Reliability of Protein Abundance and Synthesis Measurements in Human Skeletal Muscle

The repeatability of dynamic proteome profiling (DPP), which is a novel technique for measuring the relative abundance (ABD) and fractional synthesis rate (FSR) of proteins in humans, is investigated. LC–MS analysis is performed on muscle samples taken from male participants (n = 4) that consumed 4 × 50 mL doses of deuterium oxide (²H₂O) per day for 14 days. ABD is measured by label‐free quantitation and FSR is calculated from time‐dependent changes in peptide mass isotopomer abundances. One‐hundred one proteins have at least one unique peptide and are used in the assessment of protein ABD. Fifty‐four of these proteins meet more stringent criteria and are used in the assessment of FSR data. The median (M), lower‐, (Q₁) and upper‐quartile (Q₃) values for protein FSR (%/d) are M = 1.63, Q₁ = 1.07, and Q₃ = 3.24, respectively. The technical CV of ABD data has a median value of 3.6% (Q₁ 1.7% to Q₃ 6.7%), whereas the median CV of FSR data is 10.1% (Q₁ 3.5% to Q₃ 16.5%). These values compare favorably against other assessments of technical repeatability of proteomics data, which often set a CV of 20% as the upper bound of acceptability.     

Altered Intramuscular Lipid Metabolism Relates to Diminished Insulin Action in Men,but Not Women,in Progression to Diabetes

Whether sex differences in intramuscular triglyceride (IMTG) metabolism underlie sex differences in the progression to diabetes are unknown. Therefore, the current study examined IMTG concentration and fractional synthesis rate (FSR) in obese men and women with normal glucose tolerance (NGT) vs. those with prediabetes (PD). PD (n = 13 men and 7 women) and NGT (n = 7 men and 12 women) groups were matched for age and anthropometry. Insulin action was quantified using a hyperinsulinemic‐euglycemic clamp with infusion of [6,6^?2H₂]‐glucose. IMTG concentration was measured by gas chromatography/mass spectrometry (GC/MS) and FSR by GC/combustion isotope ratio MS (C‐IRMS), from muscle biopsies taken after infusion of [U^?13C]palmitate during 4 h of rest. In PD men, the metabolic clearance rate (MCR) of glucose was lower during the clamp (4.71 ± 0.77 vs. 8.62 ± 1.26 ml/kg fat‐free mass (FFM)/min, P = 0.04; with a trend for lower glucose rate of disappearance (Rd), P = 0.07), in addition to higher IMTG concentration (41.2 ± 5.0 vs. 21.2 ± 3.4 µg/mg dry weight, P ≤ 0.01), lower FSR (0.21 ± 0.03 vs. 0.42 ± 0.06 %/h, P ≤ 0.01), and lower oxidative capacity (P = 0.03) compared to NGT men. In contrast, no difference in Rd, IMTG concentration, or FSR was seen in PD vs. NGT women. Surprisingly, glucose Rd during the clamp was not different between NGT men and women (P = 0.25) despite IMTG concentration being higher (42.6 ± 6.1 vs. 21.2 ± 3.4 µg/mg dry weight, P = 0.03) and FSR being lower (0.23 ± 0.04 vs. 0.42 ± 0.06 %/h, P = 0.02) in women. Alterations in IMTG metabolism relate to diminished insulin action in men, but not women, in the progression toward diabetes.     

Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein x kg fat-free mass(-1) x h(-1) x 2.5 h) by simultaneous intravenous infusions of [5,5,5-(2)H(3)]leucine and either [ring-(13)C(6)]phenylalanine or [ring-(2)H(5)]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were approximately 20% greater (P < 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 +/- 0.005%/h; fed: 0.080 +/- 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 +/- 0.004 and 0.066 +/- 0.005%/h, respectively). The feeding-induced increase in the FSR ( approximately 20%; P = 0.011) was not different with leucine and phenylalanine tracers (P = 0.69). Furthermore, the difference between the leucine- and phenylalanine-derived FSRs was independent of the phenylalanine isotopomer used (P = 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively.     

Turnover Rates of Hepatic Collagen and Circulating Collagen-Associated Proteins in Humans with Chronic Liver Disease

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of ²H₂O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.     

Determination of muscle protein synthesis rates in fish using H2O and H NMR analysis of alanine

Following administration of deuterated water (²H₂O), the fractional synthetic rate (FSR) of a given endogenous protein can be estimated by ²H-enrichment quantification of its alanine residues. Currently, this is measured by mass spectrometry following a derivatization procedure. Muscle FSR was measured by ¹H/²H NMR analysis of alanine from seabass kept for 6 days in 5% ²H-enriched saltwater, following acid hydrolysis and amino acid isolation by cation-exchange chromatography of muscle tissue. The analysis is simple and robust, and provides precise measurements of excess alanine ²H-enrichment in the 0.1–0.4% range from 50 mmol of alanine recovered from muscle protein.     

Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.     

Leucine-enriched essential amino acid and carbohydrate ingestion following resistance exercise enhances mTOR signaling and protein synthesis in human muscle

We recently showed that resistance exercise and ingestion of essential amino acids with carbohydrate (EAA+CHO) can independently stimulate mammalian target of rapamycin (mTOR) signaling and muscle protein synthesis in humans. Providing an EAA+CHO solution postexercise can further increase muscle protein synthesis. Therefore, we hypothesized that enhanced mTOR signaling might be responsible for the greater muscle protein synthesis when leucine-enriched EAA+CHOs are ingested during postexercise recovery. Sixteen male subjects were randomized to one of two groups (control or EAA+CHO). The EAA+CHO group ingested the nutrient solution 1 h after resistance exercise. mTOR signaling was assessed by immunoblotting from repeated muscle biopsy samples. Mixed muscle fractional synthetic rate (FSR) was measured using stable isotope techniques. Muscle protein synthesis and 4E-BP1 phosphorylation during exercise were significantly reduced (P < 0.05). Postexercise FSR was elevated above baseline in both groups at 1 h but was even further elevated in the EAA+CHO group at 2 h postexercise (P < 0.05). Increased FSR was associated with enhanced phosphorylation of mTOR and S6K1 (P < 0.05). Akt phosphorylation was elevated at 1 h and returned to baseline by 2 h in the control group, but it remained elevated in the EAA+CHO group (P < 0.05). 4E-BP1 phosphorylation returned to baseline during recovery in control but became elevated when EAA+CHO was ingested (P < 0.05). eEF2 phosphorylation decreased at 1 and 2 h postexercise to a similar extent in both groups (P < 0.05). Our data suggest that enhanced activation of the mTOR signaling pathway is playing a role in the greater synthesis of muscle proteins when resistance exercise is followed by EAA+CHO ingestion.     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Measurement of skin protein breakdown in a rat model

Whereas skin protein synthesis can be measured with different approaches, no method potentially applicable in humans is available for measurement of skin protein breakdown. To that end, we measured mixed skin fractional protein breakdown (FBR) in a rat model by use of a stable isotope method (tracee release method) originally developed to measure muscle protein breakdown. Skin mixed protein and collagen fractional synthesis rates (FSR) were also measured. A primed continuous infusion of L-[ring-(2)H(5)]phenylalanine and alpha-[5,5,5-(2)H(3)]ketoisocaproate (KIC) was given for 6 h. Arterial and skin phenylalanine and leucine free enrichments were measured at plateau (5-6 h) and during the decay that followed after the infusion was stopped. Skin FBR (%/h) was 0.260 +/- 0.011 with phenylalanine and 0.201 +/- 0.032 with KIC/leucine [P = not significant (NS)]. Mixed skin FSR (%/h) was 0.169 +/- 0.055 with phenylalanine and 0.146 +/- 0.020 with KIC/leucine (P = NS). Collagen FSR was 0.124 +/- 0.023%/h (P = NS vs. mixed protein FSR). The tracee release method is a sensitive method for measurement of skin protein breakdown; however, given the high intersubject variability of FSR, the calculation of skin net balance is not advisable.     

Amino Acid Availability Regulates the Effect of Hyperinsulinemia on Skin Protein Metabolism in Pigs

The effects of amino acid supply and insulin infusion on skin protein kinetics (fractional synthesis rate (FSR), fractional breakdown rate (FBR), and net balance (NB)) in pigs were investigated. Four-month-old pigs were divided into four groups as follows: control, insulin (INS), amino acid (AA), and INS + AA groups based on the nutritional and hormonal conditions. l-[ring-¹³C₆]Phenylalanine was infused. FBR was estimated from the enrichment ratio of arterial phenylalanine to intracellular free phenylalanine. Plasma INS was increased (p < 0.05) in the INS and INS + AA groups. Plasma glucose was maintained by infusion of glucose in the groups receiving INS. The interventions did not change the NB of skin protein. However, the interventions affected the FSR and FBR differently. An infusion of INS significantly increased both FSR and FBR, although AA infusion did not. When an AA infusion was added to the infusion of insulin (INS + AA group), FSR and FBR were both lower when compared with the INS group. Our data demonstrate that in anesthetized pigs INS infusion did not exert an anabolic effect, but rather it increased AA cycling into and out of skin protein. Because co-infusion of AAs with INS ameliorated this effect, it is likely that the increased AA cycling during INS infusion was related to AA supply. Although protein kinetics were affected by both INS and AAs, none of the interventions affected the skin protein deposition. Thus, skin protein content is closely regulated under normal circumstances and is not subject to transient changes in AAs or hormonal concentrations.     

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-²H₃]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.     

Resistance-training-induced adaptations in skeletal muscle protein turnover in the fed state

Resistance training changes the balance of muscle protein turnover, leading to gains in muscle mass. A longitudinal design was employed to assess the effect that resistance training had on muscle protein turnover in the fed state. A secondary goal was investigation of the potential interactive effects of creatine (Cr) monohydrate supplementation on resistance-training-induced adaptations. Young (N = 19, 23.7 +/- 3.2 year), untrained (UT), healthy male subjects completed an 8-week resistance-training program (6 d/week). Supplementation with Cr had no impact on any of the variables studied; hence, all subsequent data were pooled. In the UT and trained (T) state, subjects performed an acute bout of resistance exercise with a single leg (exercised, EX), while their contralateral leg acted as a nonexercised (NE) control. Following exercise, subjects were fed while receiving a primed constant infusion of [d5]- and [15N]-phenylalanine to determine the fractional synthetic and breakdown rates (FSR and FBR), respectively, of skeletal muscle proteins. Acute exercise increased FSR (UT-NE, 0.065 +/- 0.025 %/h; UT-EX, 0.088 +/- 0.032 %/h; P < 0.01) and FBR (UT-NE, 0.047 +/- 0.023 %/h; UT-EX, 0.058 +/- 0.026 %/h; P < 0.05). Net balance (BAL = FSR - FBR) was positive in both legs (P < 0.05) but was significantly greater (+65%) in the EX versus the NE leg (P < 0.05). Muscle protein FSR and FBR were greater at rest following T (FSR for T-NE vs. UT-NE, +46%, P < 0.01; FBR for T-NE vs. UT-NE, +81%, P < 0.05). Resistance training attenuated the acute exercise-induced rise in FSR (T-NE vs. T-EX, +20%, P = 0.65). The present results demonstrate that resistance training resulted in an elevated resting muscle protein turnover but an attenuation of the acute response of muscle protein turnover to a single bout of resistance exercise.     

Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise

This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise.     

Protein synthesis in the whole body,liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis

Jones W. O. and Symons L. E. A. 1982. Protein synthesis in the whole body, liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis. International Journal for Parasitology12: 295–301. Tyrosine flux and the synthesis of protein in the whole body, liver, skeletal muscle and kidney cortex and of albumin in lambs infected with Trichostrongylus colubriformis and uninfected lambs fed ad libitum or pair-fed with the infected group, were measured by constant infusion of ¹⁴C-l-tyrosine. Live weight gain was lower in the infected than in pairfed lambs, but rates of whole body protein synthesis were similar in both groups. On the other hand, compared with control lambs, there was a faster rate of protein synthesis per unit of protein consumed in infected but not in pair-fed lambs. Rates of protein synthesis per unit of body weight in infected were higher than in pair-fed lambs, but similar to the rate in control lambs. The fractional synthetic rates (FSR) of albumin and liver proteins and the amount of liver protein synthesized per day were increased by infection. The FSR and amount of protein synthesized per day were depressed in skeletal muscle and kidney cortex. Anorexia did not explain any of these changes. Infection caused a loss of protein from each of these tissues, but this loss was due to anorexia in only the liver. There was generally good correlation between concentration of RNA per g fresh weight or per mg nitrogen and the FSR of protein. However, although the $\text{RNA}\text{DNA}$ ratio correlated well with synthesis in skeletal muscle, it was poorly correlated for liver proteins. The relationship between the rate of growth and protein synthesis in infected lambs is discussed.     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.     

Protein Turnover in Retina

Abstract: Rabbit retinas were exposed in vitro to 0.5-h pulses of [³H]leucine or [¹⁴C]Ieucine. Some retinas were harvested promptly after labeling to measure synthesis. These were combined, in double-labeling experiments, with retinas that had been returned to unlabeled medium for a subsequent 1 h or 3.75 h to measure degradation. All of the proteins were solubilized, and separated according to size by gel electrophoresis. The gels were cut into 95 slices, and each slice was differentially counted. The amount of protein in the slice was estimated from the Coomassie blue staining, and its molecular weight from the distribution of molecular weight (MW) standards. Turnover rates of the various sizes of proteins were calculated from these data using certain well-defined assumptions. Retinal protein contained about 32 ± 10³ nmol of polypeptide per g, with a median MW of 27,000. Total synthesis was at the rate of 103 nmol/g of protein/h, with the most rapid synthesis in the 33,000–43,000 MW range, at 2 nmol/g/h for every 1000 increment in MW. Protein renewal averaged 0.52%/h, but varied directly (p < 0.0001) with MW, so that proteins of 10,000 MW were being renewed at about 0.1%/h and proteins of 140,000 MW at about 1.4%/h. Taken together, the measurements of fractional renewal and the measurements of degradation of the newly synthesized proteins demonstrated that each slice contained proteins with markedly different breakdown coefficients, and provided enough information to characterize the proteins in the slice in terms of a fast and a slow subgroup. This analysis indicated that: breakdown coefficients varied much more than rates of synthesis and were therefore the prime determinant of the amount of each protein that was present; as MW increased, breakdown coefficients of the long-lived proteins increased (p < 0.0001), accounting in major part for the correlation between size and turnover; most staining bands were due to proteins with peculiarly long lifespans; the proteins with the slowest turnover of all appeared to be histones: there was an unusually rapid synthesis of a 138,000 MW polypeptide with a moderately short half-life (about 3 h).     

Intestinal protein synthesis in guinea pigs infected with Trichostrongylus colubriformis

The fractional synthesis rate (FSR) and daily synthesis of protein were measured in the small and large intestines of infected guinea pigs and uninfected animals fed ad libitum or quantitatively reduced rations. The FSR of the infected and parasite-free parts of the small intestine was unchanged but was increased by about 40% in the large intestine. Daily protein synthesis (mg/g body wt.) by infected guinea pig was greater by about 24% in the entire small intestine and by over 70% in the large intestine. These increases were not due to anorexia since the FSR and daily protein synthesis by the small and large intestines of the reduced ration animals were less than those of the infected group. Greater weight of the small intestine may explain increases in daily protein synthesis in the small, but not in the large intestine where weight was unchanged. Responses which may affect protein synthesis in the infected and parasite-free intestines are discussed.     

Inflexibility in Intramuscular Triglyceride Fractional Synthesis Distinguishes Prediabetes From Obesity in Humans

Whether intramuscular triglyceride (IMTG) concentration or flux is more important in the progression to type 2 diabetes is controversial. Therefore, this study examined IMTG concentration, as well as its fractional synthesis rate (FSR), in obese people with normal glucose tolerance (NGT; n = 20) vs. obese people with prediabetes (PD; n = 19), at rest and during exercise. Insulin action and secretion were assessed using an intravenous glucose tolerance test. [U‐¹³C]palmitate was infused for 4 h before and throughout 1.5 h of treadmill walking at 50% VO_2max. IMTG concentration was measured by gas chromatograph/mass spectrometer, and FSR by gas chromatography–combustion isotope ratio mass spectrometer, from muscle biopsies taken immediately before and after exercise. Basal IMTG concentration was higher (43 ± 5.7 vs. 27 ± 3.9 mg/mg dry weight, P = 0.03) and FSR trended lower (0.23 ± 0.04 vs. 0.32 ± 0.05/h, P = 0.075), as did insulin action (S_i; 2.9 ± 0.43 vs. 3.3 ± 0.35 × 10^?4/mU/ml, P = 0.07), in PD vs. NGT. IMTG concentration did not change significantly during exercise, but was no longer different in PD vs. NGT (45 ± 7.7 vs. 37 ± 5.8 mg/mg dry weight, P = 0.41). IMTG FSR suppressed during exercise in NGT (?81% to 0.06 ± 0.13/h, P = 0.02), but not PD (+4% to 0.24 ± 0.13%/h, P = 0.95). Palmitate oxidation was similar during rest (P = 0.92) and exercise (P = 0.94) between groups, but its source appeared different with more coming from muscle at rest and plasma during exercise in NGT, whereas the converse was true in PD. Altogether, higher basal IMTG concentration that is metabolically inflexible distinguishes obese people with PD from those with NGT.     

Concurrent resistance and aerobic exercise stimulates both myofibrillar and mitochondrial protein synthesis in sedentary middle-aged men

We determined myofibrillar and mitochondrial protein fractional synthesis rates (FSR), intramuscular signaling protein phosphorylation, and mRNA expression responses after isolated bouts of resistance exercise (RE), aerobic exercise (AE), or in combination [termed concurrent exercise (CE)] in sedentary middle-aged men. Eight subjects (age = 53.3 ± 1.8 yr; body mass index = 29.4 ± 1.4 kg·m(2)) randomly completed 8 × 8 leg extension repetitions at 70% of one repetition-maximum, 40 min of cycling at 55% peak aerobic power output (AE), or (consecutively) 50% of the RE and AE trials (CE). Biopsies were obtained (during a primed, constant infusion of l-[ring-(13)C(6)]phenylalanine) while fasted, and at 1 and 4 h following postexercise ingestion of 20 g of protein. All trials increased mitochondrial FSR above fasted rates (RE = 1.3-fold; AE = 1.5; CE = 1.4; P < 0.05), although only CE (2.2) and RE (1.8) increased myofibrillar FSR (P < 0.05). At 1 h postexercise, phosphorylation of Akt on Ser(473) (CE = 7.7; RE = 4.6) and Thr(308) (CE = 4.4; RE = 2.9), and PRAS40 on Thr(246) (CE = 3.8; AE = 2.5) increased (P < 0.05), with CE greater than AE for Akt Ser(473)-Thr(308) and greater than RE for PRAS40 (P < 0.05). Despite increased phosphorylation of Akt-PRAS40, phosphorylation of mammalian target of rapamycin (Ser(2448)) remained unchanged (P > 0.05), while rpS6 (Ser(235/236)) increased only in RE (10.4) (P < 0.05). CE and AE both resulted in increased peroxisome proliferator receptor-γ coactivator 1-α (PGC1α) expression at 1 h (CE = 2.9; AE = 2.8; P < 0.05) and 4 h (CE = 2.6; AE = 2.4) and PGC1β expression at 4 h (CE = 2.1; AE = 2.6; P < 0.05). These data suggest that CE-induced acute stimulation of myofibrillar and mitochondrial FSR, protein signaling, and mRNA expression are equivalent to either isolate mode (RE or AE). These results occurred without an interference effect on muscle protein subfractional synthesis rates, protein signaling, or mRNA expression.