Signal transduction and exocytosis in the pancreatic â-cell

A variety of physiologic, pharmacologic, biochemical and electrophysiologic techniques are in use to explore the regulation of pancreatic â-cell function. These include perifusion of pancreatic islets and cloned cells combined with radioimmunoassay, to measure the minute to minute release of insulin under basal, stimulated or inhibited conditions; the use of fluorescent dyes to measure the concentration of intracellular Ca2+ and other second messengers in stimulus-secretion coupling and biochemical techniques for enzyme assays or intracellular substrate determinations; electrophysiologic and patch clamp techniques for the measurement of ion channel activity; and capacitance measurements to monitor exocytosis in single â-cells. Major emphasis is currently placed on three areas.

• The action of glucose to stimulate insulin secretion is complex and includes at least two major mechanisms. One of these actions is to depolarise the cell and stimulate Ca2+ entry through voltage dependent Ca2+ channels. Increased intracellular Ca2+ is an effective stimulus to secretion. However, the major increase in secretion is due to a second action of glucose to augment the response to the Ca2+ signal. The glucose signaling pathway responsible for augmentation is thought to enter stimulus-secretion coupling at a late stage, possibly at a point close to the final exocytotic step.
• Several hormones and peptides inhibit insulin secretion. Among them are norepinephrine, somatostatin, prostaglandins, galanin, and amylin. They all exert at least four inhibitory effects on the â-cell. These include activation of K+ channels to hyperpolarize or repolarize the membrane potential, thus inactivating the cell; inhibition of voltage-dependent Ca2+-channels to block Ca2+ entry; inhibition of adenylyl cyclase to lower cyclic AMP levels; and inhibition of exocytosis by an as yet unidentified mechanism late in stimulus-secretion coupling.
• Understanding exocytosis, the process whereby the insulin-containing granules fuse with the plasma membrane and allow insulin to be released, is key to understanding the regulation of both stimulation, augmentation and inhibition. Thus we are studying exocytosis by both biochemical and electrophysiological techniques.

The group expects that the variety of approaches they are using will lead to rapid progress in our understanding of the control of pancreatic â-cell function. This, in turn, should lead to greater understanding of those diseases such as diabetes that involve disorders of insulin secretion.

• Zhao, Y., Straub, S.G., and Sharp, G.W.G. Both G_i and G_o heterotrimeric G proteins are required to exert the full effect of norepinephrine on the b-cell K_ATP channel. J. Biol. Chem. 283: 5306-5316 (2008).
• Zhao, Y., Sharp, G.W.G., and Straub, S.G. The inhibitors of protein acylation, cerulenin and tunicamycin, increase voltage-dependent Ca²⁺ currents in the insulin-secreting INS 832/13 cell. Biochemical Pharmacology 74: 273-280 (2007).
• Cheng, H., Straub, S.G. and Sharp, G.W.G. Inhibitory role of Src family tyrosine kinases on Ca²⁺-dependent insulin release. Amer. J. Physiol. (Endocrinol. Metab.) 292: 146-151 (2007).
• Straub, S.G. and Sharp G.W.G. Inhibition of insulin secretion by cerulenin might be due to impaired glucose metabolism. Diabetes/Metabolism Research and Reviews 23: E845-852 (2007).
• Komatsu, M., Schermerhorn, T, Noda, M., Straub, S.G., Aizawa, T., and Sharp, G.W.G. Glucose and nutrient-induced increase in insulin secretion in the absence of a rise in [Ca²⁺]_i. In Commentaries on Perspectives in Diabetes, Volume 2 (1993-1997). Editor, R. Paul Robertson, American Diabetes Association (2006).
• McClanahan, T., Straub, S.G., Sharp, G.W.G., and Loew, M. Automated insulin granule segmentation from electron photomicrographs of rat pancreatic beta-cells. Proceedings of SPIE Medical Imaging. Physiology, Function, and Structure from Medical Images, Amir A Amini and Armando Manduca, Editors, 5746: 706-717 (2005).
• Straub, S.G., Shanmugam, G. and G.W.G. Sharp. Stimulation of insulin release by glucose is associated with an increase in the number of docked granules in the beta-cells of rat pancreatic islets. Diabetes 53: 3179-3183 (2004).
• Straub, S.G., and G.W.G. Sharp. Massive augmentation of stimulated insulin secretion induced by fatty acid-free BSA in rat pancreatic islets. Diabetes 53: 3152-3158 (2004).
• Gunawardana, S.C., Liu, Y-J, MacDonald, M.J., Straub, S.G.,and Sharp, G.W.G. Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets. Amer. J. Physiol. (Endocrinology) 287: E828-E833 (2004).
• Straub, S.G., and G.W.G. Sharp. Hypothesis: One rate limiting step controls the magnitude of both phases of glucose-stimulated insulin secretion. Amer. J. Physiol. (Cell) 287: C565-C571 (2004).
• Cosgrove, K.E., S.G. Straub, P.D. Barnes, J. Chapman, G.W.G. Sharp, and M.J. Dunne. Y-26763: ATP-sensitive K⁺ channel activation and the inhibition of insulin release from human pancreatic b-cells. European Journal of Pharmacology 486: 133-139 (2004).
• Straub, S.G. and G.W.G. Sharp. The two phases of insulin secretion: mechanisms and controls. In Diabetes Mellitus: A Fundamental and Clinical Text. 3rd edition. Editors: LeRoith, D., Olefsky, J.M. and Taylor, S.I. Lippincott. Williams and Wilkins, pp. 3-14 (2004).
• Liu, Y-J, H. Cheng, H. Drought, M.J. MacDonald, G.W.G. Sharp, and S.G. Straub. Activation of the K_ATP channel-independent signaling pathway by the non-hydrolysable analog of leucine, BCH. Amer. J. Physiol. Endocrinol. Metab. 285: E380-E389 (2003).
• Cheng, H., S.G. Straub, and G.W.G. Sharp. Protein acylation in the inhibition of insulin secretion by norepinephrine, somatostatin, galanin, and PGE2. Amer. J. Physiol. Endocrinol. Metab. 285: E287-E294 (2003).
• Straub, S.G., Mulvaney-Musa, J., Yajima, H., Weiland, G.A., and Sharp, G.W.G. Stimulation of insulin secretion by denatonium, one of the most bitter tasting substances known. Diabetes 52: 356-364 (2003).
• Straub, S.G., and Sharp, G.W.G. Glucose-stimulated signaling pathways in biphasic insulin secretion. Diabetes/Metabolism Research and Reviews, 18: 451-463 (2002).
• Noda, M., Yamashita, S., Takahashi, N., Eto, K., Shen, L., Izumi, K., Daniel. S., Tsubamoto, Y., Nemoto, T., Iino, M., Kasai, H., Sharp, G.W.G., and Kadowacki, T. Switch to anaerobic glucose metabolism with NADH accumulation in the b-cell model of mitochondrial diabetes; characteristics of bHC9 cells deficient in mitochondrial DNA transcription. J. Biol. Chem. 277: 41817-41826 (2002).
• Straub, S.G., Daniel, S., and Sharp, G.W.G. Hypo-osmotic shock stimulates insulin secretion by two distinct mechanisms. Studies with the bHC9 cell. Amer. J. Physiol. Metabol. 282: E-1070-E1076 (2002).
• Daniel, S., Noda, M., Cerione, R.A., Sharp, G.W.G. A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release. Biochemistry 41: 9663-9671 (2002).
• Straub, S.G., H. Yajima, M. Komatsu, T. Aizawa, and G.W.G. Sharp. The effects of cerulenin, an inhibitor of protein acylation, on the two phases of glucose-stimulated insulin secretion. Diabetes 51: S91-95 (2002).
• Bratanova-Tochkova, T.K., H. Cheng, S. Daniel, S. Gunawardana, Y.-J. Liu, J. Mulvaney-Musa, T. Schermerhorn, S.G. Straub, H. Yajima, and G.W.G. Sharp. Triggering and augmentation mechanisms, granule pools and biphasic insulin secretion. Diabetes 51: S83-90 (2002).
• Gunawardana, S.C. and Sharp, G.W.G. Intracellular pH plays a critical role in glucose-Induced time-dependent potentiation of insulin release in rat islets. Diabetes 51: 105-113 (2002).
• Straub, S.G., G.W.G. Sharp, M.D. Meglasson, and C.J. De Souza. Progesterone inhibits insulin secretion by a membrane delimited, non-genomic action. Bioscience Reports 21: 653-666 (2001).
• Dunne, M.J., Ammala, C., Straub, S.G. and Sharp, G.W.G. Electrophysiology of the pancreatic b-cell and the mechanisms of inhibition of insulin release. In Handbook of Physiology: The Endocrine Pancreas and Regulation of Metabolism. Editors, Jefferson, L.S. and Cherrington, A.D. Oxford University Press (2001).
• Yajima, H., Komatsu, M., Sato, Y., Yamada, S., Yamauchi, K., Sharp, G.W.G., Aizawa, T., and Hashizume, K. Norepinephrine inhibits glucose-stimulated, Ca²⁺-independent insulin release independently from its action on adenylyl cyclase. Endocrine J.48: 647-654 (2001).
• Tsubamoto, Y., K. Eto, M. Noda, S. Daniel, S. Suga, S. Yamashita, H. Kasai, M. Wakui, G.W.G. Sharp, S. Kimura, and T. Kadowaki. Hexamminecobalt III chloride inhibits glucose-induced insulin secretion at the exocytotic process. J. Biol. Chem.276: 2979-2985 (2001).
• Straub, S.G., K.E. Cosgrove, C. Ammala, R.M. Shepherd, R.E. O'Brien, P.D. Barnes, N. Kuchinski, J.C. Chapman, M. Schaeppi, B. Glaser, K.J. Lindley, G.W.G. Sharp, A. Aynsley-Green, and M.J. Dunne. Hyperinsulinism of infancy; The regulated release of insulin by KATP channel-independent pathways. Diabetes: 329-339 (2001).
• Straub, S.G., B. Kornreich, R.E. Oswald, E.F. Nemeth, and G.W.G. Sharp. The calcimimetic R-467 potentiates insulin secretion in pancreatic b-cells by activation of a non-specific cation channel. J. Biol. Chem. 275: 18777-18784 (2000).
• Yajima, H., M. Komatsu, S. Yamada, S.G. Straub, T. Kaneko, Y. Sato, K. Yamauchi, K. Hashizume, G.W.G. Sharp, and T. Aizawa. Cerulenin, an inhibitor of protein acylation, selectively attenuates nutrient stimulation of insulin release: a study in rat pancreatic islets. Diabetes 49: 712-717 (2000).
• Schermerhorn, T., and Sharp, G.W.G. Norepinephrine acts on the KATP channel and produces different effects on [Ca²⁺]i in oscillating and non-oscillating HIT-T15 cells. Cell Calcium 27: 163-173 (2000).