Original Report: Laboratory Investigation American

Journal of

Nephrology

Am J Nephrol 2014;40:362–370 DOI: 10.1159/000366524

Received: May 20, 2014 Accepted: August 5, 2014 Published online: October 29, 2014

Acute Acidosis Attenuates Leucine Stimulated Signal Transduction and Protein Synthesis in Rat Skeletal Muscle Sumita Sood a, b Yu Chen a, b Kevin McIntire a, b Ralph Rabkin a, b a

Research Service, Veterans Affairs Health Care Palo Alto, Palo Alto, Calif., and b Medicine Department Nephrology Division, Stanford University, Stanford, Calif., USA

Abstract Background: Critical illnesses are often complicated by acute metabolic acidosis, which if persistent, adversely affects outcome. Among the harmful effects that it might cause are impaired utilization of nutrients, increased proteolysis and depressed protein synthesis, leading to muscle wasting. As the amino acid leucine stimulates protein synthesis by activating mTOR signaling, we explored whether in acidosis, impaired leucine-stimulated signaling might be a contributor to the depressed protein synthesis. Methods: Male pair-fed rats were gavaged with NH4Cl (acidosis) or NaCl (control) for 2 days and then gavaged once with leucine and sacrificed 45 min later. Extensor digitorum longus muscles were isolated, incubated with or without leucine and protein synthesis measured. The anterior tibial muscle signaling was analysed by Western immunobloting. Results: Despite pair-feeding, acidotic rats lost body and muscle weight vs. controls. Moreover, leucine-induced protein synthesis in isolated muscle from acidotic rats was impaired. Invivo, 45 min after an oral leucine load, anterior tibial muscle mTOR and 4E-BP1 phosphorylation increased significantly

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and comparably in control and acidotic rats. In contrast, leucine-stimulated phosphorylation of S6K1, a regulator of translation initiation and protein synthesis, was attenuated to approximately 56% of the control value (p < 0.05). Conclusion: This study reveals that an acute metabolic acidosis impairs leucine-stimulated protein synthesis and activation of signaling downstream of mTOR at the level of S6K1. We propose that this S6K1abnormality may account in part, for the resistance to leucine-stimulated muscle protein synthesis, and may thereby contribute to the impaired nutrient utilization and ultimately the muscle wasting that develops in aci© 2014 S. Karger AG, Basel dosis.

Introduction

Critical illnesses, such as acute kidney injury (AKI), are frequently complicated by an acute metabolic acidosis that if persistent, heralds an increase in morbidity and mortality [1–3]. However, it is often difficult to distinguish whether the adverse events arise from acidosis per se or from the underlying cause of illness. When acidosis is prolonged as in chronic kidney disease (CKD), muscle wasting can become troublesome, bone disease aggravat-

S.S. and Y.C. contributed equally to this work.

Ralph Rabkin, MD VAPAHCS (111R) 3801 Miranda Avenue Palo Alto, CA 94304 (USA) E-Mail rabkin @ stanford.edu

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Key Words Branched chain amino acids · Leucine · Mammalian target of rapamycin · 70-kDa ribosomal protein S6 kinase 1 (S6K1) · Metabolic acidosis · Protein synthesis · Skeletal muscle

Acidosis Attenuates Leucine Mediated mTOR Signaling

nating in depressed PS [20, 27, 28]. Interestingly, the defects in signal transduction are not uniform. For example, we reported that in rats with moderate non-acidotic CKD, there is partial resistance to leucine as manifested by impaired phosphorylation of 4E-BP1 [26], while in severe acidotic AKI there is complete resistance to leucinestimulated mTOR, 4E-BP1 and S6K1 phosphorylation, and failure to suppress LC3B-II protein levels, a marker of autophagy, despite an increase in signaling protein levels [25]. Whether the difference between CKD and AKI is attributable to the acute acidosis is unclear, for there is no information on the impact of acidosis on leucine-stimulated signaling, though acidosis does suppress insulin and IGF-1 signaling [1, 11, 12]. Accordingly, we tested the hypothesis that acidosis alone attenuates leucine-induced mTOR signal transduction and PS.

Materials and Methods Experimental Animals and Protocols Male Sprague-Dawley rats weighing 165–180 g were gavaged with NH4Cl (20 mmol/kg b.w. in water) twice daily to induce acidosis as before [29]. Control pair-fed rats were gavaged with water containing equimolar NaCl. Forty five hours after the first gavage and following an overnight fast, the animals were euthanised under pentobarbital anesthesia (80 mg/kg b.w. intraperitoneally). After two hours, pre-euthanasia rats received a final dose of NH4Cl or NaCl and 45 min pre-euthanasia; they were gavaged with leucine (1.35 g/kg b.w.) in water, or an equal volume of saline (Sal). Four groups were studied: Acidotic-Sal; AcidoticLeu; Control-Sal; and Control-Leu. At the time of euthanasia, arterial blood was collected and analysed for pH and acid-base balance (i-STAT® System, Abbott Inc., NJ, USA) and BCAA levels. Extensor digitorum longus (EDL) muscles were collected for in-vitro study of PS, and a muscle of similar fibre composition, namely anterior tibial (AT) [30], was collected and frozen and assayed for signaling later. Animal protocols were approved by the local Institutional Animal Care and Use Committee according to the National guidelines. Western Blotting Antibodies detecting mTOR, phospho-mTOR (Ser2448), 4E-BP1, phospho-4E-BP1 (Thr70 ), p70S6K, phospho-p70S6K1 (Thr389), rpS6, phospho-rpS6 (Ser235/236), phospho-Akt (Thr308), GAPDH and LC3B were from Cell Signaling Technology (Danvers, Mass., USA) and Akt from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Exactly as before [25], AT muscle lysates were subjected to Western imunoblot analysis after electrophoresis in a sodium dodecyl sulphate-polyacrylamide gel. First, specific phosphoproteins were detected; then after stripping, the specific protein and finally the loading control, GAPDH. For illustrative purposes, pooled samples were run in the same gel; lanes from the same gel were cut and outlined. Phosphoprotein and protein signals from each rat sample were scanned and quantitated with ImageJ software (NIH, Bethesda, Md., USA).

Am J Nephrol 2014;40:362–370 DOI: 10.1159/000366524

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ed and growth impairment worsened. Furthermore, acidosis may accelerate CKD progression [1]. Acidosis-induced muscle wasting arises largely because of increased proteolysis by the ubiquitin proteasome system [4, 5], though suppressed protein synthesis (PS) may also contribute [6–8]. Other processes activated by acidosis that cause wasting include anorexia, decreased food utilization, increased branched chain amino acid (BCAA) oxidation [9], impaired amino acid (AA) transport into cells [10], increased glucocorticoid production and resistance to and/or deficiencies of anabolic hormones [1, 11, 12]. Correction of acid/base imbalance and nutritional support are important when managing critical illness because it may provide some protection against loss of protein stores. However, as nutrient utilization is commonly impaired in critical illnesses, the impact of nutritional supplements on outcome remains to be established [13]. Nutrients of particular interest are BCAAs, which comprise >20% of total dietary protein [14]. BCAAs are actively transported into myocytes by the system A and L membrane localised transporters, including the pH sensitive sodium-coupled neutral amino-acid transporter SNAT2, that sense extracellular AA availability and regulate free intracellular AA levels [15]. Once in the cell, in addition to functioning as substrates, BCAAs, especially leucine, activate the multiprotein mammalian target of rapamycin complex 1 (mTOR) pathway in an PI3-kinase/Akt independent manner [16]. The mTOR complex 1 is a serine and threonine kinase that regulates protein translation via the phosphorylation of the 70-kDa ribosomal protein S6 kinase 1 (S6K1) and along a parallel path, the eukaryotic translation initiation factor (eIF4e) 4E-binding protein1 (4EBP1) [15, 17–19]. Phosphorylation of the translational repressor 4E-BP1, leads to its release from eIF4e, necessary for mRNA translation activation. Phosphorylation of S6K1, a regulator of translation initiation, activates S6K1 that in turn phosphorylates several proteins including 40S ribosomal protein S6 (rpS6), and PS increases [18–20]. In addition to stimulating PS, activated mTOR suppresses proteolysis by depressing autophagy [21] and possibly the ubiquitin-proteasomal system [22, 23]. Leucine also stimulates insulin secretion, which may activate the PI3-kinase/Akt pathway and in turn mTOR signaling,  thereby contributing to leucine’s anabolic actions [24]. Unfortunately, leucine resistance develops in several catabolic states including AKI [25], CKD [26], sepsis, HIV and cancer, and is manifested by impaired leucine activation of mTOR and its downstream targets culmi-

Plasma Branched-Chain Amino Acids BCAAs were assayed using a BCAA ELISA kit (BioVision Research Products; Mountain View, Calif., USA). Because animals were gavaged with saline or leucine only, the difference in plasma BCAA levels between these groups is assumed to be due to gavaged leucine [26]. In-vitro Protein Synthesis After an overnight fast, PS was assayed in paired left and right EDL muscles from each pentobarbital anesthetised, saline gavaged, acidotic and control rat [31]. Muscles from acidotic rats were incubated at 37 ° C in Krebs-Ringers buffer, pH 7.15, containing 10 mM-glucose, 1 mM CaCl2, 5 mM HEPES and other additions as indicated. Muscles from control rats were incubated in the same solution at pH 7.4. Immediately before incubation, medium was gassed with O2/CO2 (19:1). After 30-min equilibration, incubated muscles were transferred to stoppered flasks containing fresh medium supplemented with 1 mM L-[14C(U)]-Phenylalanine 0.20 uCi/ml and 500 uM/l leucine (left muscle) or vehicle (right muscle), regassed, and incubated for 2 h with gassing every 30 min. The collected muscle was homogenised in 10% trichloroacetic acid, the precipitate solubilised in 1 N NaOH and radioactivity measured. PS was taken as the rate of incorporation of [U-14C] phenylalanine into muscle divided by the specific radioactivity of phenylalanine in the medium and expressed as nmol phenylalanine/g muscle/120 min.

Blood pH Blood bicarbonate, mmol/l Initial body weight, g Body weight change, g Extensor digitorum longus w.t., mg Plantaris w.t., mg

Control

Acidotic

7.36±0.00 26.2±0.91 129±4 –1±1 45±1 224±7

6.93±0.04* 10.7±1.23* 127±4 –16±2* 41±1* 198±6*

 

Data Analysis and Statistics To eliminate errors inherent when comparing results from different Western blots, samples from any two groups to be compared were assayed in the same gel. To control for variability between blots, an internal control sample prepared from a pool of saline-treated controls and run in duplicate, was included in the assays and results normalised to this sample. Furthermore, pooled samples from each of the 4 groups were assayed in the same gel for confirmation. Relative phosphorylated protein levels were calculated by normalizing for the respective specific total protein level. The control-Sal group mean was valued at 100 and individual values were expressed relative to this value. Results are given as mean ± SEM and differences