No abstract

Glucagon-like peptide 1 (7-36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium, [Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated beta-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3',5'-phosporothioate (Rp-cAMPS, up to 3 mM) and KT5720 (up to 10 microM), did not inhibit the GLP-1-induced [Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10-40 microM), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a PKA-independent action of H-89. Similarly to H-89, the potent but unselective protein kinase inhibitor staurosporine inhibited the GLP-1-induced [Ca2+]cyt elevation only at high concentrations, at which it also inhibited glucose-induced [Ca2+]cyt elevations. The same observations as with GLP-1 were made when adenylate cyclase was stimulated with forskolin, for selective examination of signal transduction downstream of receptor and G protein. Our results suggest that the GLP-1-induced [Ca2+]cyt elevation is mediated independently of PKA and thus belongs to the yet-little-characterized ensemble of effects that are mediated by binding of cAMP to other target proteins.

No abstract

This study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINmSF, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory actlon against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells.

Abstract.Glucagon-like peptide-1(7-36)amide/(7-37)

No abstract

The incretin effect is reduced in NIDDM, although a corresponding attenuation of incretin hormone secretion does not occur. We characterized the direct interaction of GLP-I, an important incretin hormone, and leptin on insulin secretion and signal transduction in B-cells. Leptin inhibited GLP-I stimulated insulin release from the isolated perfused rat pancreas. Both phases of the biphasic insulin secretory response were inhibited. GLP-I receptor binding and GLP-I induced cAMP generation remained unchanged. Leptin reduced the GLP-I mediated increase of cytosolic Ca2+ concentration. It had similar effects on calcium elevations induced by forskolin. The effect was more pronounced during the plateau phase than during the initial peak. These effects could help to explain leptin's inhibitory effects on insulin secretion. The inhibition of GLP-I's insulinotropic effects by leptin may be an interesting aspect in the pathophysiology of NIDDM. The existence of an "adipo-insular axis" is suggested, in which leptin represents a negative feed-back signal from the adipose tissue to the endocrine pancreas.

We investigated the influence of nutrients on spontaneous cytosolic calcium oscillations in InR1-G9 glucagonoma cells, a model for pancreatic alpha-cells. The oscillations depended on calcium release from stores and on calcium influx, partly through voltage-dependent calcium channels. Oscillations required the presence of at least 1 mM glucose, 50 microM alanine, or 50 microM glutamine, but were terminated by higher nutrient concentrations (40 mM glucose, or above 2 mM alanine or glutamine). The effects depended on the metabolism of the nutrients. Glutamine and alanine hyperpolarized the cells. This effect was inhibited (glutamine) or attenuated (alanine) by 1 mM ouabain. Our findings suggest that [Ca2+]i regulation in alpha-cells is dominated by slow oscillations induced by a lack of metabolic energy, resulting in decreased calcium export and storage, as well as increased calcium influx, partly due to depolarization caused by reduced sodium pump activity. These processes, leading to an elevated cytosolic calcium concentration, may mediate oscillations by calcium-induced calcium release from intracellular stores.