[Frontiers in Bioscience 16, 1186-1196, January 1, 2011]

Alpha-Ketoglutarate and intestinal function

Yongqing Hou1, Lei Wang1, Binying Ding1, Yulan Liu1, Huiling Zhu1, Jian Liu1, Yongtang Li1, Ping Kang1,Yulong Yin2, Guoyao Wu3,4

1Hubei key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan 430023, China, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha 410125, China, State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China 100193,4Department of Animal Science, Texas A&M University, College Station, TX 77843 USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Growth performance
4. α-Ketoglutarate and gut function
4.1. Intestinal morphology
4.2. Absorptive and barrier function
4.3. antioxidative capacity
4.4. Concentrations of amino acids and protein in jejunal mucosa
4.5. Concentrations of ATP, ADP, and AMP in the intestinal mucosa
4.6. Concentrations of nitric oxide (NO) and nitric oxide synthase (NOS) in the intestinal mucosa
4.7. Heat shock protein 70 (HSP70) expression
4.8. Phosphorylation levels for mammalian target of rapamycin(mTOR)
4.9. Phosphorylation levels for adenosine monophosphate (AMP)-activated protein kinase(AMPK) and acetyl-CoA carboxylase(ACC)
5. Summary and perspective
6. Acknowledgements
7. References

1. ABSTRACT

Alpha-ketoglutarate (AKG) is an intermediate of the Krebs cycle which bridges amino acid metabolism with glucose oxidation in animals. Of particular interest is the conversion of AKG into glutamate by mitochondrial glutamate dehydrogenase in the gastrointestinal tract where glutamate has multiple physiological functions (including regulation of cell function, neurotransmission, and gastric emptying). Additionally, AKG stimulates the initiation of catabolism of branched-chain amino acids (BCAA) via BCAA transaminase in enterocytes. Oxidation of AKG also provides large amounts of ATP and modulates cellular redox state in the small intestine. Translating the basic research into practice, results of recent studies indicate that dietary supplementation with AKG alleviates oxidative stress and injury in intestinal mucosal cells, while improving intestinal mucosal integrity and absorption of nutrients in endotoxin-challenged pigs. The beneficial effects of AKG are associated with increased activation of the mTOR signaling pathway and net protein synthesis. Thus, AKG is a novel and promising supplement in diets to improve intestinal health in animals and possibly humans.

2. INTRODUCTION

The small intestine is not only the terminal organ for digestion and absorption of dietary nutrients, but is also crucial for preventing the entry of exogenous pathogens into the systemic circulation (1,2). Thus, intestinal integrity is vital to survival, growth, and health of both animals and humans (1-3). Extensive studies with experimental animals, including pigs, have demonstrated that a number of stressful factors, such as early-weaning, infection and inflammation can result in gut mucosal injury and dysfunction (3-5). The consequences are the occurrence of diarrhea, reduced growth, and even deaths, leading to a great deal of economic loss (3,5). Recent work has also identified that nutritional modulation can ameliorate damage of the small intestine in compromised neonates (6-8). Additionally, much evidence shows that glutamate and glutamine (products of AKG metabolism in animals) are main energy sources for intestinal mucosal cells under practical feeding conditions (9). At present, little attention has been paid to enteral or parenteral administration of glutamate to animals because of concerns over potential adverse effects of this amino acid on the brain. In contrast, much work has been conducted to determine the efficacy of glutamine supplementation on intestinal and whole-body homeostasis (9). However, the cost for chemical synthesis of glutamine is relatively high, which limits its use in swine production. This provides an impetus for us to identify cheaper replacements of glutamine in swine production, and one of them is α-ketoglutarate (AKG) (3,10,11).

Alpha-ketoglutarate (AKG) is a central molecule in the citric acid cycle (Krebs cycle) and an intermediate in the oxidation of key metabolic fuels. Emerging evidence shows that AKG plays a key role in systemic, intestinal and gut bacterial metabolism. Exogenous AKG can be converted to glutamate and glutamine in many tissues and be regarded as an energy substrate (12-14). Of particular interest, biochemical studies have led to the growing recognition that AKG displays remarkable metabolic and regulatory versatility in cells (3,15,16). Additionally, dietary AKG supplementation has been reported to improve mucosal morphology and function of the small intestine in endotoxin-challenged piglets (3,15,17-19). AKG can also maintain intestinal barrier integrity and attenuate gut injury through an anti-inflammatory role in weaned pigs (3). Because of ethical concerns over invasive studies with humans, the pig is often used as an animal model to study AKG metabolism in neonates and adults. Thus, much data included in this review are derived from studies with the porcine model.

3. GROWTH PERFORMANCE

Growth performance is a major criterion in animal production. Dietary supplementation with 1.0% AKG tended to increase ADG, ADFI and feed efficiency in weanling pigs, compared with the control group, 0.5% AKG group or 2.0% AKG group (Table 1). In contrast, dietary supplementation of 1.0% AKG did not affect growth performance in lipopolysaccharide(LPS)-challenged pigs likely due to low feed intake (Table 2) (3). However, AKG could relieve growth depression in weaned piglets chronically challenged by LPS when their food intake was improved (11). In the absence of stress, acute administration of AKG may not affect the growth of young pigs, as reported for rats receiving 0.5% AKG in the regular diet (20).

4. ΑKG AND GUT FUNCTION

4.1. Intestinal morphology

A high value of the villus: crypt ratio is a useful indicator of a high capacity for digestion and absorption (3, 21). There is evidence that dietary supplementation of AKG could improve intestinal histological morphology. For example, AKG supplementation resulted in: (1) increased villus height; (2) reduced crypt depth; and (3) an increased ratio of villus height to crypt depth in LPS-challenged pigs (Table 4) (3). These findings support the notion that AKG beneficially alleviates the LPS-induced damage of the intestinal structure. Similarly, dietary supplementation with 1% AKG increased the ratio of villus height to crypt depth in the gut of healthy pigs (Table 3) (10,15,17).

4.2. Absorptive and barrier function

Absorption of D-xylose from the intestinal lumen into plasma is a useful marker of in vivo intestinal function in animals. On day 14 postweaning, compared with the control group, circulating levels of D-xylose were elevated in AKG-supplemented pigs (17). Indeed, D-xylose content in plasma was the highest (quadratic, P<0.05) when the level of AKG was 1.0 %. In addition, with the increasing dose of supplemental AKG, the activity of diamine oxidase (DAO) in plasma was decreased (P<0.01). The activity of DAO was the lowest (quadratic, P<0.05) with 1% AKG in the diet (Table 5) (17).

AKG is converted into glutamate by mitochondrial glutamate dehydrogenase in the gastrointestinal tract where glutamate has multiple physiological functions (including regulation of cell function, neurotransmission, and gastric emptying) (12). AKG may spare the oxidation of glutamate and glutamine by enterocytes, thereby increasing the availability of these two amino acids to other pathways (e.g., synthesis of protein, citrulline, arginine, and glutathione). Additionally, AKG is a metabolic fuel to provide the required energy and improve the efficiency of nutrient absorption, as indicated by the entry of luminal D-xylose into the blood circulation (Table 6). This is a simple, specific, and sensitive measure of intestinal absorption ability. Similarly, Yang et al. (2005) reported that supplementation with 1% glutamine significantly increased D-xylose concentration in the plasma of pigs on days 7, 14 and 21 postweaning (22).

Diamine oxidase is present in the mammalian intestinal mucosa or the small intestine villi. This enzyme is particularly abundant in rapidly dividing cells (23). When the intestinal mucosa is damaged or replaced, DAO is released into the intestinal lymphatic and vascular space, resulting in increased DAO activity in blood but decreased enzyme activity in enterocytes (24). This occurs when intestinal permeability increases in response to LPS and inflammatory cytokines (25). Consistent with this view, dietary supplementation with 1% AKG increased DAO activity in jejunal mucosa, while decreasing DAO activity in blood (Table 6). These results indicate that AKG can alleviate intestinal injury caused by LPS challenge. When dietaty AKG enters the portal vein, it can be utilized for the synthesis of glutamine which is then released into the circulation and taken up by the small intestine. Like AKG, glutamine can serve as an energy substrate for gastrointestinal cells to promote intestinal mucosal cell proliferation (12) and decrease the expression of intestinal inflammatory factors. Taken together, these data support the notion that AKG improves the ability of the small intestine to absorb dietary nutrients under inflammatory conditions.

4.3. Antioxidative capacity

AKG enhances the intestinal antioxidative capacity by partly increasing the activity of superoxide dismutase (SOD). This effect of AKG is consistent with the decreased content of MDA content in the jejunal mucosa (Table 7). LPS can result in tissue ischemia and hypoxia, changes in cell oxidative metabolism of the microsomal enzyme system, and production of a large number of free radicals (26). Whole-body homeostasis is maintained in the oxidative stress-antioxidant balance. Usually, MDA is an important indicator to reflect the extent in the accumulation of free radicals in the body caused by oxidative damage. Superoxide dismutase functions to remove superoxide anion and is part of free radical scavenging systems. Glutathione (GSH), an antioxidant, helps protect cells from reactive oxygen species such as free radicals and peroxides (16,27). AKG can also act to detoxify excess ammonia, participate in the non-enzymatic oxidative decarboxylation during hydrogen peroxide decomposition, and facilitate the proper metabolism of fats, leading to reduced generation of oxygen radicals and preventing lipid peroxidative damages (28).

4.4. Concentrations of amino acids and protein in the jejunal mucosa

Hou et al. (3) provided a new biochemical basis to explain the beneficial effects of AKG on the intestine (3). These authors reported that dietary supplementation with AKG increased citrulline and glutamate concentration in the intestinal mucosa (Table 4), suggesting a net increase in the formation of these two amino acids in the gut of AKG-supplemented pigs. Citrulline is the immediate precursor for the endogenous synthesis of arginine, which is an essential amino acid for young mammals (3,8) and protects intestinal cells from LPS-induced injury (29). It is possible that AKG is firstly converted into glutamate, which is subsequently utilized for citrulline synthesis via pyrroline-5-carboxylate synthase (30). Indeed, it was found that 64% of the AKG disappeared from the small intestinal lumen within 1 h (31), suggesting rapid utilization of AKG by the mucosa. Alternatively, glutamine (formed from AKG in extraintestinal tissues) is a substrate for intestinal citrulline generation (32), because glutamine in arterial blood is actively taken up by the pig small intestine (33). The contribution of AKG to whole-body glutamine synthesis may be quantitatively important because 10% of the intraduodenally infused AKG is absorbed into the portal circulation (13,31,34).

4.5. Concentrations of ATP, ADP, and AMP in the intestinal mucosa

Mitochondria are key cellular organelles that regulate events related to energy production and apoptosis (35). Most of the ATP is generated by the proton gradient that develops across the inner mitochondrial membrane. ATP is a complex nanomachine that serves as the primary energy currency of the cell (36). In contrast to the traditional view that glucose is the primary fuel for the intestine, it is now known that glutamate, glutamine and aspartate are the major sources of ATP in mammalian enterocytes via mitochondrial oxidation.

AMP is a good indicator of cellular stress because an increased rate of ATP hydrolysis leads to a rapid accumulation of AMP in the cell (37). The energy charge of the adenylate pool is a better measure of the energy status in a tissue than the level of a single nucleotide. ATP hydrolysis first increases the cellular ADP concentration. The ADP is then converted by the adenylate kinase reaction (2 ADP↔ATP+AMP) to ATP and AMP (38). Therefore, during increased ATP use, AMP accumulates well before any changes in cellular ATP or ADP concentration occur (37).

As an intermediate in the citric acid cycle and an intermediate in the oxidation of glutamate and glutamine, AKG contributes substantially to ATP homeostasis and mucosal integrity in the small intestine (31). This is in keeping with our findings that AKG supplementation resulted in: 1) increased ATP concentrations; 2) reduced AMP/ATP ratio; and 3) increased AEC (Table 9) (3). These three lines of evidence indicate that dietary supplementation with AKG could modulate the adenine nucleotide pool and support the notion that AKG beneficially alleviates the LPS-induced damage of the intestinal energy metabolism. The results reveal a hitherto unrecognized role for AKG in ameliorating oxidative stress and improving energy status in the small intestine. As an intermediate in the oxidation of key gut fuels, AKG is extensively metabolized in first-pass by the intestinal mucosa (39). Thus, we suggest that AKG exerts its nutritional benefits primarily at the small-intestinal level in weanling piglets.

4.6. Concentrations of nitric oxide (NO) and nitric oxide synthase (NOS) in the intestinal mucosa

Physiological concentrations of NO are essential to the maintenance of intestinal mucosal integrity, but pathological levels are deleterious to the gut (6-9). AKG could maintain the balance of NO-NOS system in the jejunal mucosa, whereas the content of NO decreased (P<0.05) and NOS activity (P<0.05) increased in LPS group (Table 10). Nitric oxide is synthesized from L-arginine by NO synthase (NOS) in the presence of several cofactors, including NADPH, calcium, and tetrahydrobiopterin (40). The NOS has several isoforms, including inducible NOS (iNOS) and constitutive NOS (cNOS) (41). iNOS is expressed in response to inflammatory cytokines, such as tumor necrosis factor-a , interleukin-1b , and interferon-g , and endotoxin. iNOS generates a relatively large amounts of NO compared with the constitutive isoforms of NOS (40). Liu (16) reported that LPS increased the mucosal NOS activity and this effect of LPS was ameliorated by AKG (16). Dietary supplementation with 1% AKG was also beneficial for maintaining the intestinal barrier function under stressful conditions. Similarly, glutamine supplementation decreased iNOS in an experimental model of colitis in the rat (42). These results may indicate that either AKG or its metabolite (e.g., glutamine) affects expression or turnover of iNOS, thereby attenuating the intestinal injury induced by LPS. Studies indicate that iNOS-derived NO mediates LPS-induced intestinal injury and bacterial translocation from the intestinal lumen to the blood circulation (43). Thus, pathological levels of NO can be toxic and pro-inflammatory. In contrast, physiological levels of NO inhibit adhesion molecule expression, synthesis of cytokines and chemokines, as well as leukocyte adhesion and transmigration. Additionally, actions of NO are dependent on the distance of the target protein from NO sources and the initial priming of immune cells (44), as well as the activation of guanylate cyclase (45).

4.7. Heat shock protein 70 (HSP70) expression

Oral administration of glutamine, a product of AKG metabolism, can protect intestinal cells from LPS-induced damage (46). AKG may impose effects through many mechanisms. One of these putative mechanisms may involve expression of HSP70 (47). A high concentration of HSP70 is indicative of oxidative stress in intestinal cells (48), which is in agreement with our observations (Figure 1) (3). Notably, dietary supplementation with AKG reduced the elevated concentrations of the HSP70 protein in the mucosae of LPS-challenged piglets (Figure 1), indicating an important role for AKG in ameliorating oxidative stress (3). In response to stress, HSP70 is expressed at elevated levels to promote refolding and prevent aggregation of partially-denatured proteins, thereby protecting cells from injury (49). This is an adaptive mechanism to allow organisms to survive under heat shock stress.

4.8. Phosphorylation levels for mammalian target of rapamycin(mTOR)

AKG can affect expression of key proteins involved in anti-inflammatory responses via the mTOR signaling (Figure 2) (3), a major mechanism for the regulation of protein synthesis in cells (50-51), therefore supporting intestinal growth under septic conditions (11,16). In addition, mTOR plays a crucial role in the control of cell growth and proliferation (52,53). Compelling evidence shows that the mTOR signaling pathway includes the 70-kDa ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4EBP1) (54). We found that dietary supplementation with AKG increased the phosphorylated level of mTOR (the active state of mTOR) in the intestinal mucosa of piglets (Figure 2) (3). It was postulated that such an effect of AKG contributes to increased protein synthesis in the intestinal mucosa of endotoxin-treated piglets. Accordingly, mucosal protein concentration was greater in AKG-supplemented piglets than in non-supplemented piglets when they were challenged with LPS (Table 8) (3).

4.9. Phosphorylation levels for adenosine monophosphate (AMP)-activated protein kinase(AMPK) and acetyl-CoA carboxylase(ACC)

AMPK activity in mammalian cells can be regulated by stimuli that affect cellular ATP levels (55). For example, hypoxia leads to activation of AMPK via an increase in the AMP/ATP ratio (56, 57). When activated, AMPK switches on catabolic pathways for ATP regeneration, such as glucose and fatty acid β-oxidation, while switching off ATP-requiring pathways, such as fatty acid and triglyceride synthesis (58;). Our finding that AKG regulated AMPK signaling in the intestinal mucosa is novel and important (Fig 3). However, emerging evidence shows that AMPK is a target protein of mTOR in cells (59). mTOR signaling is inhibited under conditions of low nutrients, such as amino acids and low intracellular ATP levels (60). Whereas mTOR was presumed to serve as the direct cellular sensor for ATP levels (61), some evidence from in vitro cell culture studies has implicated AMPK in the regulation of mTOR activity (62-64). Thus, possibly through mTOR activation (3), AKG stimulates AMPK phosphorylation and oxidation of energy substrates (e.g., amino acids, fatty acids, and glucose) in the intestinal mucosa (65,66), thereby enhancing ATP supply and supporting cell function.

Besides activation of ATP-generating pathways, AMPK could also modulate ACC activity in cells by phosphorylating ACC-β (67-69). ACC is a rate-controlling enzyme in the conversion of acetyl-CoA to malonyl CoA (70). Malonyl CoA inhibits carnitine: palmitoyl-CoA transferase-1 (CPT1), which is a rate-limiting step for the entry of long-chain fatty acyl-CoA into mitochondria to be oxidated (71). Thus, a fall in malonyl CoA levels increases fatty acid oxidation in mitochondria (72). In cells exposed to an energy-depleting stress (such as LPS challenge), AMPK is considered to be as an energy sensor that monitor the level of total cellular ATP (namely inhibiting ATP-consuming processes and stimulating ATP-producing processes) (38). On the other hand, malonyl-CoA is the substrate for fatty acid synthesis, which is an ATP-dependent process (71). Therefore, the phosphorylation of ACC by AMPK (Figure 4) would decrease malonyl CoA levels and favors elevation of energy status in the intestinal mucosa of AKG-supplemented pigs. This novel action of AKG is nutritionally and physiologically relevant for animal growth and health.

5. SUMMARY AND PERSPECTIVE

Dietary supplementation with AKG alleviates intestinal injury and dysfunction in piglets. The actions of AKG are associated with reduced oxidative stress and increased activation of the mTOR signaling. In addition, dietary supplementation with AKG beneficially improves the energy status of the intestinal mucosa as well as AMPK activation and ACC inactivation in the intestinal mucosa of LPS-challenged pigs. AKG may directly activate mTOR and associated proteins or indirectly exert its effects via glutamate formation. Alternatively, through regulating the intestinal metabolism of branched-chain amino acids (73), including leucine (an activator of mTOR), AKG may modulate intracellular protein turnover and functions of the small intestine. Besides the small intestine, AKG may also benefit the vascular system by relieving an inhibitory action of leucine (a branched-chain amino acid) on NO synthesis by endothelial cells (74). Future studies are required to test this novel and important hypothesis.

6. ACKNOWLEDGMENTS

This work was jointly supported by National Natural Science Foundation of China (Grant No. 30871801), the Program for Innovative Research Groups of Hubei Provincial Natural Science Foundation (Grant No. 2007ABC009), National Research Initiative Competitive Grants from the Animal Growth & Nutrient Utilization Program (2008-35206-18764) of the USDA National Institute of Food and Agriculture, Texas AgriLife Research (H-82000), American Heart Association (10GRNT4480020), and the Thousand-People-Talent program at China Agricultural University.

7. REFERENCES

1.Blachier F, Mariotti F, Huneau JF, Tomé D: Effects of amino acid-derived luminal metabolites on the colonic epithelium and physiopathological consequences. Amino Acids 33, 547-562 (2007)
doi:10.1007/s00726-006-0477-9
PMid:17146590

2.Eklou-Lawson M, Bernard F, Neveux N, Chaumonte C, Bos C, Davila-Gay AM, Tomé D, Cynober L, Blachier F: Colonic luminal ammonia and portal blood L-glutamine and L-arginine concentrations: a possible link between colon mucosa and liver ureagenesis. Amino Acids 37, 751-760 (2009)
doi:10.1007/s00726-008-0218-3
PMid:19082688

3.Hou YQ, Wang L, Ding BY, Liu YL, Zhu HL, Liu J, Li YT, Wu X,Yin YL, Wu GY: Dietary α-ketoglutarate supplementation ameliorates intestinal injury in lipopolysaccharide-challenged piglets. Amino acids 29, 555-564 (2010)
doi:10.1007/s00726-010-0473-y
PMid:20127262

4.Bergen WG, Wu G: Intestinal nitrogen recycling and utilization in health and disease. J Nutr 139, 821-825 (2009)
doi:10.3945/jn.109.104497
PMid:19282369

5.Liu YL, Huang JJ, Hou YQ, Zhu HL, Zhao SJ, Ding BY, Yin YL, Yi GF, Shi JY, Fan W: Dietary arginine supplementation alleviates intestinal mucosal disruption induced by Escherichia coli lipopolysaccharide in weaned pigs. Br J Nutr 100,552-560 (2008)
doi:10.1017/S0007114508911612

6.Flynn NE, Bird JG, Guthrie AS: Glucocorticoid regulation of amino acid and polyamine metabolism in the small intestine. Amino Acids 37, 123-129 (2009)
doi:10.1007/s00726-008-0206-7
PMid:19034608

7.Han J, Liu YL, Fan W, Chao J, Hou YQ, Yin YL, Zhu HL, Meng GQ, Che ZQ: Dietary l-arginine supplementation alleviates immunosuppression induced by cyclophosphamide in weaned pigs. Amino Acids 37, 643-651 (2009)
doi:10.1007/s00726-008-0184-9
PMid:18821052

8.Wu G, Bazer FW, Davis TA, Kim SW, Li P, Rhoads JM, Satterfield MC, Smith SB, Spencer TE, Yin YL: Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37, 153-168 (2009)
doi:10.1007/s00726-008-0210-y
PMid:19030957    PMCid:2677116

9.Wu G: Intestinal mucosal amino acid catabolism. J Nutr 128, 1249-1252 (1998)
PMid:9687539
10.Hu QZ, Hou YQ, Ding BY,Zhu HL,Liu YL Wang M, Xiao HL: Effects of α-ketoglutarate on histological morphology and function of small intestine in piglets. Chin J Anim Nutr 20 (6), 662-667 (2008)

11.Liu J, Hou YQ, Ding BY, Liu YL, Zhu HL, Wang L, Li YT: Attenuating effect of α-ketoglutaric acid on growth depression in weaned pigs challenged with lipopolysaccharide. Chin J Anim Nutr 21 (4), 519-524 (2009).

12.Pierzynowski SG, Sjodin A: Perspectives of glutamine and its derivatives as feed additives for farm animals. J Anim Feed Sci 7, 79-91 (1998)

13.Kristensen NB, Jungvid H, Fernandez JA, Pierzynowski SG: Absorption and metabolism of alpha-ketoglutarate in growing pigs. J Anim Physiol Anim Nutr 86, 239-245 (2002)
doi:10.1046/j.1439-0396.2002.00380.x
PMid:15379910

14.Śliwa E, Kowalik S, Tatara MR, Krupski W, Majcher P, Łuszczewska-Sierakowska I, Pierzynowski SG, Studziński T: Effect of alpha-ketoglutarate given to pregnant sows on the development of the humerus and femur in newborns. Bull Vet Inst Pulawy 49, 117-120 (2005)

15.Hu Q, Hou Y, Ding B, Zhu H, Liu Y, Wang M, Xiao H: Effects of α-ketoglutarate on mucosal morphology and function of small intestine in piglets. J Anim Sci 87 (E-Suppl 2), 175 (2009) (Abstract)

16.Liu J: Effects of α-ketoglutaric acid on growth performance and intestinal structure and function in piglets challenged with lipopolysaccharide with reference to the mechanism(Dissertation).Wuhan:Wuhan Polytechnic University, (in Chinese) (2009)

17.Hu QZ: Effects of α-ketoglutarate on growth performance and intestinal function of weaned piglets (Dissertation).Wuhan:Wuhan Polytechnic University, (in Chinese) (2008)

18.Liu J, Hou YQ, Ding BY, Liu YL, Zhu HL, Wang L, Li YT: Effects of α-ketoglutaric acid on energy metabolism in intestinal mucosa of weaned pigs chronically challenged with lipopolysaccharide. Chin J Anim Nutr 21(6),892-896 (2009).

19.Liu J, Hou YQ, Ding BY, Liu YL, Zhu HL, Wang L, Li YT: Effects of α-ketoglutaric acid on protein synthesis and antioxidative capacity in jejunal mucosa of weaned pigs chronically challenged with lipopolysaccharide. Chin J Anim Sci 46 (11), 35-38 (2010)

20.Filip RS, Pierzynowski SG: Influence of the forms of a-ketoglutarate as feed additive on some blood indices and performance of growing rats. Medycyna Wet 63, 796-800 (2007)

21.Montagne L, Pluske JR, Hampson DJ: A review of interactions between dietary fibre and the intestinal mucosa, and their consequences on digestive health in young non-ruminant animals. Anim Feed Sci Tech 108, 95-117 (2003)
doi:10.1016/S0377-8401(03)00163-9

22.Yang CM, Xu WD, Chen AG: Glycyl-glutamine on growth performance and intestinal absorption function. Chin J Anim Sci 41 (8), 6-8 (2005)

23.Luk GD, Bayless TM, Baylin SB: Plasm a postheparin diamine oxidase: Sensitive provocative test for quantitating length of acute intestinal mucosa injury in the rat. J Clin Invest, 71, 1308-1315 (l983)

24.Wollin A, Navert H, Bounous G: Effect of intestinal ischemia on diamine oxidasc activity in rat intestinal tissue and blood. Gastroenterology 80, 349-355 (1981)

25.Ropelle ER, Pauli JR, Fernandes MFA, Rocco SA, Marin RM, Morari J, Souza KK, Dias MM, Gomes-Marcondes MC, Gontijo JAR, Franchini KG, Velloso LA, Saad MJA, José BC: Carvalheira. A central role for neuronal amp-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in high-protein diet-induced weight loss. Diabetes 57, 594-605 (2008)
doi:10.2337/db07-0573
PMid:18057094

26.Nishi K, Oda T, Takabuchi S, Oda S, Fukuda K, Adachi T, Semenza GL, Shingu K, Hirota K: LPS induces hypoxia-Inducible factor 1 activation in macrophage-differentiated cells in a reactive oxygen species-dependent manner. Antioxid Redox Signal 10, 983-995 (2008)
doi:10.1089/ars.2007.1825

27.Zhang JM, Wang LD, Gao ZC, Jiang YX, Zhang Q: Effect of dietary glutamine supplementation on antioxidant capacity of tissue in the early-weaned piglets. Acta Veterinaria et Zootechnica Sinica 33 (2), 105-109 (2002)

28.Velvizhi S, Dakshayani KB, Subramanian P: Effects of alpha-ketoglutarate on antioxidants and lipid peroxidation products in rats treated with ammonium acetate. Nutrition 18 : 747-750 (2002)
doi:10.1016/S0899-9007(02)00825-0

29.Tan BE, Yin YL, Kong XF, Li P, Li XL, Gao HJ, Li XG, Huang RL, Wu G: L-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells. Amino Acids 38, 1227-1235 (2010)
doi:10.1007/s00726-009-0334-8
PMid:19669080    PMCid:2850530

30.Wu G, Morris SM Jr: Arginine metabolism: nitric oxide and beyond. Biochem J 336, 1-17 (1998)
PMid:9806879    PMCid:1219836

31.Lambert BD, Filip R, Stoll B, Junghans P, Derno M, Hennig U, Souffrant WB, Pierzynowski S, Burrin DG: First-pass metabolism limits the intestinal absorption of enteral a-ketoglutarate in young pigs. J Nutr 136, 2779-2784 (2006)

32.Wu G, Knabe DA, Flynn NE: Synthesis of citrulline from glutamine in pig enterocytes. Biochem J 299, 115-121 (1994)
PMid:8166628    PMCid:1138029

33.Wu G, Borbolla AG, Knabe DA: The uptake of glutamine and release of arginine, citrulline and proline by the small intestine of developing pigs. J Nutr 124, 2437-2444 (1994)
PMid:16856325

34.Lambert BD, Stoll B, Niinikoski H, Pierzynowski S, Burrin DG: Net portal absorption of enterally fed a-ketoglutarate is limited in young pigs. J Nutr 132, 3383-3386 (2002)
PMid:12421855

35.Psarra AM, Solakidi S, Sekeris CE: The mitochondrion as a primary site of action of steroid and thyroid hormones: presence and action of steroid and thyroid hormone receptors in mitochondria of animal cells. Mol Cell Endocrinol 246 (1-2), 21-33 (2006)
doi:10.1016/j.mce.2005.11.025
PMid:16388892

36.Trefil, James. 1001 Things everyone should know about science. Doubleday. New York (1992)

37.Frederich M, O'Rourke MR, Furey NB, Jost JA: AMP-activated protein kinase (AMPK) in the rock crab, Cancer irroratus: an earlyindicator of temperature stress. The Journal of Experimental Biology 212, 722-730 (2009)
doi:10.1242/jeb.021998
PMid:19218524

38.Hardie DG, Scott JW, Pan DA, Hudson ER: Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett 546, 113-120 (2003)
doi:10.1016/S0014-5793(03)00560-X

39. Lambert BD, Filip R, Stoll B, Junghans P, Derno M, Hennig U, Souffrant WB, Pierzynowski S, Burrin DG: First-pass metabolism limits the intestinal absorption of enteral a-ketoglutarate in young pigs. J Nutr 136,2779-2784 (2006)

40.Jones KL, Bryan TW, Jinkins PA, Simpson KL, Grisham MB, Owens MW, Milligan SA, Markewitz BA, Robbins RA: Superoxide released from neutrophils causes a reductionin nitric oxide gas. Am J Physiol 275, 1120-1126 (1998)

41.Moncada S, Higgs A: The L-arginine-nitric oxide pathway. N Engl J  Med 329, 2002-2012 (1993)
doi:10.1056/NEJM199312303292706
PMid:7504210

42.Fillmann H, Kretzmann NA, San-Miguel B, Llesuy S, Marroni N, González-Gallego J, Tuñón MJ: Glutamine inhibits over-expression of proinflammatory genes and down-regulates the nuclear factor kappa B pathway in an experimental model of colitis in the rat. Toxicology 236, 217-226 (2007)
doi:10.1016/j.tox.2007.04.012
PMid:17543437

43.Forsythe RM, Xu DZ, Lu Q, Deitch EA: Lipopolysaccharide induced enterocyte derived nitric oxide induces intestinalmonolayer permeability in an autocrine fashion. Shock 17, 180-184 (2002)
doi:10.1097/00024382-200203000-00004
PMid:11900335

44.Guzik TJ, Korbut R, Adamek-Guzik T: Nitric Oxide and superoxe in inflammation and immune regulation. J Physiol Pharmacol 54, 469-487 (2003)
PMid:14726604

45.Barry MK, Aloisi JD, Pickering SP, Yeo CJ: Nitric oxide modulates water and electrolyte transport in the ileum. Ann Surg 219, 382-388 (1994)
doi:10.1097/00000658-199404000-00009
PMid:8161264    PMCid:1243155

46.Haynes TE, Li P, Li XL, Shimotori K, Sato H, Flynn NE, Wang JJ, Knabe DA, Wu G: L-Glutamine or L-alanyl-L-glutamine prevents oxidant- or endotoxin-induced death of neonatal enterocytes. Amino Acids 37, 131-142 (2009)
doi:10.1007/s00726-009-0243-x
PMid:19189199

47.Li N, Liboni K, Fang M, Samuelson D, Lewis P, Patel R, Neu J: Glutamine decreases lipopolysaccharide- induced intestinal inflammation in infant rats. Am J Physiol Gastrointest Liver Physiol 286, G914-G921 (2004)
doi:10.1152/ajpgi.00493.2003
PMid:14726310

48.Sepponen K, Poso AR: The inducible form of heat shock protein 70 in the serum, colon and small intestine of the pig: comparison to conventional stress markers. Vet J 171, 519-524 (2006)
doi:10.1016/j.tvjl.2005.01.005
PMid:16624719

49.Beckmann RP, Mizzen LE, Welch WJ: Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. Science 248, 850-854 (1990)
doi:10.1126/science.2188360
PMid:2188360

50. Frank JW., Escobar J, Suryawan A, Nguyen HV, Kimball SR, Jefferson LS, Davis TA: Dietary protein and lactose increase translation initiation factor activation and tissue protein synthesis in neonatal pigs. Am J Physiol Endocrinol Metab 290, E225-E233(2006)
doi:10.1152/ajpendo.00351.2005
PMid:16144813

51.Frank J, Escobar J, Nguyen HV, Jobgen SC, Jobgen WS, Davis TA, Wu G: Oral N-carbamylglutamate supplementation increases protein synthesis in skeletal muscle of pigets. J Nutr 137,315-319 (2007)
PMid:17237304

52.Davis TA, Fiorotto ML, Burrin DG, Reeds PJ, Nguyen HV, Beckett PR, Vann RC, O'Connor PMJ: Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs. Am J Physiol Endocrinol Metab 282, E880-890 (2002)

53.Sarbassov DD, Ali SM, Sabatini DM: Growing roles for the mTOR pathway. Curr Opin Cell Bio 17, 596-603 (2005)
doi:10.1016/j.ceb.2005.09.009
PMid:16226444

54.Liao XH, Majithia A, Huang XL, Kimmel AR: Growth control via TOR kinase signaling, an intracellular sensor of amino acids and energy availability, with crosstalk potential to proline metabolism. Amino Acids 35, 761-770 (2008)
doi:10.1007/s00726-008-0100-3
PMid:18651095

55.Fisslthaler B and Fleming I: Activation and signaling by the amp-activated protein kinase in endothelial cells. Circ Res 105, 114-127(2009)
doi:10.1161/CIRCRESAHA.109.201590
PMid:19608989

56.Evans, AM, Mustard KJW, Wyatt C N, Peers C, Dipp M, Kumar P, Kinnear NP, Hardie DG: Does AMP-activate protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2-sensing cells? J Biol Chem 280, 41504-41511(2005)
doi:10.1074/jbc.M510040200
PMid:16199527

57.Hardie DG, Hawley SA: AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23, 1112-1119 (2001)
doi:10.1002/bies.10009
PMid:11746230

58.Hardie DG, Carling D, Carlson M: The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67, 821-55 (1998)
doi:10.1146/annurev.biochem.67.1.821
PMid:9759505

59.Hong-Brown LQ, Brown CR, Kazi AA, Huber DS, Pruznak AM, Lang CH: Alcohol and PRAS40 knockdown decrease mTOR activity and protein synthesis via AMPK signaling and changes in mTORC1 interaction. J Cell Biochem 109, 1172-1184 (2010)
PMid:20127721

60.Shamji AF, Nghiem P, Schreiber SL: Integration of growth factor and nutrient signaling: implications for cancer biology. Mol Cell 12, 271-280 (2003)
doi:10.1016/j.molcel.2003.08.016
PMid:14536067

61.Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G: Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102-1105 (2001)
doi:10.1126/science.1063518
PMid:11691993

62.Inoki K, Li Y, Zhu T, Wu J, Guan KL: TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4, 648-657 (2002)
doi:10.1038/ncb839
PMid:12172553

63.Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K, Kemp BE, Witters LA, Mimura O, Yonezawa K: A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8, 65-79 (2003)
doi:10.1046/j.1365-2443.2003.00615.x
PMid:12558800

64.Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, Cantley LC: The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6, 91-99 (2004)
doi:10.1016/j.ccr.2004.06.007
PMid:15261145

65.Blachier F, Boutry C, Bos C, Tomé D: Metabolism and functions of L-glutamate in the epithelial cells of the small and large intestines. Am J Clin Nutr 90, 814S-821S (2009)
doi:10.3945/ajcn.2009.27462S
PMid:19571215

66.Burrin DG, Stoll B: Metabolic fate and function of dietary glutamate in the gut. Am J Clin Nutr 90, 850S-856S (2009)
doi:10.3945/ajcn.2009.27462Y
PMid:19587091

67.Carling D: The AMP-activated protein kinase cascade-a unifying system for energy control. Trends Biochem Sci 29, 18-24 (2004)
doi:10.1016/j.tibs.2003.11.005
PMid:14729328

68.Aymerich I, Foufelle F, Ferré P, Casado FJ, Pastor-Anglada M: Extracellular adenosine activates AMP-dependent protein kinase (AMPK). J Cell Sci 119, 1612-1621 (2006)
doi:10.1242/jcs.02865
PMid:16569664

69.Kemp BE, Stapleton D, Campbell DJ, Chen ZP, Murthy S, Walter M, Gupta A, Adams JJ, Katsis F, van Denderen B, Jennings IG, Iseli T, Michell BJ, Witters LA: AMP-activated protein kinase, super metabolic regulator.Biochem Soc Trans 31, 162-168 (2003)
doi:10.1042/BST0310162

70.Kahn BB, Alquier T, Carling D, Hardie DG: AMP-activated protein kinase: Ancient energy Review gauge provides clues to modern understanding of etabolism. Cell Metab 1, 15-25 (2005)
doi:10.1016/j.cmet.2004.12.003
PMid:16054041

71.Jobgen WS, Fried SK, Fu WJ, Meininger C, Wu G: Regulatory role for the arginine-nitric oxide pathway in metabolism of energy substrates. J Nutr Biochem 17, 571-588 (2006)
doi:10.1016/j.jnutbio.2005.12.001
PMid:16524713

72.Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB: Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415, 339-343 (2002)
doi:10.1038/415339a
PMid:11797013

73.Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids 37, 1-17 (2009)
doi:10.1007/s00726-009-0269-0
PMid:19301095 74. Wu G, Meininger CJ. Nitric oxide and vascular insulin resistance. BioFactors 35, 21-27 (2009)
doi:10.1002/biof.3
PMid:19319842

Abbreviations: AKG, α-ketoglutarate; ACC, acetyl-CoA carboxylase; ADFI, average daily feed intake; ADG, average daily gain; AEC, adenylate energy charge; AMPK, adenosine monophosphate (AMP)-activated protein kinase; DAO, diamine oxidase; HSP 70, Heat shock protein 70; LPS, lipopolysaccharide; mTOR, mammalian target of rapamycin; NO, nitric oxide; NOS, nitric oxide synthase; SEM, standard error of the mean. TAN, total adenine nucleotide

Key Words: Alpha-Ketoglutarate, Intestine, Gut function, Piglets, Lipopolysaccharide, Review

Send correspondence to: Yongqing Hou, Hubei key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan 430023, China, Tel: 862783956175, Fax: 862783956175, E-mail:houyq777@yahoo.com.cn