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592s Biochemical SocietyTransactions ( 1 995) 23 CCK - 8 Subhate Inhibits Intestinal Glucose Transport M. Ekhesty Tabar and K. Burdett Division of Biochemistry, The School of Biological Sciences, The University of Manchester, MI3 9PT, UK Gastrointestinal hormones and neurotransmitters regulate food intake, digestion and distribution of nutrients. We are interested in the acute regulation of non-electrolyte absorption, in particular by events early in the digestive process. Conditions in the gastric lumen as the meal is terminating stimulate the release of a novel fundic peptide that decreases intestinal glucose absorption [I]. Well characterised peptides also act at this time. Cholecystokinin (CCK) released from the proximal small intestine early after ingestion [2] has been implicated in the regulation of food intake and in the modulation of postprandial blood glucose concentrations. CCK acts by delaying gastric emptying and hence the supply of absorbable carbohydrate to the proximal intestine [3]. It could do so by classic endocrine action or by paracrine activity of peptide released from the pyloric region of the stomach [4]. In man CCK infusion delayed gastric glucose emptying but had no effect on plasma glucose concentrations when glucose was instilled directly into the duodenum [3]. There is no published evidence that CCK directly affects glucose transport from the intestine by paracrine or endocrine action, though it may modulate distribution thereafter by inducing insulin release [5]. Studying the effect of CCK on glucose transport through mucosal sheets in vitro has the advantage that any other important physiological effects such as changes in motility and insulin release do not impinge on the data. The methods used were those described previously [l]. 5 mm diameter mucosal sheets from guinea pig jejunum or ileum were equilibrated in glucose-free buffer for 30 minutes and then incubated for a further 45 minutes at 37°C with 5 mM glucose in the luminal buffer and 1 )LM CCK-8 sulphate in 0.1% albumin in the serosal buffer. The paired control sheets were exposed to 0.1% albumin. 'H polyethylene glycol 900 was added to the luminal buffer; if more than 0.5% of the luminal dose appeared on the serosal side the sheet was judged to be damaged. The difference in serosal glucose gain between each sheet and its paired control was measured. Replicate measurement of glucose transport in control sheets showed that ileal sheets absorbed approximately twice as much glucose as jejunal sheets and that the intrinsic variation between sheets taken from any one animal had a standard deviation of 3% of the mean. CCK-8 sulphate in the serosal compartment decreased the transport of glucose by 40.8 f 13.6% (n=22 pairs of sheets from 5 guinea pigs). The inhibition was consistent and highly significant when the serosal CCK-8 sulphate concentration was 1 M. Inhibition was not a non-specific peptide effect, 3 mg ml of a low molecular weight peptide mixture extracted from pig antral mucosa had no effect. r Preliminary experiments with the non-sulphated CCK octapeptide showed that it too inhibits glucose transport (41.9 f 18%, n = 11 pairs of sheets from 2 guinea pigs). The CCK-8 effect appears to be more variable and to require a higher serosal concentration (4 pM) of peptide. Whether CCK-8 sulphate is genuinely a better agonist will be investigated in future work. The sulphated octapeptide. but not CCK-8, raised plasma insulin concentrations in mice [6], so some biological systems do discriminate between the two peptides. 1 pM is a high dose of CCK-8 sulphate and may be needed in vitro to survive proteolysis and to produce a big enough gradient to drive the hormone on to mucosal receptors within the sheet. CCK-8 sulphate infused in vivo at a rate of 15 pmole/min/kg in guinea pigs [7] significantly reduces gastrointestinal glucose absorption. The inhibitory action of CCK-8 non-sulphated is being used in the design of experiments to determine the minimum amino acid sequence needed to inhibit intestinal glucose transport, and any relationship between CCK-8 and the novel inhibitory peptide from the gastric fundic mucosa ill. We gratefully acknowledge the financial support of The Islamic Republic of Iran for M.B.T. 1. Burdett, K. & Lauterbach, F. (1994) Eur. J. Physiol. 426, 491-498 2. Silver, A. J. & Morley, J. E. (1991) Prog. in Neurobiol. 36, 23-34 3. Liddle, R. A , , Rushakoff, R. J., Morita, E. T., Beccaria, L., Carter, J. D., & Goldfine, I. D. (1988) J. Clin. Invest. 81, 1675-1681 4. Michalski, S . , Herben, H., Golenhofen, K., Lepsien, G . , Nustede, R., Kohler. H., & Ludtke. F. E. (1994) Ann. N.Y. Acad. Sci. 713, 417-419 5. Rushakoff. R . J . , Goldfine, 1. D., Caner, J. D., & Liddle, R. A. (1987) J. Clin. Endocrinol. Metab. 65, 395-401 6. Reagan, J. E., Robinson, J. L., Lotti, V. J. & Goldman, M. E. (1987) Eur. J. Pharmacol 144, 241-243 - ?. Mottaghitalab, M., & Burdett. K. (1995) Unpublished results