[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


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


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.


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.


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).


4.1. 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.


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.


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.


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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