Publications by authors named "Scot R Kimball"

181 Publications

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis.

Cell Signal 2021 Aug 17;84:110010. Epub 2021 Apr 17.

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, United States of America. Electronic address:

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.
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http://dx.doi.org/10.1016/j.cellsig.2021.110010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169602PMC
August 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

A clinically relevant decrease in contractile force differentially regulates control of glucocorticoid receptor translocation in mouse skeletal muscle.

J Appl Physiol (1985) 2021 04 18;130(4):1052-1063. Epub 2021 Feb 18.

Department of Nutrition, Food and Exercise Science, Florida State University, Tallahassee, Florida.

Muscle atrophy decreases physical function and overall health. Increased glucocorticoid production and/or use of prescription glucocorticoids can significantly induce muscle atrophy by activating the glucocorticoid receptor, thereby transcribing genes that shift protein balance in favor of net protein degradation. Although mechanical overload can blunt glucocorticoid-induced atrophy in young muscle, those affected by glucocorticoids generally have impaired force generation. It is unknown whether contractile force alters the ability of resistance exercise to mitigate glucocorticoid receptor translocation and induce a desirable shift in protein balance when glucocorticoids are elevated. In the present study, mice were subjected to a single bout of unilateral, electrically induced muscle contractions by stimulating the sciatic nerve at 100 Hz or 50 Hz frequencies to elicit high or moderate force contractions of the tibialis anterior, respectively. Dexamethasone was used to activate the glucocorticoid receptor. Dexamethasone increased glucocorticoid signaling, including nuclear translocation of the receptor, but this was mitigated only by high force contractions. The ability of high force contractions to mitigate glucocorticoid receptor translocation coincided with a contraction-mediated increase in muscle protein synthesis, which did not occur in the dexamethasone-treated mice subjected to moderate force contractions. Though moderate force contractions failed to increase protein synthesis following dexamethasone treatment, both high and moderate force contractions blunted the glucocorticoid-mediated increase in LC3 II:I marker of autophagy. Thus, these data show that force generation is important for the ability of resistance exercise to mitigate glucocorticoid receptor translocation and promote a desirable shift in protein balance when glucocorticoids are elevated. Glucocorticoids induce significant skeletal muscle atrophy by activating the glucocorticoid receptor. Our work shows that muscle contractile force dictates glucocorticoid receptor nuclear translocation. We also show that blunting nuclear translocation by high force contractions coincides with the ability of muscle to mount an anabolic response characterized by increased muscle protein synthesis. This work further defines the therapeutic parameters of skeletal muscle contractions to blunt glucocorticoid-induced atrophy.
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http://dx.doi.org/10.1152/japplphysiol.01064.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262779PMC
April 2021

Intermittent Bolus Feeding Enhances Organ Growth More Than Continuous Feeding in a Neonatal Piglet Model.

Curr Dev Nutr 2020 Dec 24;4(12):nzaa170. Epub 2020 Nov 24.

United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.

Background: Orogastric tube feeding is frequently prescribed for neonates who cannot ingest food normally. In a piglet model of the neonate, greater skeletal muscle growth is sustained by upregulation of translation initiation signaling when nutrition is delivered by intermittent bolus meals, rather than continuously.

Objectives: The objective of this study was to determine the long-term effects of feeding frequency on organ growth and the mechanism by which feeding frequency modulates protein anabolism in these organs.

Methods: Eighteen neonatal pigs were fed by gastrostomy tube the same amount of a sow milk replacer either by continuous infusion (CON) or on an intermittent bolus schedule (INT). After 21 d of feeding, the pigs were killed without interruption of feeding (CON; = 6) or immediately before (INT-0; = 6) or 60 min after (INT-60; = 6) a meal, and fractional protein synthesis rates and activation indexes of signaling pathways that regulate translation initiation were measured in the heart, jejunum, ileum, kidneys, and liver.

Results: Compared with continuous feeding, intermittent feeding stimulated the growth of the liver (+64%), jejunum (+48%), ileum (+40%), heart (+64%), and kidney (+56%). The increases in heart, kidney, jejunum, and ileum masses were proportional to whole body lean weight gain, but liver weight gain was greater in the INT-60 than the CON, and intermediate for the INT-0 group. For the liver and ileum, but not the heart, kidney, and jejunum, INT-60 compared with CON pigs had greater fractional protein synthesis rates (22% and 48%, respectively) and was accompanied by an increase in ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 phosphorylation.

Conclusions: These results suggest that intermittent bolus compared with continuous orogastric feeding enhances organ growth and that in the ileum and liver, intermittent feeding enhances protein synthesis by stimulating translation initiation.
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http://dx.doi.org/10.1093/cdn/nzaa170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751947PMC
December 2020

Retinol-binding protein 4 mRNA translation in hepatocytes is enhanced by activation of mTORC1.

Am J Physiol Endocrinol Metab 2021 02 7;320(2):E306-E315. Epub 2020 Dec 7.

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.

Increased expression of the peptide hormone retinol-binding protein 4 (RBP4) has been implicated in the development of insulin resistance, type 2 diabetes, and visual dysfunction. Prior investigations of the mechanisms that influence RBP4 synthesis have focused solely on changes in mRNA abundance. Yet, the production of many secreted proteins is controlled at the level of mRNA translation, as it allows for a rapid and reversible change in expression. Herein, we evaluated mRNA translation using sucrose density gradient centrifugation. In the liver of fasted rodents, mRNA translation was low. In response to refeeding, mRNA translation was enhanced and RBP4 levels in serum were increased. In H4IIE cells, refreshing culture medium promoted mRNA translation and expression of the protein. mRNA abundance was not increased by either experimental manipulation. Enhanced mRNA translation was associated with activation of the kinase mechanistic target of rapamycin in complex 1 (mTORC1) and enhanced phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). In H4IIE cells, expression of a 4E-BP1 variant that is unable to be phosphorylated by mTORC1 or suppression of mTORC1 with rapamycin attenuated activity of a luciferase reporter encoding the mRNA 5'-untranslated region (UTR). Purine substitutions to disrupt a terminal oligopyrimidine (TOP)-like sequence in the 5'-UTR prevented the suppressive effect of rapamycin on reporter activity. Rapamycin also prevented upregulation of mRNA translation in the liver and reduced serum levels of RBP4 in response to feeding. Overall, the findings support a model in which nutrient-induced activation of mTORC1 upregulates mRNA translation to promote RBP4 synthesis. RBP4 plays a critical role in metabolic disease, yet relatively little is known about the mechanisms that regulate its production. Herein, we provide evidence for translational control of RBP4 synthesis. We demonstrate that activation of the nutrient-sensitive kinase mTORC1 promotes hepatic mRNA translation. The findings support the possibility that targeting mRNA translation represents an alternative to current therapeutic interventions that lower serum RBP4 concentration by promoting urinary excretion of the protein.
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http://dx.doi.org/10.1152/ajpendo.00494.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260359PMC
February 2021

The 2020 FASEB Virtual Science Research Conference on Nutrient Sensing and Metabolic Signaling, August 10-11, 2020.

FASEB J 2020 12 27;34(12):15627-15629. Epub 2020 Oct 27.

Department of Pediatrics, U.S. Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX, USA.

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http://dx.doi.org/10.1096/fj.202002259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8460123PMC
December 2020

Glucagon-Dependent Suppression of mTORC1 is Associated with Upregulation of Hepatic FGF21 mRNA Translation.

Am J Physiol Endocrinol Metab 2020 May 18. Epub 2020 May 18.

Department of Cellular and Molecular Physiology, Penn State College of Medicine, 500 University Drive, Hershey PA 17033, United States.

Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood, and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated post-transcriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, re-feeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.
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http://dx.doi.org/10.1152/ajpendo.00555.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468783PMC
May 2020

A time course for markers of protein synthesis and degradation with hindlimb unloading and the accompanying anabolic resistance to refeeding.

J Appl Physiol (1985) 2020 07 14;129(1):36-46. Epub 2020 May 14.

Pennsylvania State University, Department of Cellular and Molecular Physiology, College of Medicine, Hershey, Pennsylvania.

Skeletal muscle atrophy is associated with disease, aging, and disuse. Hindlimb unloading (HU) in animals provides an experimental model to study muscle atrophy. A comprehensive time course for how HU affects biomarkers of protein synthesis and degradation acutely and chronically and the associated resistance to an anabolic stimulus following disuse remain undocumented. Sixteen-week-old C57BL/6 mice underwent 0, 1, 12, 24, 72, 168, or 336 h of HU. Following 336 h of HU, mice were reloaded for 1, 24, or 72 h. Another group of mice underwent 120 h of HU, were fasted or refed, and were then compared with similar condition control animals (CTL). Protein content and phosphorylation of biomarkers of protein synthesis, degradation, and autophagy were assessed in the soleus muscle. Gastrocnemius, soleus, and plantaris muscles atrophied within 120 h of HU. Protein synthesis trended toward decrease following 24 h of HU. p70S6K phosphorylation and protein synthesis increased with reloading. Following HU, changes in MAFbx and DEPTOR expression and DEPTOR phosphorylation were consistent with development of a catabolic state. DEPTOR expression recovered following reloading. Animals that underwent 120 h of HU exhibited attenuation of refeeding-induced p70S6K phosphorylation compared with CTL counterparts. Following 120 h of HU, protein synthesis, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation, and DEPTOR, MAFbx, and Sestrin1 expression indicated a catabolic state. Following 120 h of HU, autophagy markers, including p62 expression, REDD1 expression, LC3 ratio, and Unc-51-like autophagy-activating kinase 1 (ULK1) phosphorylation, indicated impaired autophagy. HU promotes a deleterious balance between protein synthesis and degradation. The time course herein provides scientists information about when the associated biomarkers become affected. Hindlimb unloading causes significant skeletal muscle atrophy by adversely affecting the balance between protein synthesis and breakdown. This study demonstrates a more complete time course for changes in biomarkers associated with protein synthesis and breakdown and investigates the associated anabolic resistance to an anabolic stimulus following hindlimb unloading. These data in concert with information from other studies provide a basis for designing future experiments to optimally interrogate a desired cellular biomarker or pathway.
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http://dx.doi.org/10.1152/japplphysiol.00155.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469230PMC
July 2020

Nonalcoholic steatohepatitis Fitness Intervention in Thrombosis (NASHFit): Study protocol for a randomized controlled trial of a supervised aerobic exercise program to reduce elevated clotting risk in patients with NASH.

Contemp Clin Trials Commun 2020 Jun 4;18:100560. Epub 2020 Apr 4.

Department of Public Health Sciences, The Pennsylvania State University- Milton S. Hershey Medical Center, Hershey, PA, USA.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide affecting upwards of one third the global population. For reasons not fully understood, individuals with NAFLD and its more severe variant, nonalcoholic steatohepatitis (NASH), are at increased risk for venous thromboembolism which significantly increases morbidity and mortality. Lifestyle changes centering around exercise training are the mainstay of treatment for NAFLD/NASH. While exercise training can lessen venous thromboembolic risk in healthy persons and those with cardiovascular disease, whether or not this benefit is seen in patients with NAFLD/NASH remains unknown. In order to better understand how exercise training impacts thrombosis risk in NAFLD, we present the design of a thirty-two week randomized controlled clinical trial of 42 sedentary subjects age 18-69 with biopsy proven NASH. The main aim is to determine the impact of an aerobic exercise training program on the abnormal hemostatic system unique to NAFLD/NASH. The main outcome is change in plasminogen activator inhibitor one level, an established marker for venous thromboembolism. Secondary outcomes include body composition, cardiorespiratory fitness, control of comorbid metabolic conditions (e.g., obesity, hypertension, hyperlipidemia, diabetes), dietary composition, health related quality of life, liver enzymes and histology, NAFLD/NASH disease activity (e.g., biomarkers, clinical decision aids), microbiome, other markers of hemostasis, and PNPLA3 gene expression. The study represents the first clinical trial of an exercise training program to reduce elevated clotting risk in subjects with NAFLD/NASH.
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http://dx.doi.org/10.1016/j.conctc.2020.100560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154986PMC
June 2020

Exercise Attenuates Ribosomal Protein Six Phosphorylation in Fatty Liver Disease.

Dig Dis Sci 2020 11 1;65(11):3238-3243. Epub 2020 Apr 1.

Department of Cellular and Molecular Physiology, The Pennsylvania State University- College of Medicine, Hershey, PA, USA.

Introduction: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of liver disease worldwide. Nonalcoholic steatohepatitis (NASH), is a more severe type of NAFLD. Exercise improves NASH, by reversing steatosis, and may arrest fibrosis. However, the mechanisms underlying these interactions are unknown. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that is activated by energy stress. Mammalian target of rapamycin in complex 1 (mTORC1) is a nutrient sensor that regulates protein synthesis. In NASH, AMPK activity is low and mTORC1 is high. In healthy persons, exercise activates AMPK and suppresses mTORC1. We examined the effects of exercise on hepatic ribosomal protein S6 phosphorylation, a downstream target of AMPK and mTORC1 in patients with NASH.

Methods: Three subjects with biopsy-proven NASH underwent a structured, 20-week aerobic exercise intervention, five-days a week for 30-min at a moderate intensity (40-55% of VO2max). Immunofluorescence staining for rpS6 phosphorylation in hepatic tissue was quantified by ImageJ software.

Results: Following 20-weeks of aerobic exercise, rpS6 levels were significantly attenuated (3.9 ± 1.9 pre-exercise vs. 1.4 +/0.4 post-exercise, p = 0.04).

Conclusions: These findings suggest exercise modulates the AMPK/mTORC1 pathway in patients with NASH and may guide the design of future studies into the mechanism of how exercise improves NASH and possibly reverses fibrosis.
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http://dx.doi.org/10.1007/s10620-020-06226-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529701PMC
November 2020

ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.

J Nutr 2020 05;150(5):1022-1030

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA, USA.

Background: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase.

Objective: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2.

Methods: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates.

Results: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted.

Conclusions: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.
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http://dx.doi.org/10.1093/jn/nxz309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198311PMC
May 2020

Interactions of the super complexes: When mTORC1 meets the proteasome.

Int J Biochem Cell Biol 2019 12 31;117:105638. Epub 2019 Oct 31.

Department of Medicine, McGill University and the Research Institute of the McGill University Health Centre, the Montreal Diabetes Research Centre, Montréal, Quebec, H4A 3J1. Canada.

Homeostatic regulation of energy and metabolic status requires that anabolic and catabolic signaling pathways be precisely regulated and coordinated. Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is a mega protein complex that promotes energy-consuming anabolic processes of protein and nucleic acid synthesis as well lipogenesis in times of energy and nutrient abundance. However, it is best characterized as the regulator of steps leading to protein synthesis. The ubiquitin-proteasome proteolytic system (UPS) is a major intracellular proteolytic system whose activity is increased during periods of nutrient scarcity and in muscle wasting conditions such as cachexia. Recent studies have examined the impact of mTORC1 on levels and functions of the 26S proteasome, the mega protease complex of the UPS. Here we first briefly review current understanding of the regulation of mTORC1, the UPS, and the 26S proteasome complex. We then review evidence of the effect of each complex on the abundance and functions of the other. Given the fact that drugs that inhibit either complex are either in clinical trials or are approved for treatment of cancer, a muscle wasting condition, we identify studying the effect of combinatory mTORC1-proteasome inhibition on skeletal muscle mass and health as a critical area requiring investigation.
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http://dx.doi.org/10.1016/j.biocel.2019.105638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910232PMC
December 2019

Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.

Cell Rep 2019 08;28(8):1971-1980.e8

Department of Cellular & Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA. Electronic address:

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.
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http://dx.doi.org/10.1016/j.celrep.2019.07.084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716532PMC
August 2019

Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle.

Am J Physiol Endocrinol Metab 2019 05 5;316(5):E817-E828. Epub 2019 Mar 5.

Department of Cellular and Molecular Physiology, Penn State College of Medicine , Hershey, Pennsylvania.

Previous studies established that leucine stimulates protein synthesis in skeletal muscle to the same extent as a complete mixture of amino acids, and the effect occurs through activation of the mechanistic target of rapamycin in complex 1 (mTORC1). Recent studies using cells in culture showed that the Sestrins bind leucine and are required for leucine-dependent activation of mTORC1. However, the role they play in mediating leucine-dependent activation of the kinase in vivo has been questioned because the dissociation constant of Sestrin2 for leucine is well below circulating and intramuscular levels of the amino acid. The goal of the present study was to compare expression of the Sestrins in skeletal muscle to other tissues and to assess their relative role in mediating activation of mTORC1 by leucine. The results show that the relative expression of the Sestrin proteins varies widely among tissues and that in skeletal muscle Sestrin1 expression is higher than Sestrin3, whereas Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine-induced dissociation of the Sestrin·GAP activity toward Rags 2 (GATOR2) complex revealed that Sestrin1 has the highest affinity for leucine and that Sestrin3 has the lowest affinity. In agreement with the dissociation constants calculated using cells in culture, oral leucine administration promotes disassembly of the Sestrin1·GATOR2 complex but not the Sestrin2 or Sestrin3·GATOR2 complex. Overall, the results presented herein are consistent with a model in which leucine-induced activation of mTORC1 in skeletal muscle in vivo occurs primarily through release of Sestrin1 from GATOR2.
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http://dx.doi.org/10.1152/ajpendo.00522.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580170PMC
May 2019

-GlcNAcylation alters the selection of mRNAs for translation and promotes 4E-BP1-dependent mitochondrial dysfunction in the retina.

J Biol Chem 2019 04 7;294(14):5508-5520. Epub 2019 Feb 7.

From the Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033,

Diabetes promotes the posttranslational modification of proteins by linked addition of GlcNAc (GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the GlcNAc hydrolase GlcNAcase, exhibited enhanced retinal protein GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs ( mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.
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http://dx.doi.org/10.1074/jbc.RA119.007494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462503PMC
April 2019

Palmitate- and C6 ceramide-induced pre-mRNA alternative splicing occurs in a PP2A dependent manner.

Nutr Metab (Lond) 2018 17;15:87. Epub 2018 Dec 17.

1Department of Cellular and Molecular Physiology, Penn State College of Medicine, H166, 500 University Drive, Hershey, PA 17033 USA.

Background: In a previous study, we showed that consumption of diets enriched in saturated fatty acids causes changes in alternative splicing of pre-mRNAs encoding a number of proteins in rat skeletal muscle, including the one encoding skeletal muscle Troponin T (). However, whether saturated fatty acids act directly on muscle cells to modulate alternative pre-mRNA splicing was not assessed. Moreover, the signaling pathway through which saturated fatty acids act to promote changes in alternative splicing is unknown. Therefore, the objective of the present study was to characterize the signaling pathway through which saturated fatty acids act to modulate alternative splicing.

Methods: The effects of treatment of L6 myotubes with saturated (palmitate), mono- (oleate), or polyunsaturated (linoleate) fatty acids on alternative splicing of pre-mRNA was assessed using as a marker gene.

Results: Palmitate treatment caused a two-fold change ( < 0.05) in L6 myotube alternative splicing whereas treatment with either oleate or linoleate had minimal effects compared to control myotubes. Treatment with a downstream metabolite of palmitate, ceramide, had effects similar to palmitate on alternative splicing and inhibition of de novo ceramide biosynthesis blocked the palmitate-induced alternative splicing changes. The effects of palmitate and ceramide on alternative splicing were accompanied by a 40-50% reduction in phosphorylation of Akt on S473. However, inhibition of de novo ceramide biosynthesis did not prevent palmitate-induced Akt dephosphorylation, suggesting that palmitate may act in an Akt-independent manner to modulate alternative splicing. Instead, pre-treatment with okadaic acid at concentrations that selectively inhibit protein phosphatase 2A (PP2A) blocked both palmitate- and ceramide-induced changes in alternative splicing, suggesting that palmitate and ceramide act through PP2A to modulate alternative splicing.

Conclusions: Overall, the data show that fatty acid saturation level and ceramides are important factors modulating alternative pre-mRNA splicing through activation of PP2A.
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http://dx.doi.org/10.1186/s12986-018-0326-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6296074PMC
December 2018

Repressors of mTORC1 act to blunt the anabolic response to feeding in the soleus muscle of a cast-immobilized mouse hindlimb.

Physiol Rep 2018 10;6(20):e13891

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania.

We recently reported results showing that cast immobilization of a rat hindlimb rapidly leads to development of anabolic resistance as demonstrated by failure of oral leucine administration to activate the mechanistic target of rapamycin complex 1 (mTORC1) and stimulate protein synthesis in the soleus muscle. The goal of this study was to assess the possible contribution of several mTORC1 regulatory proteins to the development of anabolic resistance. To accomplish this, 14-week-old male C57BL/6 mice (n = 21) were subjected to unilateral cast immobilization of the hindlimb for either 1 or 3 days, and the immobilized limb was compared to its contralateral control. The mass of the soleus muscle was decreased in the immobilized compared to the non-immobilized limb within 72-h in association with diminished protein synthesis. In agreement with our previous report, a 24-h casting period was sufficient to induce anabolic resistance, as demonstrated by blunted re-feeding-induced activation of mTORC1. Moreover, resistance of mTORC1 activation was associated not only with upregulated expression of REDD1, but also with altered expression of other mTORC1 regulatory proteins, that is, Sestrin1 and DEP domain-containing mTOR interacting protein (DEPTOR). In addition, re-feeding-induced phosphorylation of DEPTOR was significantly impaired in the immobilized compared to the non-immobilized limb. This work builds upon previous discoveries by our laboratory to elucidate the blunted mTORC1 response to stimuli during disuse of skeletal muscle induced by cast immobilization while highlighting new potential therapeutic targets for future countermeasures against muscle atrophy.
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http://dx.doi.org/10.14814/phy2.13891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194210PMC
October 2018

Intermittent bolus feeding promotes greater lean growth than continuous feeding in a neonatal piglet model.

Am J Clin Nutr 2018 10;108(4):830-841

USDA-Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX.

Background: Orogastric tube feeding is indicated in neonates with an impaired ability to ingest food normally and can be administered with an intermittent bolus or continuous feeding schedule.

Objectives: The objectives were to 1) compare the long-term effect of continuous with intermittent feeding on growth using the newborn pig as a model, 2) determine whether feeding frequency alters lean tissue and fat mass gain, and 3) identify the signaling mechanisms by which protein deposition is controlled in skeletal muscle in response to feeding frequency.

Design: Neonatal pigs were fed the same amount of a balanced formula by orogastric tube either as an intermittent bolus meal every 4 h (INT) or as a continuous infusion (CON). Body composition was assessed at the start and end of the study by dual-energy X-ray absorptiometry, and hormone and substrate profiles, muscle mass, protein synthesis, and indexes of nutrient and insulin signaling were measured after 21 d.

Results: Body weight, lean mass, spine length, and skeletal muscle mass were greater in the INT group than in the CON group. Skeletal muscle fractional protein synthesis rates were greater in the INT group after a meal than in the CON group and were associated with higher circulating branched-chain amino acid and insulin concentrations. Skeletal muscle protein kinase B (PKB) and ribosomal protein S6 kinase phosphorylation and eukaryotic initiation factor (eIF) 4E-eIF4G complex formation were higher, whereas eIF2α phosphorylation was lower in the INT group than in the CON group, indicating enhanced activation of insulin and amino acid signaling to translation initiation.

Conclusions: These results suggest that when neonates are fed the same amounts of nutrients as intermittent meals rather than continuously there is greater lean growth. This response can be ascribed, in part, to the pulsatile pattern of amino acids, insulin, or both induced by INT, which enables the responsiveness of anabolic pathways to feeding to be sustained chronically in skeletal muscle.
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http://dx.doi.org/10.1093/ajcn/nqy133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186212PMC
October 2018

Mechanisms Underlying Muscle Protein Imbalance Induced by Alcohol.

Annu Rev Nutr 2018 08;38:197-217

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA; email: ,

Both acute intoxication and longer-term cumulative ingestion of alcohol negatively impact the metabolic phenotype of both skeletal and cardiac muscle, independent of overt protein calorie malnutrition, resulting in loss of skeletal muscle strength and cardiac contractility. In large part, these alcohol-induced changes are mediated by a decrease in protein synthesis that in turn is governed by impaired activity of a protein kinase, the mechanistic target of rapamycin (mTOR). Herein, we summarize recent advances in understanding mTOR signal transduction, similarities and differences between the effects of alcohol on this central metabolic controller in skeletal muscle and in the heart, and the effects of acute versus chronic alcohol intake. While alcohol-induced alterations in global proteolysis via activation of the ubiquitin-proteasome pathway are equivocal, emerging data suggest alcohol increases autophagy in muscle. Further studies are necessary to define the relative contributions of these bidirectional changes in protein synthesis and autophagy in the etiology of alcoholic myopathy in skeletal muscle and the heart.
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http://dx.doi.org/10.1146/annurev-nutr-071816-064642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377942PMC
August 2018

Deletion of the stress-response protein REDD1 promotes ceramide-induced retinal cell death and JNK activation.

FASEB J 2018 Jun 19:fj201800413RR. Epub 2018 Jun 19.

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania, USA.

The role of dyslipidemia in the development of retinal dysfunction remains poorly understood. Using an animal model of diet-induced obesity/pre-type 2 diabetes, we investigated molecular defects in the retina arising from consumption of a diet high in saturated fats and sugars ( i.e., a Western diet). We found that feeding mice a Western diet increased the abundance of retinal sphingolipids, attenuated protein kinase B (Akt) phosphorylation, enhanced JNK activation, and increased retinal cell death. When we used palmitate or C6-ceramide (Cer) to assess sphingolipid-mediated signaling in cultured murine and human cells, we observed similar effects on Akt, JNK, and cell death. Furthermore, both Western diet and C6-Cer exposure enhanced expression of the stress-response protein regulated in development and DNA damage response 1 (REDD1) and loss of REDD1 increased C6-Cer-induced JNK activation and cell death. Exogenous REDD1 expression repressed JNK-mediated phosphorylation in cultured cells. We found that thioredoxin-interacting protein (TXNIP) expression was elevated in REDD1-deficient cell lines and C6-Cer promoted TXNIP expression in both wild-type and REDD1-deficient cells. Likewise, TXNIP knockdown attenuated JNK activation and caspase 3 cleavage after either C6-Cer exposure or REDD1 deletion. The results support a model wherein Cer-induced REDD1 expression attenuates TXNIP-dependent JNK activation and retinal cell death.-Dai, W., Miller, W. P., Toro, A. L., Black, A. J., Dierschke, S. K., Feehan, R. P., Kimball, S. R., Dennis, M. D. Deletion of the stress-response protein REDD1 promotes ceramide-induced retinal cell death and JNK activation.
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http://dx.doi.org/10.1096/fj.201800413RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219834PMC
June 2018

A Unique ISR Program Determines Cellular Responses to Chronic Stress.

Mol Cell 2017 Dec;68(5):885-900.e6

Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address:

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.
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http://dx.doi.org/10.1016/j.molcel.2017.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730339PMC
December 2017

Leucine-Induced Upregulation of Terminal Oligopyrimidine mRNA Translation in Skeletal Muscle: Just the Tip of the Iceberg?

Authors:
Scot R Kimball

J Nutr 2017 09 2;147(9):1603-1604. Epub 2017 Aug 2.

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA

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http://dx.doi.org/10.3945/jn.117.256289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572500PMC
September 2017

Dietary Fat Quantity and Type Induce Transcriptome-Wide Effects on Alternative Splicing of Pre-mRNA in Rat Skeletal Muscle.

J Nutr 2017 09 2;147(9):1648-1657. Epub 2017 Aug 2.

Departments of Entomology and Biology, Penn State University, University Park, State College, PA

Fat-enriched diets produce metabolic changes in skeletal muscle, which in turn can mediate changes in gene regulation. We examined the high-fat-diet-induced changes in skeletal muscle gene expression by characterizing variations in pre-mRNA alternative splicing. Affymetrix Exon Array analysis was performed on the transcriptome of the gastrocnemius/plantaris complex of male obesity-prone Sprague-Dawley rats fed a 10% or 60% fat (lard) diet for 2 or 8 wk. The validation of exon array results was focused on troponin T (). splice form analyses were extended in studies of rats fed 10% or 30% fat diets across 1- to 8-wk treatment periods and rats fed 10% or 45% fat diets with fat sources from lard or mono- or polyunsaturated fats for 2 wk. Nuclear magnetic resonance (NMR) was used to measure body composition. Consumption of a 60% fat diet for 2 or 8 wk resulted in alternative splicing of 668 and 726 pre-mRNAs, respectively, compared with rats fed a 10% fat diet. transcripts were alternatively spliced in rats fed a 60% fat diet for either 2 or 8 wk. The high-fat-diet-induced changes in alternative splicing were observed in rats fed a 30% fat diet across 1- to 8-wk treatment periods. Moreover, this effect depended on fat type, because alternative splicing occurred in response to 45% fat diets enriched with lard but not in response to diets enriched with mono- or polyunsaturated fatty acids. Fat mass (a proxy for obesity as measured by NMR) did not differ between groups in any study. Rat skeletal muscle responds to overconsumption of dietary fat by modifying gene expression through pre-mRNA alternative splicing. Variations in alternative splicing occur independently of obesity and are dependent on dietary fat quantity and suggest a role for saturated fatty acids in the high-fat-diet-induced modifications in alternative splicing.
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http://dx.doi.org/10.3945/jn.117.254482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572497PMC
September 2017

Dietary Methionine Restriction Regulates Liver Protein Synthesis and Gene Expression Independently of Eukaryotic Initiation Factor 2 Phosphorylation in Mice.

J Nutr 2017 06 26;147(6):1031-1040. Epub 2017 Apr 26.

Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ;

The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR). We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance. Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 () or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase () alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA ( < 0.05). Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in mice. Finally, feeding the MR diet to mice with deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls. Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. status influences body fat loss but not protein balance when Met is restricted.
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http://dx.doi.org/10.3945/jn.116.246710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443467PMC
June 2017

Regulation of protein and mRNA expression of the mTORC1 repressor REDD1 in response to leucine and serum.

Biochem Biophys Rep 2016 Dec 8;8:296-301. Epub 2016 Oct 8.

Intercollege Graduate Degree Program in Physiology, Penn State College of Medicine, Hershey, PA; Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA.

Expression of the mTORC1 repressor, Regulated in DNA Damage and Development 1 (REDD1), is elevated in skeletal muscle during various catabolic conditions including fasting, hindlimb immobilization, and sepsis. Conversely, REDD1 expression is suppressed by anabolic stimuli such as resistance exercise or nutrient consumption following a fast. Though it is known that nutrient consumption reduces REDD1 expression, it is largely unknown how nutrients and hormones individually contribute to the reduction in REDD1 expression. Therefore, the purpose of the present study was to determine how nutrients and hormones individually regulate REDD1 expression. HeLa cells were deprived of leucine or serum for 10 hours, after which either leucine or serum was reintroduced to cell culture medium for 60 minutes. Re-supplementation of either leucine or serum resulted in a reduction in REDD1 protein levels by 34.8 ± 5.8% and 54.1 ± 3.4%, respectively, compared to the deprived conditions. Re-supplementation of leucine or serum to deprived cells also led to a reduction in REDD1 mRNA content by 49.1% ± 2.7% and 65.0 ± 1.4%, respectively, compared to the deprived conditions. Interestingly, rates of REDD1 protein degradation were unaffected by either leucine or serum re-supplementation, as assessed in cells treated with cycloheximide to block protein synthesis. Likewise, addition of leucine or serum to cells treated with Actinomycin D to inhibit gene transcription failed to alter the rate of REDD1 mRNA degradation. The data indicate that the leucine or serum-induced suppression of REDD1 expression occurs independent of changes in the rate of degradation of either the REDD1 protein or mRNA. Thus, the leucine- or serum-induced suppression likely occurs through alternative mechanism(s) such as reduced REDD1 gene transcription and/or mRNA translation.
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http://dx.doi.org/10.1016/j.bbrep.2016.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5370564PMC
December 2016

Activation of the Stress Response Kinase JNK (c-Jun N-terminal Kinase) Attenuates Insulin Action in Retina through a p70S6K1-dependent Mechanism.

J Biol Chem 2017 02 13;292(5):1591-1602. Epub 2016 Dec 13.

From the Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033. Electronic address:

Despite recent advances in therapeutics, diabetic retinopathy remains a leading cause of vision impairment. Improvement in the treatment of diabetic retinopathy requires a better understanding of the molecular mechanisms that cause neurovascular complications, particularly in type 2 diabetes. Recent studies demonstrate that rodents fed a high fat diet exhibit retinal dysfunction concomitant with attenuated Akt phosphorylation. The purpose of the present study was to evaluate the impact of a high fat/high sucrose diet on retinal insulin signaling and evaluate the mechanism(s) responsible for the changes. Mice fed a high fat/sucrose diet exhibited attenuated Akt phosphorylation in the retina as compared with mice fed normal chow. Retinas of mice fed a high fat/sucrose diet also exhibited elevated levels of activated JNK as well as enhanced p70S6K1 autoinhibitory domain phosphorylation. In cells, JNK activation enhanced p70S6K1 phosphorylation and mTORC1-dependent activation of the kinase, as evidenced by enhanced phosphorylation of key substrates. Rictor phosphorylation by p70S6K1 was specifically enhanced by the addition of phosphomimetic mutations in the autoinhibitory domain and was more sensitive to inhibition of the kinase as compared with rpS6. Notably, rictor and IRS-1 phosphorylation by p70S6K1 attenuate insulin action through a negative feedback pathway. Indeed, p70S6K1 inhibition prevented the repressive effect of JNK activation on insulin action in retinas. Overall, the results identify the JNK/S6K1 axis as a key molecular mechanism whereby a high fat/sucrose diet impairs insulin action in retina.
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http://dx.doi.org/10.1074/jbc.M116.760868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290937PMC
February 2017

Loss of REDD1 augments the rate of the overload-induced increase in muscle mass.

Am J Physiol Regul Integr Comp Physiol 2016 09 27;311(3):R545-57. Epub 2016 Jul 27.

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania.

The overload-induced increase in muscle mass is accompanied by protein accretion; however, the initiating events are poorly understood. Regulated in Development and DNA Damage 1 (REDD1), a repressor of the mechanistic target of rapamycin in complex 1 (mTORC1), blunts the elevation in protein synthesis induced by acute muscle contractions. Therefore, this study was designed to determine whether REDD1 alters the rate of the overload-induced increase in muscle mass. Wild-type (WT) and REDD1-null mice underwent unilateral functional overload (OV) of the plantaris, while the contralateral sham leg served as a control. After 3 and 5 days of OV, puromycin incorporation was used as a measurement of protein synthesis. The percent increase in plantaris wet weight and protein content was greater in REDD1-null mice after 3, 5, and 10 days OV. The overload-stimulated rate of protein synthesis in the plantaris was similar between genotypes after 3 days OV, but translational capacity was lower in REDD1-null mice, indicating elevated translational efficiency. This was likely due to elevated absolute mTORC1 signaling [phosphorylation of p70S6K1 (Thr-389) and 4E-BP1 (Ser-65)]. By 5 days of OV, the rate of protein synthesis in REDD1-null mice was lower than WT mice with no difference in absolute mTORC1 signaling. Additionally, markers of autophagy (LC3II/I ratio and p62 protein) were decreased to a greater absolute extent after 3 days OV in REDD1-null mice. These data suggest that loss of REDD1 augments the rate of the OV-induced increase in muscle mass by altering multiple protein balance pathways.
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http://dx.doi.org/10.1152/ajpregu.00159.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142228PMC
September 2016

Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

F1000Res 2016 1;5:250. Epub 2016 Mar 1.

Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA, USA.

REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1 (-/-) mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin.
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http://dx.doi.org/10.12688/f1000research.7691.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893971PMC
June 2016

Emerging role for regulated in development and DNA damage 1 (REDD1) in the regulation of skeletal muscle metabolism.

Am J Physiol Endocrinol Metab 2016 Jul 17;311(1):E157-74. Epub 2016 May 17.

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; and.

Since its discovery, the protein regulated in development and DNA damage 1 (REDD1) has been implicated in the cellular response to various stressors. Most notably, its role as a repressor of signaling through the central metabolic regulator, the mechanistic target of rapamycin in complex 1 (mTORC1) has gained considerable attention. Not surprisingly, changes in REDD1 mRNA and protein have been observed in skeletal muscle under various physiological conditions (e.g., nutrient consumption and resistance exercise) and pathological conditions (e.g., sepsis, alcoholism, diabetes, obesity) suggesting a role for REDD1 in regulating mTORC1-dependent skeletal muscle protein metabolism. Our understanding of the causative role of REDD1 in skeletal muscle metabolism is increasing mostly due to the availability of genetically modified mice in which the REDD1 gene is disrupted. Results from such studies provide support for an important role for REDD1 in the regulation of mTORC1 as well as reveal unexplored functions of this protein in relation to other aspects of skeletal muscle metabolism. The goal of this work is to provide a comprehensive review of the role of REDD1 (and its paralog REDD2) in skeletal muscle during both physiological and pathological conditions.
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http://dx.doi.org/10.1152/ajpendo.00059.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967146PMC
July 2016

Leucine induced dephosphorylation of Sestrin2 promotes mTORC1 activation.

Cell Signal 2016 08 21;28(8):896-906. Epub 2016 Mar 21.

The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.

The studies described herein were designed to explore the role of Sestrin2 in mediating the selective action of leucine to activate mTORC1. The results demonstrate that Sestrin2 is a phosphoprotein and that its phosphorylation state is responsive to the availability of leucine, but not other essential amino acids. Moreover, leucine availability-induced alterations in Sestrin2 phosphorylation correlated temporally and dose dependently with the activation state of mTORC1, there being a reciprocal relationship between the degree of phosphorylation of Sestrin2 and the extent of repression of mTORC1. With leucine deprivation, Sestrin2 became more highly phosphorylated and interacted more strongly with proteins of the GATOR2 complex. Notably, in cells lacking the protein kinase ULK1, the activation state of mTORC1 was elevated in leucine-deficient medium, such that the effect of re-addition of the amino acid was blunted. In contrast, overexpression of ULK1 led to hyperphosphorylation of Sestrin2 and enhanced its interaction with GATOR2. Neither rapamycin nor Torin2 had any effect on Sestrin2 phosphorylation, suggesting that leucine deprivation-induced repression of mTORC1 was not responsible for the action of ULK1 on Sestrin2. Mass spectrometry analysis of Sestrin2 revealed three phosphorylation sites that are conserved across mammalian species. Mutation of the three sites to phospho-mimetic amino acids in exogenously expressed Sestrin2 promoted its interaction with GATOR2 and dramatically repressed mTORC1 even in the presence of leucine. Overall, the results support a model in which leucine selectively promotes dephosphorylation of Sestrin2, causing it to dissociate from and thereby activate GATOR2, leading to activation of mTORC1.
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http://dx.doi.org/10.1016/j.cellsig.2016.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899281PMC
August 2016
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