Leucine-enriched amino acids maintain peripheral mTOR-Rheb localization independent of myofibrillar protein synthesis and mTORC1 signaling postexercise

Hannaian, Sarkis J.; Hodson, Nathan; Sawan, Sidney Abou; Mazzulla, Michael; Kato, Hiroyuki; Matsunaga, Keiko; Waskiw-Ford, Marcus; Duncan, Justin; Kumbhare, Dinesh; Moore, Daniel R.

doi:10.1152/japplphysiol.00241.2020

Cited by 20 publications

(26 citation statements)

References 62 publications

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“…Changes in LAT1 and SNAT2 transporter expression have also been observed in isolated membrane fractions during periods of low amino acid availability following acute resistance exercise [22]. Collectively, these data suggest that feeding and resistance exercise influence the gene and protein expression of LAT1 and SNAT2 that could contribute to enhanced amino acid transport and a greater protein synthetic response within skeletal muscle [1,2,8], although we [23] and others [24] have failed to replicate these acute protein expression changes. However, extracellular essential amino acid availability [1,2] and the inward transport of amino acids in skeletal muscle [25] is increased early after feeding and, as a result, muscle protein synthesis rates are stimulated within 2 h post-feeding/exercise [5][6][7].…”

Section: Introductionmentioning

confidence: 58%

“…This influenced our decision to include two metabolic tracers of different essential amino acids, one of which (leucine) is generally considered to be the preeminent anabolic amino acid [11,30,31]. We hypothesized that, in line with our previous findings [23], amino acid ingestion at rest and after resistance exercise would not increase LAT1 and SNAT2 protein expression nor LAT1 membrane localization in the early postprandial period but that there would be a positive association between basal transporter expression/membrane localization and dietary amino acid incorporation into myofibrillar protein.…”

Section: Introductionmentioning

confidence: 80%

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LAT1 and SNAT2 Protein Expression and Membrane Localization of LAT1 Are Not Acutely Altered by Dietary Amino Acids or Resistance Exercise Nor Positively Associated with Leucine or Phenylalanine Incorporation in Human Skeletal Muscle

et al. 2021

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The influx of essential amino acids into skeletal muscle is primarily mediated by the large neutral amino acid transporter 1 (LAT1), which is dependent on the glutamine gradient generated by the sodium-dependent neutral amino acid transporter 2 (SNAT2). The protein expression and membrane localization of LAT1 may be influenced by amino acid ingestion and/or resistance exercise, although its acute influence on dietary amino acid incorporation into skeletal muscle protein has not been investigated. In a group design, healthy males consumed a mixed carbohydrate (0.75 g·kg−1) crystalline amino acid (0.25 g·kg−1) beverage enriched to 25% and 30% with LAT1 substrates L-[1-13C]leucine (LEU) and L-[ring-2H5]phenylalanine (PHE), respectively, at rest (FED: n = 7, 23 ± 5 y, 77 ± 4 kg) or after a bout of resistance exercise (EXFED: n = 7, 22 ± 2 y, 78 ± 11 kg). Postprandial muscle biopsies were collected at 0, 120, and 300 min to measure transporter protein expression (immunoblot), LAT1 membrane localization (immunofluorescence), and dietary amino acid incorporation into myofibrillar protein (ΔLEU and ΔPHE). Basal LAT1 and SNAT2 protein contents were correlated with each other (r = 0.55, p = 0.04) but their expression did not change across time in FED or EXFED (all, p > 0.05). Membrane localization of LAT1 did not change across time in FED or EXFED whether measured as outer 1.5 µm intensity or membrane-to-fiber ratio (all, p > 0.05). Basal SNAT2 protein expression was not correlated with ΔLEU or ΔPHE (all, p ≥ 0.05) whereas basal LAT1 expression was negatively correlated with ΔPHE in FED (r = −0.76, p = 0.04) and EXFED (r = −0.81, p = 0.03) but not ΔLEU (p > 0.05). Basal LAT1 membrane localization was not correlated with ΔLEU or ΔPHE (all, p > 0.05). Our results suggest that LAT1/SNAT2 protein expression and LAT1 membrane localization are not influenced by acute anabolic stimuli and do not positively influence the incorporation of dietary amino acids for de novo myofibrillar protein synthesis in healthy young males.

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Section: Introductionmentioning

confidence: 58%

Section: Introductionmentioning

confidence: 80%

LAT1 and SNAT2 Protein Expression and Membrane Localization of LAT1 Are Not Acutely Altered by Dietary Amino Acids or Resistance Exercise Nor Positively Associated with Leucine or Phenylalanine Incorporation in Human Skeletal Muscle

et al. 2021

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“…In agreement with recent in vitro data ( 22), p-RPS6 Ser240/244 was observed to localize with focal adhesion complexes, purported to be central regulators of anabolic signal transduction. RPS6 Ser240/244 phosphorylation, when measured by immunoblot, is elevated upwards of 5-fold following anabolic stimuli (5,19,31,32), with these elevations often greater than other commonly assessed mTORC1-regulated sites such as p-S6K1 Thr389 and p-4EBP1 Thr37/46 (33). Furthermore, RPS6 Ser240/244 phosphorylation has consistently been shown to be rapamycinsensitive in vitro (25,26) and in rodent and human skeletal muscle (5,27,28).…”

Section: Discussionmentioning

confidence: 98%

“…Immunoblotting was completed as described previously (19). Briefly, a small piece of skeletal muscle tissue (20mg) was homogenized by mechanical pulverization in radioimmunoprecipitation assay (RIPA) buffer (65 mM Tris-base, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with added protease and phosphatase inhibitors (Roche Applied Science, Mannheim, GER).…”

Section: Immunoblottingmentioning

confidence: 99%

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Mechanistic target of rapamycin complex 1 (mTORC1) activity occurs predominantly in the periphery of human skeletal muscle fibers, in close proximity to focal adhesion complexes, following anabolic stimuli

Hodson

Mazzulla

Kumbhare

et al. 2021

Preprint

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Following anabolic stimuli (e.g. mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1 activity occurs prior to or following this translocation. We therefore aimed to determine the cellular location of mTORC1 activity in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7, 23±5yrs, 76.8±3.6kg, 13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7, 22±2yrs, 78.1±3.6kg, 12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. The spatial regulation of mTORC1 activity was assessed through immunofluorescent staining of p-RPS6Ser240/244, an mTORC1-specific phosphorylation event. p-RPS6Ser240/244 measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining was displayed a similar pattern, suggesting mTORC1 activity occurs predominantly in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that mTORC1 activity occurs in the region of human muscle fibers to which mTORC1 translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 activation in vivo.

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Resistance Exercise, Aging, Disuse, and Muscle Protein Metabolism

McKendry

Stokes

Mcleod

et al. 2021

Comprehensive Physiology

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Leucine-enriched amino acids maintain peripheral mTOR-Rheb localization independent of myofibrillar protein synthesis and mTORC1 signaling postexercise

Cited by 20 publications

References 62 publications

LAT1 and SNAT2 Protein Expression and Membrane Localization of LAT1 Are Not Acutely Altered by Dietary Amino Acids or Resistance Exercise Nor Positively Associated with Leucine or Phenylalanine Incorporation in Human Skeletal Muscle

LAT1 and SNAT2 Protein Expression and Membrane Localization of LAT1 Are Not Acutely Altered by Dietary Amino Acids or Resistance Exercise Nor Positively Associated with Leucine or Phenylalanine Incorporation in Human Skeletal Muscle

Mechanistic target of rapamycin complex 1 (mTORC1) activity occurs predominantly in the periphery of human skeletal muscle fibers, in close proximity to focal adhesion complexes, following anabolic stimuli

Resistance Exercise, Aging, Disuse, and Muscle Protein Metabolism

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