique which can trace metabolic processes, like serotonin synthesis. Tracers developed for this purpose are α-[11C]methyltryptophan ([11C]AMT) and 5-hydroxy-L-[β-11C]tryptophan ([11C]5-HTP). Both tracers have advantages and disadvantages. [11C]AMT can enter the kynurenine pathway under inflammatory conditions (and thus provide a false signal), but this tracer has been used in many studies leading to novel insights regarding antidepressant action. [11C]5-HTP is difficult to produce, but trapping of this compound may better represent serotonin synthesis. AMT and 5-HTP kinetics are differently affected by tryptophan depletion and changes of mood. This may indicate that both tracers are associated with different enzymatic processes. In conclusion, PET with radiolabelled substrates for the serotonergic pathway is the only direct way to detect changes of serotonin synthesis in the living brain.

Differential effects of insulin deprivation and systemic insulin treatment on plasma protein synthesis in type 1 diabetic people

It remains to be determined whether systemic insulin replacement normalizes synthesis rates of different plasma proteins and whether there are differential effects on various plasma proteins. We tested a hypothesis that insulin deprivation differentially affects individual plasma protein synthesis and that systemic insulin treatment may not normalize synthesis of all plasma proteins. We measured synthesis rates of 41 plasma proteins in seven each of type 1 diabetic (T1DM) and nondiabetic participants (ND) using [ring-(13)C(6)]phenylalanine as a tracer. T1DM were studied while on chronic insulin treatment and during 8 h insulin deprivation. Insulin treatment normalized glucose levels, but plasma insulin levels were higher during insulin treatment than during insulin deprivation in T1DM and ND. Individual plasma proteins were purified by affinity chromatography and two-dimensional gel electrophoresis. Only 41 protein gel spots from over 300 were chosen based on their protein homogeneity. Insulin deprivation and hyperglycemia either significantly increased (n = 12) or decreased (n = 12) synthesis rates of 24 of 41 plasma proteins in T1DM compared with ND. Insulin treatment normalized synthesis rates of 13 of these 24 proteins, which were altered during insulin deprivation. However, insulin treatment significantly altered the synthesis of 14 additional proteins. In conclusion, acute insulin deprivation caused both a decrease and increase in synthesis rates of many plasma proteins with various functions. Moreover, chronic systemic insulin treatment not only did not normalize synthesis of all plasma proteins but also altered synthesis of several additional proteins that were unaltered during insulin deprivation.

Measuring synthesis rates of different proteins – clues to training adaptations

Over the past 15–20 years several laboratories have been investigating the regulation of muscle protein synthesis by diet and exercise. It is clear that resistance exercise stimulates muscle protein synthesis. The greatest stimulation comes from resistance exercise plus a source of amino acids (for review see Tipton & Ferrando, 2008). Interestingly, similar increases in muscle protein synthesis are observed following endurance exercise (Tipton et al. 1996); protein ingestion also enhances this response (Howarth et al. 2009). Given the differences in adaptations to these types of exercise, this similarity of response of muscle protein synthesis may seem somewhat paradoxical. The explanation may lie in the methodology. The response of mixed muscle protein synthesis was determined in these studies (Tipton & Ferrando, 2008). Measurement of mixed muscle protein synthesis is essentially a weighted average of the rates of synthesis of all proteins in the muscle, i.e. it does not distinguish the responses of different proteins. Intuitively, given the differences in adaptations to different types of training, it is not difficult to accept that resistance and endurance exercise, for example, should differentially influence different proteins. Recent advances in methodology have allowed us to begin to address these questions. The acute response to exercise and nutritional interventions may be measured and extrapolated to represent the potential for long-term adaptive changes. Adaptations to any exercise training regime result from changes in the type, quantity and activity of proteins in various tissues. For example, muscle growth in response to resistance exercise training is determined by increases in the myofibrillar proteins. Muscle protein synthesis rate has been shown to be predictive of long-term changes in muscle (Tipton, 2008). For example, a recent metabolic study demonstrated that milk ingestion resulted in greater muscle protein synthesis than soy following resistance exercise (Wilkinson et al. 2007). A follow-up longitudinal study by the same research group demonstrated that milk supplementation resulted in greater increases in muscle mass and strength than soy supplements (Hartman et al. 2007). Thus, it is clear that the acute metabolic response to various exercise and nutritional combinations is a useful way to determine optimal training and diet regimes. The results of a study published in this issue of The Journal of Physiology by Moore et al. (2009) offer interesting and important advances toward our understanding of the influence of nutrition and exercise on different types of proteins. This study helps pave the way for future studies that will further our understanding of training adaptations. Moore et al. (2009) used a unilateral exercise model to simultaneously measure protein synthesis rates in response to whey protein ingestion at rest and following resistance exercise. Moreover, the time course of the response to each was determined. The results demonstrate a differential response of the two protein types to feeding. The rate of sarcoplasmic protein synthesis was increased following protein ingestion to the same extent in both the resting and the exercised leg. On the other hand, myofibrillar protein synthesis was increased to a greater extent and the response lasted longer in the exercised leg. These novel findings illustrate that the response of proteins to exercise and nutrition is not uniform. Moreover, and perhaps more importantly, these results underline the value of measuring the response of various protein fractions to nutritional and exercise interventions. The present study examined the response only to resistance exercise (Moore et al. 2009). However, another recent study by this research group provides additional information on differential responsiveness of various protein fractions. Wilkinson et al. (2008) report that myofibrillar protein synthesis and mitochondrial protein synthesis rates both increased in response to resistance exercise. Only mitochondrial protein synthesis increased in response to endurance exercise. Furthermore, exercise training changed these responses (Wilkinson et al. 2008). These data were gathered with a background of hyperaminoacidaemia for all conditions, so the interactive effect of exercise type and nutrition on the different types of protein could not be determined. Clearly, these types of studies provide us with the ability to investigate the potential for exercise and nutrition to influence training results. Further investigation of the impact of different types of exercise on the synthesis of different protein fractions and the interaction with nutritional interventions must follow. Furthermore, studies should delineate responses in various populations. These studies will provide important information for athletes practicing various types of sports. More importantly, these types of studies will offer information critical to designing optimal nutrition and exercise interventions to diminish muscle wasting and metabolic disease.

In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial proteins

Skeletal muscle mitochondrial dysfunction occurs in many conditions including aging and insulin resistance, but the molecular pathways of the mitochondrial dysfunction remain unclear. Presently, no methodologies are available to measure synthesis rates of individual mitochondrial proteins, which limits our ability to fully understand the translational regulation of gene transcripts. Here, we report a methodology to measure synthesis rates of multiple muscle mitochondrial proteins, which, along with large-scale measurements of mitochondrial gene transcripts and protein concentrations, will enable us to determine whether mitochondrial alteration is due to transcriptional or translational changes. The methodology involves in vivo labeling of muscle proteins with l-[ring-(13)C(6)]phenylalanine, protein purification by two-dimensional gel electrophoresis of muscle mitochondrial fraction, and protein identification and stable isotope abundance measurements by tandem mass spectrometry. Synthesis rates of 68 mitochondrial and 23 nonmitochondrial proteins from skeletal muscle mitochondrial fraction showed a 10-fold range, with the lowest rate for a structural protein such as myosin heavy chain (0.16 +/- 0.04%/h) and the highest for a mitochondrial protein such as dihydrolipoamide branched chain transacylase E2 (1.5 +/- 0.42%/h). This method offers an opportunity to better define the translational regulation of proteins in skeletal muscle or other tissues.

Measuring synthesis rates of nitrogen-containing polymers by using stable isotope tracers1,2

The major N-containing polymer compounds in the body include protein, RNA, and DNA. The endogenous gastrointestinal secretions as well as the portal-drained visceral and peripheral immune responses are basic physiological functions. Elevated endogenous secretions and immune activities, as affected by developmental stages, diets, and management factors, decrease the availability of dietary nutrients for peripheral muscle synthesis and deposition. Measurements of in vivo protein, RNA, and DNA synthesis rates associated with the viscera, peripheral immune cells, and skeletal muscles should, in principle, be the sensitive biochemical and cellular endpoints for studying factors affecting nonruminant nutrition, metabolism, and growth. The selection of stable isotope tracers for precursors, routes of tracer delivery, and mass spectrometric analyses of tracer enrichments are the major methodological considerations. To measure in vivo protein, RNA, and DNA synthesis rates, oral feeding with heavy water (2H2O), and continuous infusion of [U-13C]glucose and [15N]Gly intravenously for labeling the sugar moieties ribose and deoxyribose and de novo purine base synthesis have been established. Flooding doses of tracer Phe, for example, L-[ring-2H5]Phe, via the i.p. route are reliable and cost-effective for measuring in vivo protein synthesis rates, especially for the viscera in small nonruminants. Therefore, measurements of the major N-containing polymer synthesis rates in the viscera, the peripheral immune cells, and muscles through oral feeding with 2H2O and/or i.p. flooding doses of Phe tracers are the emerging tools for studying nonruminant nutrition, metabolism, and growth under research and field test conditions.

Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647 u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [ 2 H 3 ]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M +1 and M +2 mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3–10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.

Acute effects of moderate dehydration on the hepatic conversion of amino nitrogen into urea nitrogen in healthy men

Intracellular hydration may play a role in the regulation of protein and nitrogen metabolism. The hepatic removal of nitrogen by urea synthesis has a key regulatory role in nitrogen balance. The purpose of the present study was to establish the acute effects of dehydration on the hepatic kinetics of urea synthesis, quantified by functional hepatic nitrogen clearance (FHNC), in healthy volunteers. Seven healthy men were studied twice in random order. On both study days, a primed continuous infusion of alanine was given. On the day of dehydration an intravenous bolus injection of a loop diuretic (furosemide, 1 mg/kg) was superimposed. FHNC was calculated as the ratio between measured synthesis rates of urea nitrogen and blood alanine concentrations. Furosemide induced a weight loss of 1 kg. During dehydration, FHNC decreased by approx. 25% (41+/-11 to 54+/-10 litres/h; P<0.02). On both occasions individual FHNC and glucagon values were positively correlated (r(2)>0.6). In addition, dehydration more than halved the linear slope of the relationship (P<0.05). The FHNC values were correlated with the urinary excretion of both potassium and sodium (r(2)=0.68, P<0.01 and r(2)=0.62, P<0.02 respectively). Changes in the reactivity of urea synthesis to glucagon (i.e. the ratio between FHNC and glucagon concentration) was negatively correlated with an indirectly estimated change in intracellular water (r(2)=0.79, P<0.05). We conclude that acute moderate dehydration down-regulates both total urea synthesis and its sensitivity to glucagon. The latter was related to estimated intracellular water loss. Dehydration may thus have nitrogen-saving consequences with regard to the hepatic contribution to whole-body nitrogen homoeostasis. The mechanism of this effect and the relationship with sodium and potassium fluxes is not known.

In vitro method for measuring synthesis rates in the intervertebral disc.

A method for culturing intervertebral disc tissue in vitro under conditions that control changes in hydration and minimize loss of proteoglycans has been developed. Disc slices were enclosed in small-pore dialysis tubing and incubated in medium containing polyethylene glycol (PEG). By varying the PEG concentration, different external swelling pressures could be applied to the tissue. The rate of glycosaminoglycan synthesis in rabbit and dog disc was then measured using [35S]sulphate as a precursor. Synthesis rates varied with final hydration of the tissue, and the highest rates were found at hydrations close to those found in vivo. Under conditions of controlled hydration, rabbit nucleus has a higher rate of glycosaminoglycan synthesis than annulus. However, the rates measured in sagittal slices of dog disc decreased from the outer to the inner regions of the anterior annulus, reaching a minimum value in the nucleus and then increasing again in the posterior part of the annulus. The results show that in the intervertebral disc, measured rates depend on culture conditions.