In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.
References
[1]
Wu M, Falasca M, Blough ER (2010) Akt/Protein kinase B in skeletal muscle physiology and pathology. J Cell Physiol 226: 29–36.
[2]
Glass DJ (2010) Signaling pathways perturbing muscle mass. Curr Opin Clin Nutr Metab Care 13: 225–229.
[3]
Medeiros C, Frederico MJ, da Luz G, Pauli JR, Silva AS, et al. (2011) Exercise training reduces insulin resistance and upregulates the mTOR/p70S6k pathway in cardiac muscle of diet-induced obesity rats. J Cell Physiol 226: 666–674.
[4]
Whiting DR, Guariguata L, Weil C, Shaw J (2011) IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract 94: 311–321.
[5]
Newsholme P, Abdulkader F, Rebelato E, Romanatto T, Pinheiro CH, et al. (2011) Amino acids and diabetes: implications for endocrine, metabolic and immune function. Front Biosci 1: 315–339.
[6]
Wolfe RR (2002) Regulation of muscle protein by amino acids. J Nutr 132: 3219S–3224S.
[7]
Pallafacchina G, Calabria E, Serrano AL, Kalhovde JM, Schiaffino S (2001) A protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type specification. Proc Natl Acad Sci USA 99: 9213–9218.
[8]
Ohanna M, Sobering AK, Lapointe T, Lorenzo L, Praud C, et al. (2005) Atrophy of S6K1(？/？) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control. Nat Cell Biol 7: 286–294.
[9]
Hara K, Yonezawa K, Kozlowski MT, Sugimoto T, Andrabi K, et al. (1997) Regulation of eIF-4E BP1 phosphorylation by mTOR. J Biol Chem 272: 26457–26463.
[10]
Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, et al. (2001) Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704–1708.
[11]
Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL (2001) Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci U S A 98: 14440–14445.
[12]
Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, et al. (2004) Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117: 399–412.
[13]
Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, et al. (2004) The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 14: 395–403.
[14]
Greer EL, Brunet A (2005) FOXO transcription factors at the interface between longevity and tumor supression. Oncogene 24: 7410–7425.
[15]
Latres E, Amini AR, Amini AA, Griffiths J, Martin FJ, et al. (2005) Insulin-like growth factor-1 (IGF-1) inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. J Biol Chem 280: 2737–2744.
[16]
Nicklin P, Bergman P, Zhang B, Triantafellow , Wang H, et al. (2009) Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136: 521–534.
[17]
Curi R, Lagranha CJ, Doi SQ, Sellitti DF, Procopio J, et al. (2005) Molecular mechanisms of glutamine action. J Cell Physiol 204: 392–401 Review.
[18]
Pithon-Curi TC, Schumacher RI, Freitas JJ, Lagranha C, Newsholme P, et al. (2003) Glutamine delays spontaneous apoptosis in neutrophils. Am J Physiol Cell Physiol 284: 1355–1361.
[19]
Xi P, Jiang Z, Zheng C, Lin Y, Wu G (2011) Regulation of protein metabolism by glutamine: implications for nutrition and health. Front Biosci 1: 578–597.
[20]
Lagranha CJ, Senna SM, de Lima TM, Silva EP, Doi SQ, et al. (2004) Beneficial effect of glutamine on exercise-induced apoptosis of rat neutrophils. Med Sci Sports Exerc 36: 210–217.
[21]
Windmueller HG, Spaeth AE (1974) Uptake and metabolism of plasma glutamine by the small intestine. J Biol Chem 249: 5070–5079.
[22]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
[23]
Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y) 10: 413–417.
[24]
Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8: R19.
[25]
Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56: 2907–2914.
[26]
Peters SJ, van Helvoort A, Kegler D, Argilès JM, Luiking YC, et al. (2011) Dose-dependent effects of leucine supplementation on preservation of muscle mass in cancer cachectic mice. Oncol Rep 26: 247–254.
[27]
Curi R, Newsholme P, Procopio J, Lagranha C, Gorj？o R, et al. (2007) Glutamine, gene expression, and cell function. Front Biosci 1 12: 344–357.
[28]
Wijekoon EP, Skinner C, Brosnan ME, Brosnan JT (2004) Amino acid metabolism in the Zucker diabetic fatty rat: effects of insulin resistance and of type 2 diabetes. Can J Physiol Pharmacol 82: 506–514.
[29]
Sugita H, Kaneki M, Sugita M, Yasukawa T, Yasuhara S, et al. (2005) Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle. Am J Physiol Endocrinol Metab 288: E585–591.
[30]
Saha AK, Xu XJ, Lawson E, Deoliveira R, Brandon AE, et al. (2010) Downregulation of AMPK accompanies leucine- and glucose-induced increases in protein synthesis and insulin resistance in rat skeletal muscle. Diabetes 59: 2426–2434.
[31]
Vary TC (2007) Acute oral leucine administration stimulates protein synthesis during chronic sepsis through enhanced association of eukaryotic initiation factor 4G with eukaryotic initiation factor 4E in rats. J Nutr 137: 2074–2079.
[32]
O'Neill ED, Wilding JP, Kahn CR, Van Remmen H, McArdle A, et al. (2010) Absence of insulin signalling in skeletal muscle is associated with reduced muscle mass and function: evidence for decreased protein synthesis and not increased degradation. Age 32: 209–222.
[33]
Baptista IL, Leal ML, Artoli GG, Aoki MS, Fiamoncini J, et al. (2010) Leucine attenuates skeletal muscle wasting via inhibition of ubiquitin ligases. Muscle Nerve 41: 800–808.
Kim J, Guan KL (2011) Amino acid signaling in TOR activation. Annu Rev Biochem 80: 1001–1032.
[36]
Jope RS, Johnson GV (2004) The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 2: 95–102.
[37]
Dokken BB, Sloniger JA, Henriksen EJ (2005) Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle. Am J Physiol Endocrinol Metab 288: 1188–1194.
[38]
Semiz S, Orvig C, McNeill JH (2002) Effects of diabetes, vanadium, and insulin on glycogen synthase activation in Wistar rats. Mol Cell Biochem 231: 23–35.
[39]
Kimball SR, Jefferson LS (2004) Amino acids as regulators of gene expression. Nutr Metab (Lond) 1: 3.
[40]
Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, et al. (2005) Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OHkinase. Proc Natl Acad Sci U S A 102: 14238–14243.
