Fractional Synthesis Rate Research Articles

Effects of Whey Protein Combined with Amylopectin/Chromium on the Muscle Protein Synthesis and mTOR Phosphorylation in Exercised Rats.

This study aimed to examine the impact of varying doses of whey protein (WP) and amylopectin/chromium complex (ACr) supplementation on muscle protein synthesis (MPS), amino acid and insulin levels, and the rapamycin (mTOR) signaling pathways in exercised rats. A total of 72 rats were randomly divided into nine groups: (1) Exercise (Ex), (2) Ex + WPI to (5) Ex + WPIV with various oral doses of whey protein (0.465, 1.55, 2.33, and 3.1g/kg) and (6) Ex + WPI + ACr to (9) Ex + WPIV + ACr with various doses of whey protein combined with 0.155g/kg ACr. On the day of single-dose administration, the products were given by oral gavage after exercise. To measure the protein fractional synthesis rate (FSR), a bolus dose of deuterium-labeled phenylalanine was given, and its effects were evaluated 1h after supplementation. Rats that received 3.1g/kg of whey protein (WP) combined with ACr exhibited the most significant increase in muscle protein synthesis (MPS) compared to the Ex group (115.7%, p < 0.0001). In comparison to rats that received the same dose of WP alone, those given the combination of WP and ACr at the same dosage showed a 14.3% increase in MPS (p < 0.0001). Furthermore, the WP (3.1g/kg) + ACr group exhibited the highest elevation in serum insulin levels when compared to the Ex group (111.9%, p < 0.0001). Among the different groups, the WP (2.33g/kg) + ACr group demonstrated the greatest increase in mTOR levels (224.2%, p < 0.0001). Additionally, the combination of WP (2.33g/kg) and ACr resulted in a 169.8% increase in 4E-BP1 levels (p < 0.0001), while S6K1 levels rose by 141.2% in the WP (2.33g/kg) + ACr group (p < 0.0001). Overall, supplementation with various doses of WP combined with ACr increased MPS and enhanced the mTOR signaling pathway compared to WP alone and the Ex group.

Achilles Tendon Tissue Turnover Before and Immediately After an Acute Rupture

Background: An Achilles tendon rupture (ATR) is a frequent injury and results in the activation of tendon cells and collagen expression, but it is unknown to what extent turnover of the tendon matrix is altered before or after a rupture. Purpose/Hypothesis: The purpose of this study was to characterize tendon tissue turnover before and immediately after an acute rupture in patients. It was hypothesized that a rupture would result in pronounced collagen synthesis in the early phase (first 2 weeks) after the injury. Study Design: Cross-sectional study; Level of evidence, 3. Methods: The study included patients (N = 18) eligible for surgery after an ATR. At the time of inclusion, the patients ingested deuterium oxide (2H2O) orally, and on the day of surgery (within 14 days of the injury), they received a 3-hour flood-primed infusion of an 15N-proline tracer. During surgery, the patients had 1 biopsy specimen taken from the ruptured part of the Achilles tendon and 1 that was 3 to 5 cm proximal to the rupture as a control. The biopsy specimens were analyzed for carbon-14 (14C) levels in the tissue to calculate long-term turnover (years), incorporation of 2H-alanine (from 2H2O) into the tissue to calculate the fractional synthesis rate (FSR) of proteins in the short term (days), and incorporation of 15N-proline into the tissue to calculate the acute FSR (hours). Results: Both the rupture and the control samples showed consistently lower levels of 14C compared with the predicted level of 14C in a healthy tendon, which indicated increased tendon turnover in a fraction (48% newly synthesized) of the Achilles tendon already for a prolonged period before the rupture. Over the first days after the rupture, the synthesis rate for collagen was relatively constant, and the average synthesis rate on the day of surgery (2-14 days after the rupture) was 0.025% per hour, irrespective of the length of time after a rupture and the site of sampling (rupture vs control). No differences were found in the FSR between the rupture and control samples in the days after the rupture. Conclusion: Higher than normal tissue turnover in the Achilles tendon before a rupture indicated that changes in the tendon tissue preceded the injury. In addition, we observed no increase in tendon collagen tissue turnover in the first 2 weeks after an ATR. This favors the view that an increase in the formation of new tendon collagen is not an immediate phenomenon during the regeneration of ruptured tendons in patients. Registration: NCT03931486 (ClinicalTrials.gov identifier).

The acute removal of metformin induces mitochondrial remodeling and increases mitochondrial respiration

Metformin is a candidate to slow biological aging and extend healthspan, as it reduces the risk of morbidity and mortality in people with type 2 diabetes. However, it is not clear how metformin impacts individuals absent of chronic disease. This unknown is important to answer before prescribing metformin to healthy individuals to prolong the healthspan. It has been shown that metformin creates an inefficiency in energy transfer by accumulating within the mitochondria. Therefore, we hypothesized that the effects of metformin on healthspan are context specific; beneficial in the context of low energy demand/mitochondrial capacity but detrimental in the context of high energy demand/mitochondrial capacity. To test this hypothesis, we used 18-month, male rats selectively bred for low and high treadmill running capacity (LCR/HCR) as models of low and high intrinsic mitochondrial function. For both HCR and LCR, we used high-resolution respirometry (Oroboros O2K) on skeletal muscles from controls (CON), 4 weeks of metformin-treatment (MET), 4 weeks of metformin-treatment with a 48-hour metformin washout (MET-WO), and 48-hour washout with metformin added back to the respirometry chamber (MET-WO-SPIKE). A week prior to sacrifice, we labeled the rats with deuterium oxide to assess protein turnover as an indicator of mitochondrial remodeling. In gastrocnemius muscle, the HCR MET-WO had greater Complex I (CI), CI+Complex II (CII), and maximal respiration (MaxR) (115 ± 23, 164 ± 38, 157 ± 35 pmol/(s✕mg), respectively) compared to the CON (70 ± 24, p = 0.02, 114 ± 32, p = 0.01, 115 ± 40 pmol/(s✕mg), p = 0.04, respectively), and MET (63 ± 54, p = 0.007, 90 ± 50, p &lt; 0.001, 91 ± 48 pmol/(s✕mg), p = 0.002, respectively). The HCR MET-SPIKE group had greater CII (83 ± 33 pmol/(s✕mg)) and MaxR (86 ± 35 pmol/(s✕mg)) compared to the MET (p = 0.007 and p = 0.002, respectively). In the soleus, MET-WO (4.15 ± 0.33%/day) had greater mitochondrial fractional synthesis rates (FSRs) compared to MET (3.12 ± 0.62%/day, p = 0.001) in the HCRs. In the LCRs, MET-WO (4.61 ± 0.42%/day) had greater FSRs compared to CON (p &lt; 0.001, 3.07 ± 0.67%/day) and MET (3.12 ± 0.61%/day, p = 0.001). In the tibialis anterior, MET-WO (3.47 ± 0.80%/day) had greater FSRs compared to the MET (2.41 ± 0.47%/day, p = 0.04) within the HCRs with no differences in the LCR. These data show that metformin reduces mitochondrial respiration in HCR rats but not in LCR rats. The presence of metformin produced a greater subsequent response in HCR compared to LCR as shown by its acute removal revealing metformin-induced mitochondrial remodeling. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of pea/rice and amylopectin/chromium as an alternative protein source to improve muscle protein synthesis in rats.

A preclinical study reported that the combination of an amylopectin/chromium complex (ACr) of branched-chain amino acids (BCAA) significantly enhanced muscle protein synthesis (MPS). This study was conducted to determine the effects of the addition of ACr complex to a pea/rice (PR) protein on MPS, insulin, muslin levels, and the mTOR pathway in exercised rats. Twenty-four rats were divided into three groups: (i) exercise (Ex); (ii) Ex + PR 1:1 blend (0.465g/kg BW); (iii) Ex + PR + ACr (0.155g/kg BW). On the day of single-dose administration, after the animals were exercised at 26/m/min for 2h, the supplement was given by oral gavage. The rats were injected with a bolus dose (250mg/kg BW, 25g/L) of deuterium-labeled phenylalanine to determine the protein fractional synthesis rate (FSR) one h after consuming the study product. The combination of PR and ACr enhanced MPS by 42.55% compared to the Ex group, while Ex + PR alone increased MPS by 30.2% over the Ex group (p < 0.0001) in exercised rats. Ex + PR plus ACr significantly enhanced phosphorylation of mTOR and S6K1 (p < 0.0001), and 4E-BP1 (p < 0.001) compared to the Ex (p < 0.0001). PR to ACr also significantly increased insulin and musclin levels (p < 0.0001) in exercised rats. Additionally, compared to Ex + PR alone, Ex + PR + ACr enhanced mTOR (p < 0.0001) and S6K1 (p < 0.0001) levels. These data suggested that PR + ACr may provide an alternative to animal proteins for remodeling and repairing muscle by stimulating MPS and mTOR signaling pathways in post-exercised rats. More preclinical and clinical human studies on combining pea/rice and amylopectin/chromium complex are required.

Mainly Muscle, Jejunum, And Lung Fractional Protein Synthesis Rates Are Related To Whole-Body Protein Synthesis In The Pig

Mainly Muscle, Jejunum, And Lung Fractional Protein Synthesis Rates Are Related To Whole-Body Protein Synthesis In The Pig

A novel dynamic proteomics approach for the measurement of broiler chicken protein fractional synthesis rate.

The study of protein synthesis in farm animals is uncommon despite its potential to increase knowledge about metabolism and discover new biomarkers of health and growth status. The present study describes a novel dynamic proteomics approach for the measurement of protein fractional synthesis rate (FSR) in broiler chickens. Chickens received a 10g/kg oral dose of 2 H2 O at day 21 of their life. Body water 2 H abundance was measured in plasma samples using a portable Fourier transform infrared spectrometer. Free and protein-bound amino acids (AAs) were isolated and had their 2 H enrichment measured by gas chromatography with mass spectrometry (GC/MS). Peptide 2 H enrichment was measured by proteomics analysis of plasma and muscle samples. Albumin, fibrinogen and muscle protein FSR were calculated from GC/MS and proteomics data. Ala appeared to be more enriched at the site of protein synthesis than in the AA free pools. Glu was found to be the AA closest to isotopic equilibrium between the different AA pools. Glu was used as an anchor to calculate n(AA) values necessary for chicken protein FSR calculation in dynamic proteomics studies. FSR values calculated using proteomics data and GC/MS data showed good agreement as evidenced by a Bland-Altman residual plot. A new dynamic proteomics approach for the measurement of broiler chicken individual protein FSR based on the administration of a single 2 H2 O oral bolus has been developed and validated. The proposed approach could facilitate new immunological and nutritional studies on free-living animals.

Albumin Synthesis Rates Measured with [2H5ring]Phenylalanine are not Responsive to Short-Term Intravenous Nutrients in Healthy Humans

The response of albumin synthesis rates to infusion of either an intravenous nutrient mixture containing adequate nitrogen and energy or an isotonic saline infusion was compared in adult men. In a crossover study, nine healthy male subjects received a short-term (13.5-h) infusion of either the nutrient mixture or the saline solution, and albumin synthesis rates were measured by a stable isotope technique employing [2H5ring]phenylalanine. Fractional rates of albumin synthesis did not differ significantly between treatments (saline, 7.3 ± 1.0%/d; intravenous nutrients, 7.8 ± 1.0%/d). The secretion time of albumin was significantly lower with intravenous nutrients infusion than with saline infusion. Plasma albumin concentration as well as prealbumin and transferrin concentrations decreased over time when subjects were infused with either saline or the nutrient solution as a result of dilution rather than a change in synthesis. These results suggest that albumin synthesis rate does not respond to short-term intravenous nutrients.

Combined enteral infusion of glutamine, carbohydrates, and antioxidants modulates gut protein metabolism in humans

Background:Available data suggest that nutrients can affect intestinal protein metabolism, which contributes to the regulation of gut barrier function. Objective:We aimed to assess whether an oral nutritional supplement (ONS) containing glutamine (as the dipeptide Ala-Gln), carbohydrates, and antioxidants would modulate duodenal protein metabolism in healthy humans. Design:Thirty healthy control subjects were included and, over a period of 5 h, received by nasogastric tube either saline or ONS providing 11.7 kcal/kg as 0.877 g Ala-Gln/kg, 3.9 g carbohydrates/kg, and antioxidants (29.25 mg vitamin C/kg, 9.75 mg vitamin E/kg, 195 μg β-carotene/kg, 5.85 mg Se/kg, and 390 μg Zn/kg) or glutamine (0.585 g/kg, 2.34 kcal/kg). Simultaneously, a continuous intravenous infusion of l-[1-13C]-leucine was done until endoscopy. Leucine enrichment was assessed by using gas chromatography–mass spectrometric analysis, and mucosal fractional synthesis rate was calculated by using intracellular amino acid enrichment as precursor. Mucosal proteolytic pathways were also evaluated. Results:ONS infusion resulted in a doubling increase (P < 0.01) of duodenal fractional synthesis rate and a significant (P < 0.05) decrease in cathepsin D–mediated proteolysis compared with saline, whereas proteasome and Ca2+-dependent activities were unaffected. ONS infusion significantly (P < 0.01) decreased duodenal glutathione but not glutathione disulfide concentrations or the ratio of glutathione to glutathione disulfide. Insulinemia increased after ONS infusion, whereas plasma essential amino acids decreased. Infusion of glutamine alone did not reproduce ONS effects. Conclusions:ONS infusion improves duodenal protein balance in healthy humans. Further investigations are needed to study the origin of these effects and to evaluate ONS supply in stressed persons.

Insulin Treatment Affects Brain Protein Synthesis Rate in Streptozotocin-Induced Diabetic Rats

The purpose of this study was to determine whether insulin treatment affected the rate of brain protein synthesis in diabetic rats. Experiments were conducted on three groups of rats given streptozotocin without insulin treatment (diabetes), treated with streptozotocin + insulin (diabetes + insulin) or treated with neither streptozotocin nor insulin (control). The fractional rates of protein synthesis in brain, liver and kidney of diabetic rats treated with insulin were significantly greater than in diabetic rats without insulin treatment. In brain, liver and kidney, the RNA activity [g protein synthesized/(g RNA·d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that insulin treatment of diabetic rats is likely to increase the rate of protein synthesis in the brain, and that RNA activity is related to the fractional rate of brain protein synthesis.

Protein Synthesis Is Stimulated in Nutritionally Obese Rats

To investigate the tissue growth and the protein synthesis in vivo in nutritional obesity we used lipid-rich multichoice diet feeding. Young male rats of the Wistar strain were divided in two groups: control and obese. Control rats were fed pelleted nonpurified diet. Obese rats were fed a multichoice diet based on a variety of highly palatable energy-rich human foods for 30 d. Protein intake was kept equal in the groups to avoid its influence on protein turnover. The tissue growth pattern was evaluated by protein, DNA and RNA contents of liver, kidney, heart, skeletal muscles and small intestine. Protein synthesis in vivo was measured in these tissues by the phenylalanine flooding-dose technique. Rats fed the multichoice diet showed significantly greater body growth when compared with rats fed the nonpurified diet. Adipose and other tissue weights were significantly greater in the obese rats. The tissue growth pattern was characterized mainly by hyperplasia. In most tissues the net protein accretion found in obese rats resulted from an enhancement in the fractional rate of protein synthesis. The greater protein synthesis was due to an increase in the efficiency of ribosomes in kidney, heart and skeletal muscle and to an increase in the synthetic capacity in liver and small intestine. These data suggest that excess energy intake when protein intake is adequate stimulates tissue growth and protein synthesis in rats.

Relationship of Protein Synthesis to mRNA Levels in the Muscle of Chicks under Various Nutritional Conditions

The relationship between the rate of protein synthesis and the level of messenger RNA (mRNA) in the breast muscle (pectoralis major) was studied to examine the mechanisms regulating muscle protein synthesis in chicks under various nutritional conditions. The fractional synthesis rate (FSR) of protein was measured by a large-dose injection of L-[4-3H]phenylalanine. Poly(U)-Sepharose 4B affinity chromatography was used to extract Poly(A)+ RNA as mRNA. The first study examined the effect of dietary protein levels (0–60%) on protein synthesis rate and the contents of mRNA and total RNA. The fractional synthesis rate increased with increasing dietary protein levels from 0 to 20%. When chicks were fed high protein diets (40 and 60% of dietary protein), both the FSR and absolute synthesis rate (ASR) of protein were reduced compared with the 20% protein group. Multiple regression analysis indicated that for graded dietary protein levels below the dietary protein requirement, the response of muscle protein synthesis is largely regulated by variation in the ASR per unit RNA (the efficiency of RNA in synthesizing protein). At dietary protein levels above 20%, synthesis rates are also related to changes in the RNA:mRNA ratio (ribosome number relative to mRNA), the mRNA:DNA ratio (mRNA availability) and the DNA:protein ratio (DNA concentration). When chicks were food deprived for 2 d, FSR was reduced to about half that of well-fed controls. Upon refeeding of these chicks with the control diet, the FSR returned to the normal level within 0.5 h. When food-deprived chicks were refed with protein, carbohydrate or fat alone, the FSR returned to the control level within 0.5 h after consumption of each nutrient. The increase in muscle protein synthesis of unfed chicks following refeeding of various nutrients was explained by an increased ASR per unit RNA. Our results suggest that nutrient intake can change muscle protein synthesis rates in chicks largely by altering the ASR per unit RNA, in some cases associated also with changes in the levels of RNA, mRNA and DNA.

Effects of amino acids and albumin administration on albumin metabolism in surgically stressed rats: A basic nutritional study.

Nutrition therapy and administration of albumin preparations are common in postsurgical patients. However, the effects of these interventions on albumin metabolism are unclear. We elucidated the effect of postoperative albumin and/or parenteral nutrition administration on it. Sprague-Dawley rats underwent surgery involving intestinal rubbing followed by intestinal exposure. Subsequently, they were administered experimental solutions for 48 h, their blood samples were collected at 24 and 48 h, and livers were excised at 48 h. Based on experimental solutions, rats were divided into five groups: non-surgical (Non-surg); glucose and electrolyte solution (GE); amino acid, glucose, and electrolyte solution (AGE); GE + rat serum albumin (Alb) (GE + Alb); and AGE + Alb. Their plasma albumin concentrations; albumin fractional synthesis rate (ALB FSR); mercaptoalbumin/total albumin ratio (MA ratio); and messenger RNA (mRNA) expressions of albumin and hepatocyte nuclear factor-1 (HNF-1) in the liver were measured. The GE and AGE groups showed significant decline in albumin concentrations. ALB FSR was significantly enhanced in the AGE group compared with the GE group. The mRNA expression of albumin was similar to ALB FSR in all groups and that of HNF-1 was significantly decreased in the GE + Alb and AGE + Alb groups compared with the Non-surg group. The MA ratio in the AGE group was similar to the Non-surg group. The administration of amino acids comprising parenteral nutrition after surgery augmented ALB FSR and maintained the MA ratio only without simultaneous albumin administration.

Soy-Dairy Protein Blend or Whey Protein Isolate Ingestion Induces Similar Postexercise Muscle Mechanistic Target of Rapamycin Complex 1 Signaling and Protein Synthesis Responses in Older Men

Background: Previous work demonstrated that a soy-dairy protein blend (PB) prolongs hyperaminoacidemia and muscle protein synthesis in young adults after resistance exercise.Objective: We investigated the effect of PB in older adults. We hypothesized that PB would prolong hyperaminoacidemia, enhancing mechanistic target of rapamycin complex 1 (mTORC1) signaling and muscle protein anabolism compared with a whey protein isolate (WPI).Methods: This double-blind, randomized controlled trial studied men 55–75 y of age. Subjects consumed 30 g protein from WPI or PB (25% soy, 25% whey, and 50% casein) 1 h after leg extension exercise (8 sets of 10 repetitions at 70% one-repetition maximum). Blood and muscle amino acid concentrations and basal and postexercise muscle protein turnover were measured by using stable isotopic methods. Muscle mTORC1 signaling was assessed by immunoblotting.Results: Both groups increased amino acid concentrations (P < 0.05) and mTORC1 signaling after protein ingestion (P < 0.05). Postexercise fractional synthesis rate (FSR; P ≥ 0.05), fractional breakdown rate (FBR; P ≥ 0.05), and net balance (P = 0.08) did not differ between groups. WPI increased FSR by 67% (mean ± SEM: rest: 0.05% ± 0.01%; postexercise: 0.09% ± 0.01%; P < 0.05), decreased FBR by 46% (rest: 0.17% ± 0.01%; postexercise: 0.09% ± 0.03%; P < 0.05), and made net balance less negative (P < 0.05). PB ingestion did not increase FSR (rest: 0.07% ± 0.03%; postexercise: 0.09% ± 0.01%; P ≥ 0.05), tended to decrease FBR by 42% (rest: 0.25% ± 0.08%; postexercise: 0.15% ± 0.08%; P = 0.08), and made net balance less negative (P < 0.05). Within-group percentage of change differences were not different between groups for FSR, FBR, or net balance (P ≥ 0.05).Conclusions: WPI and PB ingestion after exercise in older men induced similar responses in hyperaminoacidemia, mTORC1 signaling, muscle protein synthesis, and breakdown. These data add new evidence for the use of whey or soy-dairy PBs as targeted nutritional interventions to counteract sarcopenia. This trial was registered at clinicaltrials.gov as NCT01847261.

Skeletal Muscle Nuclei in Mice are not Post-mitotic.

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (ie, myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST), and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202±0.0093 in PLA, 0.0239±0.0040 in TA, 0.0076±0. 0058 in GAST, and 0.0138±0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions, although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication, which results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth.

Abstract 10408: Insulin Resistance in Type 2 Diabetes is Moderated by Saturated Fatty Acids

Background: Increased intramyocellular lipid storage occurs in skeletal muscle of both athletes and type 2 diabetes patients (T2D) but their metabolic phenotypes are at opposite ends of insulin sensitivity and cardiometabolic risk. Hypothesis: Intramyocellular lipid saturation and flux are determinants of insulin resistance. Methods: Age-matched athletes (n=28) and T2D (n=27) underwent 1 H-magnetic resonance spectroscopy for analysis of saturated (fSL) and unsaturated lipid (fUL) fractions of vastus lateralis, blood lipids, glucose and insulin sensitivity, skeletal muscle biopsy for lipidomics/insulin signalling, cardiopulmonary exercise peak VO 2 and intravenous administration of stable isotopes of [U- 13 C]-palmitate and -linoleate for skeletal muscle saturated/unsaturated fatty acid fluxes (fractional synthesis rate, FSR). Tests were performed at baseline and after controlled insulin sensitivity changes (4-week athletes deconditioning and endurance exercise training at ≥65% of baseline peakVO 2, 5 hours/week x 8 weeks in T2D). Results: We demonstrate higher intramyocellular fSL and lower fUL in athletes compared to T2D (p=0.0004). Trained athletes have a 4-fold (palmitate) and 2.2-fold (linoleate) higher FSR compared to T2D (p=0.005 and 0.006, respectively). Athletes’ deconditioning results in reductions in skeletal muscle lipid saturation, palmitate and linoleate FSR. Conversely, 8-week endurance training of T2D results in intramyocellular lipid saturation increase (p&lt;0.0001) as well as a relatively larger (2.2-fold) palmitate FSR increase (p=0.04) than the 1.8-fold linoleate FSR increase (p=0.03), re-aligning the T2D skeletal muscle phenotype to that of deconditioned athletes. These adaptations were synchronous with significant insulin sensitivity improvement (p&lt;0.05), decrease in serum cholesterol (p=0.02) and triglycerides (p=0.0005), improved glycaemic control (HBA1c, p=0.0003), increased peak VO 2 (p&lt;0.0001) and upregulation of skeletal muscle phosphorylated AKT (p=0.01) and AMPK (p=0.005) in T2D. Conclusions: Maladapted intramyocellular lipid storage and fluxes in T2D patients show reversibility after endurance training through increased contributions of the saturated fatty acids pools.

Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Tryptophan oxidation in young children with environmental enteric dysfunction classified by the lactulose rhamnose ratio

ABSTRACTBackgroundIn young children, associations between linear growth faltering, environmental enteric dysfunction (EED), and the plasma kynurenine (Kyn)/tryptophan (Trp) ratio (KTR) have led to the proposal that higher Trp catabolism in response to intestinal/systemic inflammation limits Trp availability for protein synthesis, resulting in impaired growth.ObjectivesWe sought to estimate the Trp oxidation rate and the Trp conversion rate to Kyn in young children with and without EED.MethodsChildren aged 18–24 mo, from urban slums, were assigned to EED (n = 19) or no-EED (n = 26) groups on the basis of a urinary lactulose/rhamnose ratio (LRR) cutoff based on mean + 2 SDs of LRR (≥0.068) in normal age- and sex-matched, high–socioeconomic status children. Plasma KTR and fecal biomarkers of EED were measured. Trp oxidation in the fed state was measured using 13C1-Trp in an oral plateau feeding protocol.ResultsThe median (quartile 1, quartile 3) fasted KTR was 0.089 (0.066, 0.110) in children with EED compared with 0.070 (0.050, 0.093) in children with no EED (P = 0.077). However, there was no difference in fed-state Trp oxidation [median (quartile 1, quartile 3) 3.1 (1.3, 5.8) compared with 3.9 (1.8, 6.0) µmol/kg FFM/h, respectively, P = 0.617] or Trp availability for protein synthesis [42.6 (36.5, 45.7) compared with 42.5 (37.9, 46.9) µmol/kg FFM/h, respectively, P = 0.868] between the groups. In contrast, the median (quartile 1, quartile 3) fractional synthesis rates of Kyn [12.5 (5.4, 20.0) compared with 21.3 (16.1, 24.7) %pool/h, P = 0.005] and the fraction of Ala derived from Trp [0.007 (0.005, 0.015) compared with 0.012 (0.008, 0.018), P = 0.037], respectively, in the plasma compartment were significantly slower in the EED group. Fecal biomarkers of EED did not differ between the groups.ConclusionsThe static plasma KTR value is not a good indicator of the dynamic Trp flux down its oxidative pathway. In a poor sanitary environment, children without EED actually have a faster Kyn synthesis rate, which might be beneficial, because of the cytoprotective and anti-inflammatory functions of downstream metabolites. This study was registered in the Clinical Trials Registry of India as CTRI/2017/02/007921.

Role of liver AMPK and GCN2 kinases in the control of postprandial protein metabolism in response to mid-term high or low protein intake in mice.

Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

Anabolic Characterization Of Immortalized Murine L6, And Canine Myok9 Skeletal Myotubes

Healthy skeletal muscle is an important site of metabolic regulation. Metabolic dysregulation of skeletal muscle contributes to the development of obesity, the metabolic syndrome, and muscle degeneration. Interest in developing an in vitro canine model has been increasing as canine skeletal muscle is more pathologically similar to human skeletal muscle than rodent. In vitro models are useful to control variables, reduce clinical costs, and assess molecular characteristics of tissue. Therefore, characterization of Myok9 myotube anabolic function and comparison to an established cell line is necessary to understand metabolic function of this novel cell line for its use in the study of skeletal muscle dysfunction and disease. PURPOSE: To describe anabolic signaling and determine fractional synthesis rates of novel Myok9 myotubes when compared to established murine L6 myotubes. METHODS: L6 and Myok9 myoblasts were grown in vitro in standard culture medium until 70% confluent. Differentiation was induced by switching to a low serum medium. Media containing 4% deuterium oxide (stable isotope label) supplemented with the appropriate pharmacological treatment (DMSO, rapamycin, and torin1) was added to myotubes during the final 24-hours of the 7-day differentiation period. Cells and media samples were harvested for fractional synthesis rates, and protein content of key anabolic markers using Western blotting techniques. RESULTS: Myofibrillar protein synthesis rates (MPS) were similar between species, but slightly lower in canine versus rat cells. Both cell lines exhibited significantly lower MPS in the myotubes treated with torin1. The phosphorylated to total protein ratios of 4EBP1 were significantly lower in both L6 and Myok9 myotubes treated with torin1. The phosphorylated to total protein ratios of p70S6K were significantly lower in L6 and Myok9 myotubes treated with rapamycin or torin1. CONCLUSION: Similar anabolic responses to pharmacologic inhibition occurred in both L6 and Myok9 myotubes. Interestingly, MPS rates in Myok9 myotubes were slightly lower compared to L6 myotubes. These results suggest Myok9 cell line could be used in the development of pharmacologic treatments of skeletal muscle dysfunction or disease while remaining cognizant of lower overall protein synthesis rates.

A Randomized Trial of the Effects of Dietary n3-PUFAs on Skeletal Muscle Function and Acute Exercise Response in Healthy Older Adults.

Skeletal muscle is critical for maintaining mobility, independence, and metabolic health in older adults. However, a common feature of aging is the progressive loss of skeletal muscle mass and function, which is often accompanied by mitochondrial impairments, oxidative stress, and insulin resistance. Exercise improves muscle strength, mitochondrial health, and cardiorespiratory fitness, but older adults often exhibit attenuated anabolic responses to acute exercise. Chronic inflammation associated with aging may contribute to this “anabolic resistance” and therapeutic interventions that target inflammation may improve exercise responsiveness. To this end, we conducted a randomized controlled trial to determine the effect of 6 months of dietary omega-3 polyunsaturated fatty acids (n3-PUFA) supplementation on skeletal muscle function (mass, strength), mitochondrial physiology (respiration, ATP production, ROS generation), and acute exercise responsiveness at the level of the muscle (fractional synthesis rate) and the whole-body (amino acid kinetics) in healthy older adults. When compared with a corn oil placebo (n = 33; 71.5 ± 4.8 years), older adults treated with 4 g/day n3-PUFA (n = 30; 71.4 ± 4.5 years) exhibited modest but significant increases in muscle strength (3.1 ± 14.7% increase in placebo vs. 7.5 ± 14.1% increase in n3-PUFA; p = 0.039). These improvements in muscle strength with n3-PUFA supplementation occurred in the absence of any effects on mitochondrial function and a minor attenuation of the acute response to exercise compared to placebo. Together, these data suggest modest benefits of dietary n3-PUFAs to muscle function in healthy older adults. Future studies may elucidate whether n3-PUFA supplementation improves the exercise response in elderly individuals with co-morbidities, such as chronic inflammatory disease or sarcopenia.