p70S6K Thr389 Phosphorylation Research Articles

Co-ingestion of cluster dextrin carbohydrate does not increase exogenous protein-derived amino acid release or myofibrillar protein synthesis following a whole-body resistance exercise in moderately trained younger males: a double-blinded randomized controlled crossover trial

PurposeThis study investigates if co-ingestion of cluster dextrin (CDX) augments the appearance of intrinsically labeled meat protein hydrolysate-derived amino acid (D5-phenylalanine), Akt/mTORC1 signaling, and myofibrillar protein fractional synthetic rate (FSR).MethodsTen moderately trained healthy males (age: 21.5 ± 2.1 years, body mass: 75.7 ± 7.6 kg, body mass index (BMI): 22.9 ± 2.1 kg/m2) were included for a double-blinded randomized controlled crossover trial. Either 75 g of CDX or glucose (GLC) was given in conjunction with meat protein hydrolysate (0.6 g protein * FFM−1) following a whole-body resistance exercise. A primed-continuous intravenous infusion of L-[15N]-phenylalanine with serial muscle biopsies and venous blood sampling was performed.ResultsA time × group interaction effect was found for serum D5-phenylalanine enrichment (P < 0.01). Serum EAA and BCAA concentrations showed a main effect for group (P < 0.05). Tmax serum BCAA was greater in CDX as compared to GLC (P < 0.05). However, iAUC of all serum parameters did not differ between CDX and GLC (P > 0.05). Tmax serum EAA showed a trend towards a statistical significance favoring CDX over GLC. The phosphorylation of p70S6KThr389, rpS6Ser240/244, ERK1/2Thr202/Tyr204 was greater in CDX compared to GLC (P < 0.05). However, postprandial myofibrillar FSR did not differ between CDX and GLC (P = 0.17).ConclusionIn moderately trained younger males, co-ingestion of CDX with meat protein hydrolysate does not augment the postprandial amino acid availability or myofibrillar FSR as compared to co-ingestion of GLC during the recovery from a whole-body resistance exercise despite an increased intramuscular signaling.Trial registrationClinicalTrials.gov ID: NCT03303729 (registered on October 3, 2017).

Moderate-Intensity Strength Exercise to Exhaustion Results in More Pronounced Signaling Changes in Skeletal Muscles of Strength-Trained Compared With Untrained Individuals.

Lysenko, EA, Popov, DV, Vepkhvadze, TF, Sharova, AP, and Vinogradova, OL. Moderate-intensity strength exercise to exhaustion results in more pronounced signaling changes in skeletal muscles of strength-trained compared with untrained individuals. J Strength Cond Res 34(4): 1103-1112, 2020-The aim of our investigation was to compare the response pattern of signaling proteins and genes regulating protein synthesis and degradation in skeletal muscle after strength exercise sessions performed to volitional fatigue in strength-trained and untrained males. Eight healthy recreationally active males and 8 power-lifting athletes performed 4 sets of unilateral leg presses to exhaustion (65% 1 repetition maximum). Biopsy samples of m. vastus lateralis were obtained before, 1 and 5 hours after cessation of exercise. Phosphorylation of p70S6k, 4EBP1, and ACC increased, whereas phosphorylation of eEF2 and FOXO1 decreased only in the trained group after exercise. Expression of DDIT4, MURF1, and FOXO1 mRNAs increased and expression of MSTN mRNA decreased also only in the trained group after exercise. In conclusion, moderate-intensity strength exercise performed to volitional fatigue changed the phosphorylation status of mTORC1 downstream signaling molecules and markers of ubiquitin-proteasome system activation in trained individuals, suggesting activation of protein synthesis and degradation. In contrast to the trained group, signaling responses in the untrained group were considerably less pronounced. It can be assumed that the slowdown in muscle mass gain as the athletes increase in qualification cannot be associated with a decrease in the sensitivity of systems regulating protein metabolism, but possibly with inadequate intake or assimilation of nutrients necessary for anabolism. Perhaps, the intake of highly digestible protein or protein-carbohydrate dietary supplements could contribute to the increase in muscle mass in strength athletes.

Mammalian target of rapamycin and p70S6K mediate thrombin-induced nuclear factor-κB activation and IL-8/CXCL8 release in human lung epithelial cells

Thrombin plays a crucial role in lung inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Thrombin induces the release of interleukin-8 (IL-8)/CXCL8 by lung epithelial cells, and this phenomenon plays a vital role in lung inflammation. Our previous studies have indicated that thrombin stimulates IL-8/CXCL8 expression through PI3K/Akt/IκB kinase (IKK)α/β/nuclear factor-κB (NF-κB) and p300 pathways in human lung epithelial cells. In the present study, we explored the roles of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K) in thrombin-induced NF-κB activation and IL-8/CXCL8 release in human lung epithelial cells. In this study, we found that rapamycin (an mTOR inhibitor) and p70S6K siRNA diminished thrombin-induced IL-8/CXCL8 release. Thrombin induced mTOR Ser2448 phosphorylation and p70S6K Thr389 phosphorylation in a time-dependent manner. Moreover, rapamycin attenuated thrombin-stimulated p70S6K phosphorylation. We also found that transfection of cells with the dominant negative mutant of Akt (Akt DN) reduced the thrombin-induced increase in mTOR phosphorylation and p70S6K phosphorylation. Moreover, thrombin-stimulated p300 phosphorylation was attenuated by Akt DN, rapamycin, and p70S6K siRNA. Thrombin triggered p70S6K translocation from the cytosol to the nucleus in a time-dependent manner. Thrombin induced the complex formation of p70S6K, p300, and p65; acetylation of p65 Lys310, and recruitment of p70S6K, p300, and p65 to the κB-binding site of the IL-8/CXCL8 promoter region. In conclusion, these results indicate that thrombin initiates the Akt-dependent mTOR/p70S6K signaling pathway to promote p300 phosphorylation and NF-κB activation and finally induces IL-8/CXCL8 release in human lung epithelial cells.

Type 2 diabetes causes skeletal muscle atrophy but does not impair resistance training-mediated myonuclear accretion and muscle mass gain in rats.

New Findings What is the central question of this study? Type 2 diabetes mellitus (T2DM) causes skeletal muscle atrophy; does it affect resistance training (RT)‐mediated molecular adaptations and subsequent muscle hypertrophy? What is the main finding and its importance? Although skeletal muscle mass and regulation were not preserved under conditions of T2DM, the response of RT‐induced skeletal muscle hypertrophy was not impaired in T2DM rat skeletal muscle. These findings suggest that the capacity of RT‐mediated muscle mass gain is not diminished in the T2DM condition. Type 2 diabetes mellitus (T2DM) is known to cause skeletal muscle atrophy. However, it is not known whether T2DM affects resistance training (RT)‐mediated molecular adaptations and subsequent muscle hypertrophy. Therefore, we investigated the effect of T2DM on response of skeletal muscle hypertrophy to chronic RT using a rat resistance exercise mimetic model. T2DM and healthy control rats were subjected to 18 bouts (3 times per week) of chronic RT on unilateral lower legs. RT significantly increased gastrocnemius muscle mass and myonuclei in both T2DM and healthy control rats to the same extent, even though T2DM caused muscle atrophy in the resting condition. Further, T2DM significantly reduced mechanistic target of rapamycin complex 1 (mTORC1) activity (phosphorylation of p70S6KThr389 and 4E‐BP1Thr37/46) to insulin stimulation and the number of myonuclei in the untrained basal condition, but RT‐mediated adaptations were not affected by T2DM. These findings suggested that although the skeletal muscle mass and regulation were not preserved under basal conditions of T2DM, the response of RT‐induced skeletal muscle hypertrophy was not impaired in T2DM rat skeletal muscle.

Signaling responses to high and moderate load strength exercise in trained muscle.

We examined signaling responses in the skeletal muscle of strength athletes after strength exercises under high and moderate load. Eight trained male powerlifters were recruited. The volunteers performed four sets of leg presses to volitional fatigue using a moderate load (65% 1‐repetition maximum [1RM]) for one leg, and a high load (85% 1RM) for the contralateral leg. The work volume performed by the leg moving a moderate load was higher than that of the contralateral leg moving a high load. Biopsy of the m. vastus lateralis was performed before, and at 1, 5, and 10 h after, cessation of exercise. Phosphorylation of p70S6kThr389, 4E‐BP1Thr37/46, and ACCS er79 increased after moderate load exercises, whereas phosphorylation of ERK1/2Thr202/Tyr204 increased, and that of eEF2Thr56 decreased, after high load exercises. Exercise under a moderate load and a high work volume activated mTORC1‐dependent signaling in trained skeletal muscle, whereas exercise under a high load but lower work volume activated the MEK‐ERK1/2 signaling cascade and eEF2.

Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells

Leucine is a key amino acid for initiating translation in muscle cells, but the dose-dependent effects of leucine on intracellular signaling are poorly characterized. This study examined the effect that increasing doses of leucine would have on changes in mechanistic target of rapamycin (mTOR)–mediated signaling, rates of protein synthesis, and cell size in C2C12 cells. We hypothesized that a leucine “threshold” exists, which represents the minimum stimulus required to initiate mTOR signaling in muscle cells. Acute exposure to 1.5, 3.2, 5.0, and 16.1 mM leucine increased phosphorylation of mTORSer2448 (~1.4-fold; P < .04), 4E-BP1 Thr37/46 (~1.9-fold; P < .001), and rpS6Ser235/6 (~2.3-fold; P < .001). However, only p70S6kThr389 exhibited a dose-dependent response to leucine with all treatments higher than control (~4-fold; P < .001) and at least 5 mM higher than the 1.5-mM concentration (1.2-fold; P < .02). Rates of protein synthesis were not altered by any treatment. Seven days of exposure to 0.5, 1.5, 5.0, and 16.5 mM leucine resulted in an increase in cell size in at least 5 mM treatments (~1.6-fold, P < .001 vs control). Our findings indicate that even at low leucine concentrations, phosphorylation of proteins regulating translation initiation signaling is enhanced. The phosphorylation of p70S6kThr389 follows a leucine dose-response relationship, although this was not reflected by the acute protein synthetic response. Nevertheless, under the conditions of the present study, it appears that leucine concentrations of at least 5 mM are necessary to enhance cell growth.

Acute low-load resistance exercise with and without blood flow restriction increased protein signalling and number of satellite cells in human skeletal muscle

To investigate hypertrophic signalling after a single bout of low-load resistance exercise with and without blood flow restriction (BFR). Seven subjects performed unilateral knee extensions at 30 % of their one repetition maximum. The subjects performed five sets to failure with BFR on one leg, and then repeated the same amount of work with the other leg without BFR. Biopsies were obtained from m. vastus lateralis before and 1, 24 and 48 h after exercise. At 1-h post-exercise, phosphorylation of p70S6KThr389 and p38MAPKThr180/Tyr182 was elevated in the BFR leg, but not in the free-flow leg. Phospho-p70S6KThr389 was elevated three- to fourfold in both legs at 24-h post-exercise, but back to baseline at 48 h. The number of visible satellite cells (SCs) per muscle fibre was increased for all post-exercise time points and in both legs (33–53 %). The proportion of SCs with cytoplasmic extensions was elevated at 1-h post in the BFR leg and the number of SCs positive for myogenin and/or MyoD was increased at 1- and 24-h post-exercise for both legs combined. Acute low-load resistance exercise with BFR resulted in early (1 h) and late (24 h) enhancement of phospho-p70S6KThr389, an early response of p38MAPK, and an increased number of SCs per muscle fibre. Enhanced phospho-p70S6KThr389 at 24-h post-exercise and increases in SC numbers were seen also in the free-flow leg. Implications of these findings for the hypertrophic effects of fatiguing low-load resistance exercise with and without BFR are discussed.

Pattern of protein ingestion to maximise muscle protein synthesis after resistance exercise

Pattern of protein ingestion to maximise muscle protein synthesis after resistance exercise

Preexercise Aminoacidemia and Muscle Protein Synthesis after Resistance Exercise

We have previously shown that the aminoacidemia caused by the consumption of a rapidly digested protein after resistance exercise enhances muscle protein synthesis (MPS) more than the amino acid (AA) profile associated with a slowly digested protein. Here, we investigated whether differential feeding patterns of a whey protein mixture commencing before exercise affect postexercise intracellular signaling and MPS. Twelve resistance-trained males performed leg resistance exercise 45 min after commencing each of three volume-matched nutrition protocols: placebo (PLAC, artificially sweetened water), BOLUS (25 g of whey protein + 5 g of leucine dissolved in artificially sweetened water; 1 × 500 mL), or PULSE (15 × 33-mL aliquots of BOLUS drink every 15 min). The preexercise rise in plasma AA concentration with PULSE was attenuated compared with BOLUS (P < 0.05); this effect was reversed after exercise, with two-fold greater leucine concentrations in PULSE compared with BOLUS (P < 0.05). One-hour postexercise, phosphorylation of p70 S6K(thr389) and rpS6(ser235/6) was increased above baseline with BOLUS and PULSE, but not PLAC (P < 0.05); furthermore, PULSE > BOLUS (P < 0.05). MPS throughout 5 h of recovery was higher with protein ingestion compared with PLAC (0.037 ± 0.007), with no differences between BOLUS or PULSE (0.085 ± 0.013 vs. 0.095 ± 0.010%.h(-1), respectively, P = 0.56). Manipulation of aminoacidemia before resistance exercise via different patterns of intake of protein altered plasma AA profiles and postexercise intracellular signaling. However, there was no difference in the enhancement of the muscle protein synthetic response after exercise. Protein sources producing a slow AA release, when consumed before resistance exercise in sufficient amounts, are as effective as rapidly digested proteins in promoting postexercise MPS.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser9 and, to a lesser extent, Thr390, the dephosphorylation of p70S6K at Thr389, and the phosphorylation of p70S6K at Thr421 and Ser424. The specific p38 inhibitor SB203080 reduced the p-GSK3βSer9 and autophagy through the phosphorylation of p70S6KThr389; however, it augmented the levels of p-ERK, p-GSK3βThr390, and p-70S6KThr421/Ser424 induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSKSer9/p70S6KThr380 and ERK/GSK3βThr390/p70S6KThr421/Ser424 kinase signaling, which is involved in cell survival and cell death.

Timing Of Post-exercise Protein Ingestion Alters Anabolic Signaling During Prolonged Recovery

Muscle protein synthesis (MPS) is synergistically enhanced by resistance exercise and feeding with mRNA translation initiation being a critical regulatory step. Previous studies indicate 20 g of protein immediately after resistance exercise maximally stimulates MPS during the early recovery period. However, no studies have examined the effect of manipulating the quantity and timing of protein ingestion on anabolic signaling over a whole day. PURPOSE:To determine the time-course of anabolic signaling during prolonged recovery from resistance exercise. METHODS: 24 resistance trained men were randomly assigned to one of three groups. Each reported to the laboratory following 72 h diet/activity control and an overnight fast. Immediately following leg extension exercise (4 × 10 @ 80% 1repetition maximum, 3 min recovery) one group (PULSE) consumed an 8 × 10 g (125 mL) whey protein isolate drink at 1.5 h intervals; an intermediate group (INT) consumed 4 × 20 g (250 mL) every 3 h; and the final group (BOLUS) received 2 × 40 g (500 mL) 6 h apart during 12 h passive recovery. Muscle biopsy samples from vastus lateralis were taken at rest before exercise and 1, 4, 6, 7 and 12 h post-exercise. p70S6K Thr389 phosphorylation was measured as a marker of translation initiation for MPS. RESULTS: BOLUS ingestion increased phosphorylation of p70S6K Thr389 above rest only at 1 h (∼20 fold) and 7 h post-exercise (∼12 fold; both p<0.05). INT feeding also increased Thr389 phosphorylation compared to rest at the 1 h time-point during recovery (∼12 fold, p<.05) and this effect was still evident 4 h post-exercise (∼8 fold, p<0.05) but not at any later time points. In contrast, there were no differences in phosphorylation with PULSE. Moreover, the phopshorylation with BOLUS and INT ingestion was greater than PULSE 1 h post-exercise (P<0.05). CONCLUSIONS: Pulse feeding (10 g) appears to have only modest capacity for activating translation. In contrast, Bolus (40 g) feeding may induce transient, repeatable activation of translational machinery while Intermediate ingestion (20 g) extends the early, but not late, anabolic response. We show that altering the quantity and timing of protein ingestion generates different anabolic signaling responses during extended recovery from resistance exercise. This project was funded by ARC project grant LP100100010.

Abstract 252: Evaluating the role of STK33 kinase in mutant KRAS cells

Abstract Tumors harboring KRAS mutations remain an elusive target for oncology therapeutics. A recent publication (Scholl et al 2009) described a high throughput cellular RNAi screen which suggested a synthetic lethal relationship between STK33 and mutant KRAS. STK33 is a member of the calcium/calmodulin kinase family with a poorly characterized function. Although no genetic alterations in the STK33 gene have been reported in cancer, inhibiting STK33 kinase activity could offer a novel therapeutic approach to target mutant KRAS tumors. Thus, we sought to validate whether the survival of cancer cell lines with mutant KRAS is dependent on STK33, in parallel with a program to identify small molecule inhibitors of STK33 (Zhang Y et al AACR 2011). We focused on mutant KRAS leukemia cells because of their reported dependence on STK33. Using a panel of siRNA, 50-80% knockdown of STK33 at the RNA and protein level was achieved in NOMO-1KRASG13D and SKM-1KRASK117N cells. However we did not observe any alteration in cell viability. In addition, putative STK33 downstream signaling, (phosphorylation of p70 S6K thr389 and RPS6 ser235/236) was unaltered by modulation of STK33 expression. In contrast, knockdown of KRAS caused a significant reduction in both viability and signaling in these cell lines, confirming their dependence on KRAS. A panel of 27 cancer cell lines was screened with an siRNA library representing 1500 druggable genes. STK33 siRNA had no significant effect on viability regardless of KRAS mutational status. As expected, knockdown of KRAS significantly reduced cell viability, especially in mutant KRAS cells. A kinase dead mutant STK33 was used to test the dependence on STK33 kinase activity in mutant KRAS cell lines. Transient over-expression of kinase dead STK33 in PANC-1KRASG12D and DLD-1KRASG13D cells had no effect on viability, nor on the phosphorylation of p70 S6K thr389 and RPS6 ser235/236. In summary we have been unable to confirm a synthetic lethal relationship between STK33 and KRAS. Our data do not support inhibition of STK33 as a promising therapeutic approach for targeting mutant KRAS tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2011-252

Quantitative Analysis of Anti-apoptotic Function of Akt in Akt1 and Akt2 Double Knock-out Mouse Embryonic Fibroblast Cells under Normal and Stressed Conditions

Quantitative Analysis of Anti-apoptotic Function of Akt in Akt1 and Akt2 Double Knock-out Mouse Embryonic Fibroblast Cells under Normal and Stressed Conditions