Absolute Synthesis Rates Research Articles

Palmitoyl-carnitine production by blood cells associates with the concentration of circulating acyl-carnitines in healthy overweight women

Circulating acyl-carnitines (acyl-CNTs) are associated with insulin resistance (IR) and type 2 diabetes (T2D) in both rodents and humans. However, the mechanisms whereby circulating acyl-CNTs are increased in these conditions and their role in whole-body metabolism remains unknown. The purpose of this study was to determine if, in humans, blood cells contribute in production of circulating acyl-CNTs and associate with whole-body fat metabolism. Eight non-diabetic healthy women (age: 47±19y; BMI: 26±1kg·m-2) underwent stable isotope tracer infusion and hyperinsulinemic-euglycemic clamp study to determine invivo whole-body fatty acid flux and insulin sensitivity. Blood samples collected at baseline (0min) and after 3h of clamp were used to determine the synthesis rate of palmitoyl-carnitine (palmitoyl-CNT) invitro. The fractional synthesis rate of palmitoyl-CNT was significantly higher during hyperinsulinemia (0.788±0.084 vs. 0.318±0.012%·hr-1, p=0.001); however, the absolute synthesis rate (ASR) did not differ between the periods (p=0.809) due to ∼30% decrease in blood palmitoyl-CNT concentration (p=0.189) during hyperinsulinemia. The ASR of palmitoyl-CNT significantly correlated with the concentration of acyl-CNTs in basal (r=0.992, p<0.001) and insulin (r=0.919, p=0.001) periods; and the basal ASR significantly correlated with plasma palmitate oxidation (r=0.764, p=0.027). In women, blood cells contribute to plasma acyl-CNT levels and the acyl-CNT production is linked to plasma palmitate oxidation, a marker of whole-body fat metabolism. Future studies are needed to confirm the role of blood cells in acyl-CNT and lipid metabolism under different physiological (i.e., in response to meal) and pathological (i.e., hyperlipidemia, IR and T2D) conditions.

Palmitoyl‐carnitine production by blood cells is associated with plasma palmitate oxidation in women

Circulating acyl‐carnitines (acyl‐CNTs) are associated with insulin resistance (IR) and type 2 diabetes (T2D) in both rodents and humans. However, the mechanisms whereby circulating acyl‐CNTs are increased in these conditions and its implications for whole‐body metabolism have not been determined. Here we aimed to determine if blood cells contribute in production of circulating acyl‐CNTs and whether such production is correlated with whole‐body fatty acid fluxes.Methods and ResultsEight non‐diabetic healthy women (Age: 47±19 y; BMI: 26±1 kg•m−2) underwent stable isotope tracer infusion and hyperinsulinemic‐euglycemic clamp study to determine in vivo whole‐body fatty acid kinetics and insulin sensitivity. At baseline (0 min) and after 3 h of hyperinsulinemic‐euglycemic clamp, blood samples were collected for measurement of in vitro synthesis rates of palmitoyl‐carnitine (palmitoyl‐CNT). The fractional synthesis rate of palmitoyl‐CNT was significantly higher during hyperinsulinemia (0.788±0.084 vs. 0.318±0.012 %•hr−1, p=0.001); however, the absolute synthesis rate did not differ between the periods (p=0.809) due to decreased (~30%) palmitoyl‐CNT concentration (p=0.189) during hyperinsulinemia. Regression analyses demonstrated that the absolute synthesis of palmitoyl‐CNT significantly correlated with the concentration of acyl‐CNTs in basal (r=0.992, p&lt;0.001) and insulin (r=0.919, p=0.001) periods; and the basal absolute synthesis significantly correlated with plasma palmitate oxidation rate (r=0.764, p=0.027).ConclusionIn women, blood cells contribute to plasma acyl‐CNT levels and basal whole‐body plasma palmitate oxidation is associated with acyl‐CNT production. Future studies investigating the role of blood cells in acyl‐CNT and lipid metabolism under different physiological (i.e., hyperlipidemia) and pathological (i.e., IR and T2D) conditions are warranted.Support or Funding InformationClaude D. Pepper OAIC grant (# P30‐AG024832), the Institute for Translational Sciences at the UTMB, supported in part by a Clinical and Translational Science Award (#UL1 TR001439) from the NCATS/NIH, and the Shriners Grant #84090, Metabolism Unit, Shriners Hospitals for Children.

Novel Method for Measuring Hepatic Collagen Turnover Rate in Nonalcoholic Fatty Liver Disease (NAFLD): Comparison of Blood and Liver Biopsy Biomarkers of Fibrogenesis

Background and ObjectivesExcess collagen synthesis in the liver plays a causal role in progression from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). Markers that identify a patient's risk for progression, characterize disease activity and provide early assessment of treatment efficacy are needed. In particular, the availability of a non‐invasive metric of the rates of liver collagen synthesis (fibrogenesis) and turnover (fibrolysis) would be extremely valuable. We evaluated novel clinical biomarkers that directly quantify hepatic fibrogenesis rate in well‐characterized patients with biopsy‐proven NAFLD.MethodsPatients with suspected NAFLD and a clinical indication for liver biopsy received the stable, non‐radioactive tracer heavy water (2H2O, 50 ml, 2–3 times daily) by mouth for 20–35 days prior to biopsy. A plasma sample was collected weekly. Collagen fractional synthesis rate (FSR) from liver biopsy tissue and FSR of lumican (a proteoglycan involved in collagen fibril assembly that exhibits increased synthesis when collagen synthesis is increased) from plasma were measured by 2H incorporation, using tandem mass spectrometry (LC‐MS/MS). Magnetic resonance elastography (MRE) measurements of liver stiffness were obtained within 2 weeks of biopsy.ResultsFSR of hepatic type I collagen (Col1a1) was significantly elevated in NASH relative to NAFL (0.97% vs 0.43 %/day, p=0.03). Liver Col1a1 FSR also significantly correlated with fibrosis severity by histological score (r= 0.63), with liver stiffness by MRE (r=0.65), and with HbA1c levels (r=0.47). FSRs of fibrillar hepatic collagen subtypes (I and III) were highly correlated (r= 0.94). FSR of circulating plasma lumican correlated strongly with liver collagen FSR (r=0.69) and with liver stiffness (r=0.71).ConclusionWe conclude that hepatic collagen synthesis rates increase with fibrosis severity in NAFLD patients, indicating not only markedly increased absolute rates of collagen synthesis with disease progression but also suggesting that hepatic scar is dynamic even in advanced disease and may therefore be potentially reversible. Importantly, plasma lumican FSR correlates closely with liver collagen FSR and with liver stiffness. Moreover, established clinical risk factors for disease progression (NASH diagnosis, histologic fibrosis score, diabetes) were associated with higher collagen FSR. Non‐invasive kinetic measurements of fibrogenesis, such as plasma lumican FSR, may be helpful as markers of risk for disease progression and as early read outs of treatment response in anti‐fibrotic trials in NASH. Prospective studies are required to establish the predictive power of fibrogenesis rates for disease progression or reversal.Support or Funding InformationKineMed, Inc.

Individuals Maintain Similar Rates of Protein Synthesis over Time on the Same Plane of Nutrition under Controlled Environmental Conditions.

Consistent individual differences in animal performance drive individual fitness under variable environmental conditions and provide the framework through which natural selection can operate. Underlying this concept is the assumption that individuals will display consistent levels of performance in fitness-related traits and interest has focused on individual variation and broad sense repeatability in a range of behavioural and physiological traits. Despite playing a central role in maintenance and growth, and with considerable inter-individual variation documented, broad sense repeatability in rates of protein synthesis has not been assessed. In this study we show for the first time that juvenile flounder Platichthys flesus reared under controlled environmental conditions on the same plane of nutrition for 46 days maintain consistent whole-animal absolute rates of protein synthesis (As). By feeding meals containing 15N-labelled protein and using a stochastic end-point model, two non-terminal measures of protein synthesis were made 32 days apart (d14 and d46). As values (mass-corrected to a standard mass of 12 g) showed 2- to 3-fold variation between individuals on d14 and d46 but individuals showed similar As values on both days with a broad sense repeatability estimate of 0.684 indicating significant consistency in physiological performance under controlled experimental conditions. The use of non-terminal methodologies in studies of animal ecophysiology to make repeat measures of physiological performance enables known individuals to be tracked across changing conditions. Adopting this approach, repeat measures of protein synthesis under controlled conditions will allow individual ontogenetic changes in protein metabolism to be assessed to better understand the ageing process and to determine individual physiological adaptive capacity, and associated energetic costs of adaptation, to global environmental change.

Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

SummaryThe assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

Reduced invivo hepatic proteome replacement rates but not cell proliferation rates predict maximum lifespan extension in mice.

Combating the social and economic consequences of a growing elderly population will require the identification of interventions that slow the development of age-related diseases. Preserved cellular homeostasis and delayed aging have been previously linked to reduced cell proliferation and protein synthesis rates. To determine whether changes in these processes may contribute to or predict delayed aging in mammals, we measured cell proliferation rates and the synthesis and replacement rates (RRs) of over a hundred hepatic proteins invivo in three different mouse models of extended maximum lifespan (maxLS): Snell Dwarf, calorie-restricted (CR), and rapamycin (Rapa)-treated mice. Cell proliferation rates were not consistently reduced across the models. In contrast, reduced hepatic protein RRs (longer half-lives) were observed in all three models compared to controls. Intriguingly, the degree of mean hepatic protein RR reduction was significantly correlated with the degree of maxLS extension across the models and across different Rapa doses. Absolute rates of hepatic protein synthesis were reduced in Snell Dwarf and CR, but not Rapa-treated mice. Hepatic chaperone levels were unchanged or reduced and glutathione S-transferase synthesis was preserved or increased in all three models, suggesting a reduced demand for protein renewal, possibly due to reduced levels of unfolded or damaged proteins. These data demonstrate that maxLS extension in mammals is associated with improved hepatic proteome homeostasis, as reflected by a reduced demand for protein renewal, and that reduced hepatic protein RRs hold promise as an early biomarker and potential target for interventions that delay aging in mammals.

Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.)

For the first time, use of the flooding dose technique using 3H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of 3H-phenylalanine into the posterior adductor muscle. Incorporation of 3H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1–6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d−1 for the gill, 2.5±0.3%d−1 for the mantle and 2.6±0.3%d−1 for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day−1. The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

Albumin Synthesis in States of Inflammation

per day of the intravascular albumin pool, respectively (p < 0.001). Conclusions A more than 50% elevation of de novo albumin absolute synthesis rate was seen in patients with acute abdominal inflammation compared to volunteers and patients scheduled for pancreatic surgery. The synthesis rate for albumin did not differ between the volunteers and patients scheduled for surgery, despite the lower plasma albumin concentrations in the latter group. This might relate to their underlying pathology associated with a chronic inflammation.

The stoichiometric divisome: a hypothesis.

Dividing Escherichia coli cells simultaneously constrict the inner membrane, peptidoglycan layer, and outer membrane to synthesize the new poles of the daughter cells. For this, more than 30 proteins localize to mid-cell where they form a large, ring-like assembly, the divisome, facilitating division. Although the precise function of most divisome proteins is unknown, it became apparent in recent years that dynamic protein–protein interactions are essential for divisome assembly and function. However, little is known about the nature of the interactions involved and the stoichiometry of the proteins within the divisome. A recent study (Li et al., 2014) used ribosome profiling to measure the absolute protein synthesis rates in E. coli. Interestingly, they observed that most proteins which participate in known multiprotein complexes are synthesized proportional to their stoichiometry. Based on this principle we present a hypothesis for the stoichiometry of the core of the divisome, taking into account known protein–protein interactions. From this hypothesis we infer a possible mechanism for peptidoglycan synthesis during division.

Albumin synthesis in very low birth weight infants is enhanced by early parenteral lipid and high-dose amino acid administration

Albumin is one of the most important plasma proteins and plays a key role in many physiologic processes, such as preserving colloid osmotic pressure, scavenging radicals, and binding and transporting bilirubin, hormones, and drugs. However, albumin concentrations are often low in preterm infants during the first days of life. We hypothesized that early parenteral lipid and high-dose amino acid (AA) administration to very low birth weight (VLBW) infants from birth onwards increases hepatic albumin synthesis rates. Inborn VLBW infants were randomized to receive from birth onwards either 2.4g amino acids/(kg(·)d) (control group), 2.4g amino acids/(kg(·)d) plus 2g lipids/(kg(·)d) (AA+lipid group), or 3.6g amino acids/(kg(·)d) plus 2g lipids/(kg(·)d) (high AA+lipid group). On postnatal day 2, infants received a primed continuous infusion of [U-(13)C6,(15)N]leucine. Mass spectrometry was used to determine the fractional and absolute albumin synthesis rates (FSR and ASR, respectively). In total, 28 infants (median gestational age 27 weeks (IQR 25-28), median birth weight 810g (IQR 679-998) were studied. The median FSR was 6.5%/d in the control group, 10.6%/d in the AA group, and 12.3%/d in the high AA+lipid group, while the median was 84 mg/(kg(·)d) in the control group, 138 mg/(kg(·)d) in the AA group, and 160 mg/(kg(·)d) in the high AA+lipid group. A group of VLBW infants given parenteral nutrition containing lipids and high-dose amino acids showed a higher rate of albumin synthesis compared to infants receiving no lipids and standard amounts of amino acids during the first two days of life.

Double blind exploratory study on de novo lipogenesis in preterm infants on parenteral nutrition with a lipid emulsion containing 10% fish oil

Provision of long chain polyunsaturated fatty acids (LCP) both of the omega-3 and omega-6 families is recommended for preterm infants (PI). Fish oil (FO) contains omega-3 and omega-6 LCP and it is incorporated in the fat blend of the new generation lipid emulsions (LE). Omega-3 LCP have been shown to reduce the expression of genes involved in lipogenesis, which could be important for several organs development. The aim of this study was to ascertain if the use of intravenous FO has an effect on lipogenesis in PI. Forty PI were randomized to receive two LE: MSF (50:40:10 Medium Chain Triglycerides (MCT): Soybean oil (SO): FO) or MS (50:50 MCT:SO). We measured plasma lipids on day 7 and the fractional and absolute synthesis rates (FSR and ASR) of cholesterol and of selected fatty acids (FA) after (2)H2O body water labeling. Plasma phospholipids (PL), free cholesterol (FC), and cholesterol esters (CE) concentrations were all lower in MSF than in MS. In spite of lower plasma FC and CE concentrations, cholesterol biosynthesis was similar between the two study groups (FC: FSR 16.0±1.4 vs 14.1±1.1%/d, p=0.74; ASR 6.8±0.6 vs 7.1±0.6mgkg(-1)d(-1), p=0.93; CE: FSR 3.6±0.5 vs 4.2±0.4%/d, p=0.38; ASR: 3.3±0.4 vs 4.4±0.5mgkg(-1)d(-1), p=0.13, in MSF and MS respectively). FSR and ASR of selected FA were, or tended to be, lower in MSF than in MS. ASR of PL palmitate (4.0±0.3 vs 4.8±0.4mgkg(-1)d(-1), p=0.045), PL oleate (0.2±0.04 vs 0.4±0.05mgkg(-1)d(-1), p=0.02) and CE oleate (0.5±0.1 vs 0.9±0.1mgkg(-1)d(-1), p=0.03) were significantly lower in MSF than in MS. There were no differences in plasma TG FA biosynthesis. Cholesterol biosynthesis was not affected by 10% FO during neonatal parenteral nutrition. Ten percent FO caused a statistically significant reduction in the lipogenesis of selected FA and an overall tendency towards a reduced lipogenesis. The magnitude seems to be limited and the biological significance is unknown. Our data warrant follow-up studies in PI who receive intravenous FO, especially in those infants who receive larger doses than in the present study. Since this trial started in 2007, trial registration was not required.

Triglycerides produced in the livers of fasting rabbits are predominantly stored as opposed to secreted into the plasma

ObjectiveThe liver plays a central role in regulating fat metabolism; however, it is not clear how the liver distributes the synthesized triglycerides (TGs) to storage and to the plasma. Materials and methodsWe have measured the relative distribution of TGs produced in the liver to storage and the plasma by means of U-13C16-palmitate infusion in anesthetized rabbits after an overnight fast. ResultsThe fractional synthesis rates of TGs stored in the liver and secreted into the plasma were not significantly different (stored vs. secreted: 31.9±0.8 vs. 27.7±2.6%∙h−1, p>0.05). However, the absolute synthesis rates of hepatic stored and secreted TGs were 543±158 and 27±7nmol∙kg−1∙min−1 respectively, indicating that in fasting rabbits the TGs produced in the liver were predominately stored (92±3%) rather than secreted (8±3%) into the plasma. This large difference was mainly due to the larger pool size of the hepatic TGs which was 21±9-fold that of plasma TGs. Plasma free fatty acids (FFAs) contributed 47±1% of the FA precursor for hepatic TG synthesis, and the remaining 53±1% was derived from hepatic lipid breakdown and possibly plasma TGs depending on the activity of hepatic lipase. Plasma palmitate concentration significantly correlated with hepatic palmitoyl-CoA and TG synthesis. ConclusionIn rabbits, after an overnight fast, the absolute synthesis rate of hepatic stored TGs was significantly higher than that of secreted due to the larger pool size of hepatic TGs. The net synthesis rate of TG was approximately half the absolute rate. Plasma FFA is a major determinant of hepatic TG synthesis, and therefore hepatic TG storage.

Determination of in vivo RNA kinetics using RATE-seq.

The abundance of a transcript is determined by its rate of synthesis and its rate of degradation; however, global methods for quantifying RNA abundance cannot distinguish variation in these two processes. Here, we introduce RNA approach to equilibrium sequencing (RATE-seq), which uses in vivo metabolic labeling of RNA and approach to equilibrium kinetics, to determine absolute RNA degradation and synthesis rates. RATE-seq does not disturb cellular physiology, uses straightforward normalization with exogenous spike-ins, and can be readily adapted for studies in most organisms. We demonstrate the use of RATE-seq to estimate genome-wide kinetic parameters for coding and noncoding transcripts in Saccharomyces cerevisiae.

Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources

Quantitative views of cellular functions require precise measures of rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here, we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli data set transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. Members of multiprotein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles for optimizing design. These include how the level of different types of transcription factors is optimized for rapid response and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. Our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from quantitative analyses of protein synthesis.

Protein Accounting in the Cellular Economy

Knowing the copy number of cellular proteins is critical for understanding cell physiology. By being able to measure the absolute synthesis rates of the majority of cellular proteins, Li etal. gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the functional needs.

A Dietary Supplementation with Leucine and Antioxidants Is Capable to Accelerate Muscle Mass Recovery after Immobilization in Adult Rats

Prolonged inactivity induces muscle loss due to an activation of proteolysis and decreased protein synthesis; the latter is also involved in the recovery of muscle mass. The aim of the present work was to explore the evolution of muscle mass and protein metabolism during immobilization and recovery and assess the effect of a nutritional strategy for counteracting muscle loss and facilitating recovery. Adult rats (6–8 months) were subjected to unilateral hindlimb casting for 8 days (I0–I8) and then permitted to recover for 10 to 40 days (R10–R40). They were fed a Control or Experimental diet supplemented with antioxidants/polyphenols (AOX) (I0 to I8), AOX and leucine (AOX + LEU) (I8 to R15) and LEU alone (R15 to R40). Muscle mass, absolute protein synthesis rate and proteasome activities were measured in gastrocnemius muscle in casted and non-casted legs in post prandial (PP) and post absorptive (PA) states at each time point. Immobilized gastrocnemius protein content was similarly reduced (-37%) in both diets compared to the non-casted leg. Muscle mass recovery was accelerated by the AOX and LEU supplementation (+6% AOX+LEU vs. Control, P<0.05 at R40) due to a higher protein synthesis both in PA and PP states (+23% and 31% respectively, Experimental vs. Control diets, P<0.05, R40) without difference in trypsin- and chymotrypsin-like activities between diets. Thus, this nutritional supplementation accelerated the recovery of muscle mass via a stimulation of protein synthesis throughout the entire day (in the PP and PA states) and could be a promising strategy to be tested during recovery from bed rest in humans.

Roles of Insulin, Age, and Asymmetric Dimethylarginine on Nitric Oxide Synthesis In Vivo

We tested the effects of insulin on production of nitrous oxide (NO)-related substances (nitrites and nitrates [NOx]) after 15N-arginine intravenous infusion and on asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) concentrations in conditions reportedly associated with altered NO availability, i.e., aging, hypertension, hypercholesterolemia, and type 2 diabetes mellitus (T2DM). A total of 26 male subjects (age 23–71 years, BMI 23–33 kg/m2), some of whom were affected by mixed pathologic features, were enrolled. NOx fractional synthesis rate (FSR) was lower in elderly (P < 0.015) and T2DM subjects (P < 0.03) than in matched control subjects. Hyperinsulinemia generally increased both NOx FSR and absolute synthesis rate (ASR) and reduced NOx, ADMA, and SDMA concentrations. Insulin sensitivity was impaired only in T2DM. With use of simple linear regression analysis across all subjects, age was inversely correlated with both NOx FSR (R2 = 0.23, P < 0.015) and ASR (R2 = 0.21, P < 0.02). NOx FSR inversely correlated with both ADMA and SDMA. With use of multiple regression analysis and various models, NOx FSR remained inversely associated with age and ADMA, whereas ASR was inversely associated with age and diabetes. No association with insulin sensitivity was found. We conclude that whole-body NOx production is decreased in aging and T2DM. Age, ADMA concentration, and T2DM, but not insulin resistance, appear as negative regulators of whole-body NOx production.

Large increases in adipose triacylglycerol flux in Cushingoid CRH-Tg mice are explained by futile cycling

Glucocorticoids are extremely effective anti-inflammatory therapies, but their clinical use is limited due to severe side effects, including osteoporosis, muscle wasting, fat redistribution, and skin thinning. Here we use heavy water labeling and mass spectrometry to measure fluxes through metabolic pathways impacted by glucocorticoids. We combine these methods with measurements of body composition in corticotropin-releasing hormone (CRH)-transgenic (Tg)(+) mice that have chronically elevated, endogenously produced corticosterone and a phenotype that closely mimics Cushing's disease in humans. CRH-Tg(+) mice had increased adipose mass, adipose triglyceride synthesis, and greatly increased triglyceride/fatty acid cycling in subcutaneous and abdominal fat depots and increased de novo lipogenesis in the abdominal depot. In bone, CRH-Tg(+) mice had decreased bone mass, absolute collagen synthesis rates, and collagen breakdown rate. In skin, CRH-Tg(+) mice had decreased skin thickness and absolute collagen synthesis rates but no decrease in the collagen breakdown rate. In muscle, CRH-Tg(+) mice had decreased muscle mass and absolute protein synthesis but no decrease in the protein breakdown rate. We conclude that chronic exposure to endogenous glucocorticoid excess in mice is associated with ongoing decreases in bone collagen, skin collagen, and muscle protein synthesis without compensatory reduction (coupling) of breakdown rates in skin and muscle. Both of these actions contribute to reduced protein pool sizes. We also conclude that increased cycling between triglycerides and free fatty acids occurs in both abdominal and subcutaneous fat depots in CRH-Tg(+) mice. CRH-Tg mice have both increased lipolysis and increased triglyceride synthesis in adipose tissue.

The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

341 Cholesterol Biosynthesis in Preterm Infants on Parenteral Nutrition with a Lipid Emulsion Containing 10% Fish Oil (FO): a Pilot Study

Background and AimsLimited data are available on the metabolic effects of intravenous fish oil (FO) in premature infant. We reported on the plasma cholesterol lowering effect of an intravenous lipid...