Alpha-ketoglutarate reduces duodenal myoelectric disturbances induced by E. coli enterotoxin in pigs1

Pigs with bipolar electrodes implanted on the antrum and duodenum were exposed to heat-labile enterotoxin from E. coli (LT) infused intraduodenally. The exposure was repeated after three days of supplementing feed with α-ketoglutarate (AKG). 0.5 μg/kg of LT induced prolonged changes in the migrating myoelectric complex (MMC) without inducing clinical signs of enterotoxaemia. AKG had no effect on the basic MMC pattern, but prevented LT-induced changes. The possible mechanism of AKG and LT-induced alteration may involve gamma-aminobutyric acid (GABA) synthesis and activation of GABAergic neurotransmission in the gut.


INTRODUCTION
Gastrointestinal tract motility is crucial for its functions.Heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli is a major virulent factor responsible for infectious diarrhoea in young animals and children.Enterotoxaemia-induced ileus can lead to death due to endotoxaemia and septicaemia.It has been shown that various bacterial toxins influence the migrating myoelectric complex (MMC) in pigs, rats and calves.After administration of endotoxins, MMC cycles are shortened and more frequent.In pigs, elevated MMC migration velocity and cycling frequency are maintained one day after endotoxin administration during feeding and return to basal values 4 days later (Bruins et al., 2003).
Glutamine has a beneficial effect on gut morphology (Potsic et al., 2002).However it is debated whether glutamine has beneficial effects on the gut during disease.Since glutamine is unstable in solutions, α-ketoglutarate (AKG) can be used as a glutamine precursor.No studies have been performed on the influence of AKG on motility or MMC during enterotoxaemia.Therefore, the present study was designed to evaluate the effect of heat-labile enterotoxin on MMC in weaned pigs, followed by investigating the effect of AKG on parameters of MMC in animals exposed to enterotoxin.

MATERIAL AND METHODS
Castrated male pigs (10.5-13 kg BW) had surgically implanted bipolar silver electrodes on serosal membrane of the stomach antrum and duodenum, as described by Gacsalyi et al. (2000).The electrodes were connected to a telemetric transmitter permitting constant measurement of GI tract electrical activity.The animals were also fitted with a duodenal cannula.
Ten pigs were subjected to intraduodenal infusion of E. coli LT (Sigma, USA) in doses 0.1 and 0.5 µg/kg BW.LT was injected in a 2 ml bolus at the end of phase I of MMC between 2 and 3 p.m.Each dose was tested on the same pig with a one-or two-day interval.A further 8 pigs were used for studying the effect of AKG.The MMC in each animal was recorded under control conditions, after infusion of LT at 0.5 µg/kg BW, after 1-3 days of feeding AKT at 10 mmol/kg BW, and after LT infusion at 0.5 µg/kg BW given on the 5 th day after starting AKT supplementation.

RESULTS
The pigs were in good condition and stable during the entire experiment.LT had no effect on the duration or signal power of electromyographic events in the antrum.In the duodenum after LT infusion, the duration of the MMC cycle increased at night (postprandially).The increase was due to elongation of phase II (Figure 1).The velocity of phase III migration in the duodenum was significantly increased following LT administration (Figure 2).
Feed supplementation with AKT had no effect on basic electrical activity in the antrum.The velocity of phase III in the duodenum was unchanged after 3 days of AKG supplementation.Administration of LT on the 5 th day of AKT supplementation did not affect the velocity of phase III (Figure 3)

DISCUSSION
In the present study we have shown that low doses of LT induce disturbances in gut motility without evoking other clinical signs.Therefore, exposure to bacterial toxins even without inducing disease may cause alterations in gut function that are followed by a decrease in feed utilization.Feed supplementation with AKG has no effect on basic motility, but prevents changes induced by LT.It has been assumed that enhanced gamma-aminobutyric acid formation (GABA) in the intestinal mucosa by ornithine α-ketoglutarate treatment might be of physiologic importance in the regulatory processes of cell growth and differentiation (Raul, 1995).The presence of GABA-positive neurons in the submucosal and myenteric plexus has been shown in pigs (Timmermans and Scheuermann, 1998).Spontaneous relaxations of the rat gastroduodenum include responses that involve a GABAergic nitric oxide-related pathway, which is targeted by VIP (Krantis et al., 1998).

CONCLUSIONS
Feed supplementation with α-ketoglutarate may have a beneficial effect on subclinical forms of enterotoxaemia, decreasing diarrhoea and improving feed utilization that leads to better health.

Figure 1 .
Figure 1.Duration of duodenal MMC phases in control (white columns) and after intraduodenal administration of E. coli heat-labile enterotoxin (LT) 0.5 µg/kg BW (black columns) recorded in 10 pigs.Data expressed as mean ± SE. * different from respective control; P<0.05 (paired t-test)