Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells

Abstract

α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.

Functional amino acids in nutrition and health

“…Based on the foregoing lines of compelling evidence from animal and human studies, Wu (2010) proposed the new concept of functional AA, which are defined as those AA that participate in and regulate key metabolic pathways to improve health, survival, growth, development, lactation, and reproduction of the organisms. Metabolic pathways include: (1) intracellular protein turnover (synthesis and degradation) and associated events (Bertrand et al 2012;Kong et al 2012;Wauson et al 2013;Xi et al 2011Xi et al , 2012Yao et al 2012), (2) AA synthesis and catabolism (Brosnan and Brosnan 2012;Lei et al 2012a, b), (3) generation of small peptides, nitrogenous metabolites, and sulfur-containing substances [e.g., H 2 S (Mimoun et al 2012)], (4) urea cycle and uric acid synthesis (Wu 2013), (5) lipid and glucose metabolism Go et al 2012;Satterfield et al 2011Satterfield et al , 2012, (6) one-carbon unit metabolism (Wang et al 2012), and (7) cellular redox signaling (Hou et al 2012a). Functional AA can be nutritionally ''essential'', ''nonessential'', or conditionally essential AA (Table 1).…”

Glutamine metabolism in advanced age

“…59,60 Glutamine is a key regulator of amino acidcontrolled cell growth through the mammalian target of rapamycin (mTOR) signaling pathway in rat intestinal epithelial cells. 27,28,31,39,42,61,62 Amino acids are important signaling regulators, especially for p70 S6 kinase and eIF-4E binding protein 1 via mTOR, which have 2 structurally and functionally distinct complexes termed mTORC1 and mTORC2. mTORC1 is activated by nutrients (amino acids), growth factors, and cellular energy, while mTORC2 is activated by growth factors alone.…”

Effects of reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in weaned piglets

“…After the quantity of complementary DNA (cDNA) was determined by a NanoDrop ® ND-1000 (NanoDrop Technologies, Rockland, DE, USA), real-time quantitative polymerase chain reaction (PCR) analyses were performed on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems, CA, USA) with a total volume of 10 μl containing 5 ng of cDNA, 5 μl SYBR Green mix, 0.2 μl ROX Reference Dye (50×), and 0.2 μl each of the forward and reverse primers (Yao et al, 2012). The following protocol was used: (1) .…”