299 PYROGLUTAMYL- PROLINE AMIDE (EFP}INIIIBITSTHYROTROPIN.RELEA~NG HORMONE (TRII) STIMULATED BUT NOT GLP-I(7-36)AMIDE, ARGININE, OR GLIBENCLAMIDE STIMULATED INSULIN SECRETION IN HIT-TI5 CELIa. RN KULEARNI, M HAOU£, DM SMITH, MA GHATEI & SR BLOOM. Dept. of Medlcln#, HammersmlOl Ho~i~l, Du Cane Road, London WI2 0NN, U£. TRH and the TRH-rolated peptide, EFP, have been shown to be present in the insulin secreting clonal/~-cell line HIT-T15. Although TRH has been shown to stimulate insulin secretion from HIT cells, its role in the autocrine control of insulin secretion in the adult pancreatic islet is uncertain. In the present study, we demonstrate that TRH (1/zM) significantly stimulates insulin secretion from the HIT cells, in a dose-dependent manner (n = 11; p < 0.01) while EFP, at a concentration of 1 #M, completely suppressed TRH-stimulated (1 /~M) insulin secretion (n = I0, p = ns). We also demonstrate that EFP alone suppressed insulin secretion dose-dependently, reaching a maximum at 1 /zM (n = 12; p < 0.01). In addition, EFP failed to reverse the stimulatory effect of 100 nM GLP-l(7- 36)amide, 10 mM arginine and t/zM of the oral hypoglycaemic agent glibenclamide (each n = 5). These results suggest that EFP, specifically antagonises the stimulatory effect of TRH and therefore can be used as a tool, to study the role of endogenously produced TRH from the pancreatic islets or the insulin-producing clonal cell lines. The failure of EFP to reverse the stimulatory effect of the other seeretagogues, indicates that EFP probably acts by a mechanism independent of cAMP, nitric oxide or via the ATP-dependent K* channel respectively. INVESTIGATION INTO THE PRESENCE OF REGULATORY PEPTIDES IN RAT ISLETS OF LANGERHANS LINDA STEVENS, PHIL JONES & STEPHEN BLOOM Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN. A number of regulatory peptides have been shown to be produced locally by the islets of Langerhans and their potential role in regulating islet function through an autocrine/paracrine system is becoming increasingly apparent. Studies have shown that the mRNA and concentrations of NPY,VIP and p-CGRP in the islets of Langerhans are low in comparison to insulin and glucagon. As a result, investigation of the synthesis of regulatory peptides in this tissue requires separation of islets from the exocrine tissue in order to obtain a purified preparation. We have previously achieved this by hand picking islets from collagenase digested pancreata using a light microscope. Apart from being time consuming, this method cannot produce preparations free from exocrine tissue. For this reason we have evaluated the technique of BSA density gradient centrifugation in islet preparation and compared it to hand picking. Analysis of this method of islet purification showed at least 60% purification, with a yield of approx. 1000 islets/rat. Northern blot analysis employing a rat insulin cDNA probe and a rat NPY cRNA probe revealed that the mRNA content of both peptides from islets obtained by the BSA density gradient technique was approx. 6 - 7 fold greater than the mRNA content of the peptides analysed from islets obtained by the standard hand picking method. Analysis of the exocrine tissue, which can be isolated as a single layer, was found to be free from islet material. Preliminary work has shown that islets isolated by this technique are functional and can be used in perfusion studies. These results demonstrate that islet purification is greatly improved using the BSA density gradient method, and that this technique should greatly facilitate studies into the role of these peptides in this tissue.

Investigation into the presence of regulatory peptides in rat islets of Langerhans

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Page 1: Investigation into the presence of regulatory peptides in rat islets of Langerhans

299

PYROGLUTAMYL- PROLINE AMIDE (EFP} INIIIBITS THYROTROPIN.RELEA~NG HORMONE (TRII) STIMULATED BUT NOT GLP-I(7-36)AMIDE, ARGININE, OR GLIBENCLAMIDE STIMULATED INSULIN SECRETION IN HIT-TI5 CELIa.

RN KULEARNI, M HAOU£, DM SMITH, MA GHATEI & SR BLOOM. Dept. of Medlcln#, HammersmlOl Ho~i~l, Du Cane Road, London WI2 0NN, U£.

TRH and the TRH-rolated peptide, EFP, have been shown to be present in the insulin secreting clonal/~-cell line HIT-T15. Although TRH has been shown to stimulate insulin secretion from HIT cells, its role in the autocrine control of insulin secretion in the adult pancreatic islet is uncertain. In the present study, we demonstrate that TRH (1/zM) significantly stimulates insulin secretion from the HIT cells, in a dose-dependent manner (n = 11; p < 0.01) while EFP, at a concentration of 1 #M, completely suppressed TRH-stimulated (1 /~M) insulin secretion (n = I0, p = ns). We also demonstrate that EFP alone suppressed insulin secretion dose-dependently, reaching a maximum at 1 /zM (n = 12; p < 0.01). In addition, EFP failed to reverse the stimulatory effect of 100 nM GLP-l(7- 36)amide, 10 mM arginine and t /zM of the oral hypoglycaemic agent glibenclamide (each n = 5). These results suggest that EFP, specifically antagonises the stimulatory effect of TRH and therefore can be used as a tool, to study the role of endogenously produced TRH from the pancreatic islets or the insulin-producing clonal cell lines. The failure of EFP to reverse the stimulatory effect of the other seeretagogues, indicates that EFP probably acts by a mechanism independent of cAMP, nitric oxide or via the ATP-dependent K* channel respectively.

INVESTIGATION INTO THE PRESENCE OF REGULATORY PEPTIDES IN RAT ISLETS OF LANGERHANS

LINDA STEVENS, PHIL JONES & STEPHEN BLOOM Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN.

A number of regulatory peptides have been shown to be produced locally by the islets of Langerhans and their potential role in regulating islet function through an autocrine/paracrine system is becoming increasingly apparent. Studies have shown that the mRNA and concentrations of NPY,VIP and p-CGRP in the islets of Langerhans are low in comparison to insulin and glucagon. As a result, investigation of the synthesis of regulatory peptides in this tissue requires separation of islets from the exocrine tissue in order to obtain a purified preparation. We have previously achieved this by hand picking islets from collagenase digested pancreata using a light microscope. Apart from being time consuming, this method cannot produce preparations free from exocrine tissue. For this reason we have evaluated the technique of BSA density gradient centrifugation in islet preparation and compared it to hand picking. Analysis of this method of islet purification showed at least 60% purification, with a yield of approx. 1000 islets/rat. Northern blot analysis employing a rat insulin cDNA probe and a rat NPY cRNA probe revealed that the mRNA content of both peptides from islets obtained by the BSA density gradient technique was approx. 6 - 7 fold greater than the mRNA content of the peptides analysed from islets obtained by the standard hand picking method. Analysis of the exocrine tissue, which can be isolated as a single layer, was found to be free from islet material. Preliminary work has shown that islets isolated by this technique are functional and can be used in perfusion studies. These results demonstrate that islet purification is greatly improved using the BSA density gradient method, and that this technique should greatly facilitate studies into the role of these peptides in this tissue.