Marco A. Pineyro

Glucagon-Like Peptide-1 Regulates the Beta Cell Transcription Factor, PDX-1, in Insulinoma Cells

Glucagon-like peptide-1 (GLP-1) enhances insulin biosynthesis and secretion as well as transcription of the insulin, GLUT2 and glucokinase genes. The latter are also regulated by the PDX-1 homeoprotein. We investigated the possibility that GLP-1 may be having its long-term pleiotropic effects through a hitherto unknown regulation of PDX-1. We found that PDX-1 mRNA level was significantly increased (p<0.01) after 2 hours and insulin mRNA level was subsequently increased (p<0.01) after 3 hours of treatment with GLP-1 (10 nM) in RIN 1046-38 insulinoma cells. Under these experimental conditions, there was also a 1.6-fold increase in the expression of PDX-1 protein in whole cell and nuclear extracts. Overexpression of PDX-1 in these cells confirmed the finding of the wild type cells such that GLP-1 induced a 2-fold increase in whole cell extracts and a 3-fold increase in nuclear extracts of PDX-1 protein levels. The results of electrophoretic mobility shift experiments showed that PDX-1 protein binding to the Al element of the rat insulin II promoter was also increased 2 h post treatment with GLP-1. In summary, we have uncovered a previously unknown aspect to the regulation of PDX-1 in beta cells. This has important implications in the physiology of adult beta cells and the treatment of type 2 diabetes mellitus with GLP-1 or its analogs.

Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: Involvement of PDX-1 and HNF3β transcription factors†‡

Exendin‐4 (EX‐4), a long acting agonist of GLP‐1, induces an endocrine phenotype in Capan‐1 cells. Under culture conditions which include serum, ∼10% of the cells contain insulin and glucagon. When exposed to EX‐4 (0.1 nM, up to 5 days), the number of cells containing insulin and glucagon increased to ∼40%. Western blot analysis detected a progressive increase in protein levels of glucokinase and GLUT2 over 3 days of EX‐4 treatment. We explored the sequence of activation of certain transcription factors known to be essential for the beta cell phenotype: PDX‐1, Beta2/NeuroD, and hepatocyte nuclear factor 3β (HNF3β). Double immunostaining showed that PDX‐1 coexisted with insulin and glucagon in EX‐4‐treated cells. Treatment caused an increase in PDX‐1 protein levels by 24 h and induced its nuclear translocation. Beta2/NeuroD protein levels also increased progressively over 24 h. HNF3β protein level increased twofold as early as 6 h after EX‐4 treatment. EMSA results indicated that EX‐4 caused a 12‐fold increase in HNF3β binding to PDX‐1 promoter area II. Beta2/NeuroD protein levels progressively increased after 24 h treatment. Differentiation to insulin‐producing cells was also seen when Capan‐1 cells were transfected with pdx‐1, with 80% of these cells expressing insulin 3 days after transfection. PDX‐1 antisense totally inhibited such conversion. During the differentiation of duct cells to endocrine cells, cAMP levels (EX‐4 is a ligand for the GLP‐1, G‐protein coupled receptor) and MAP kinase activity increased. Our results indicate that EX‐4 activates adenylyl cyclase and MAP kinase which, in turn, may lead to activation of transcription factors necessary for an endocrine phenotype. Published 2002 Wiley‐Liss, Inc.

GLUCAGON-LIKE PEPTIDE-1 DOES NOT MEDIATE AMYLASE RELEASE FROM AR42J CELLS

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon‐like peptide‐1 (GLP‐1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP‐1 receptors on AR42J cells by reverse trasncriptase‐polymerase chain reaction (RT‐PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP‐1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP‐1 increased intracellular calcium. Similar to GLP‐1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK‐mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP‐1 treatment, where no such increased phosphorylation was seen. Instead, GLP‐1 decreased such protein phosphorylations. Genestein blocked CCK‐induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP‐1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470–478, 1999. Published 1999 Wiley‐Liss, Inc.

Adenylyl cyclase activation by halide anions other than fluoride

Abstract Adenylyl cyclase of rat liver and fat cells is activated by chloride, bromide, and iodide in addition to fluoride, previously believed to be uniquely effective among the halide anions. Liver homogenates are activated approximately 6 fold by fluoride while chloride and bromide increase cyclase by 3 fold and iodide about 2 fold. Optimal concentrations of chloride, bromide and iodide are about 100 times higher than those required for activation by fluoride. The cyclase of fat cell ghosts is activated some 9 fold by fluoride, but the other halide anions produced effects very similar in magnitude to those seen with liver, although for fat the optimally effective concentrations were lower. These observations appear to relate adenylate cyclase to a number of other anion activated enzymes, some of which have already been studied in pure form by a number of physico-chemical techniques, and which may serve as models for understanding the action of fluoride and other anions on adenylyl cyclase.

Partial purification from rat and pig liver of cytosolic stimulators of hormone-sensitive adenylate cyclase: quantitation and resolution of two components.

Procedures were carried out to isolate from liver cytosol the protein activators of hormone-sensitive adenylate cyclase. A method for quantifying amounts of activator protein was used to monitor recovery after each isolation step. The activator proteins were precipitable by ammonium sulfate (30-60% saturation) and partially recoverable from the precipitate. On gel filtration of cytosol, stimulatory activity for glucagon-sensitive adenylate cyclase was recovered in two peaks representing proteins with molecular weights of 49,000 and 25,500. Exposure to GTP-Sepharose reduced liver cytosols content of stimulatory factors for glucagon-sensitive adenylate cyclase by up to 70%. However, soluble protein adenylate cyclase activators distinct from GTP could not be subsequently eluted from the affinity matrix. Purification efforts were thwarted by factor instability and large losses during simple and conventional steps despite the use of a variety of protein stabilizers and protease inhibitors. If the problem of stimulator instability can be overcome, large-scale purification should be possible using pig liver as a starting material.

Membrane Association of Soluble Protein Activators of Rat Liver Adenylate Cyclase Evidence for Distinctness from the Guanine Nucleotide-binding Stimulating Protein (NS)

Sonication of a crude rat liver membrane preparation and centrifugation at 100,000 X g yielded a supernatant which activated basal and hormone-sensitive adenylate cyclases [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. The membrane origin of the stimulatory activity was confirmed by the use of lactate dehydrogenase as a marker for contamination by cytosol. The solubility of the activating factors was verified by their passage through 0.05 micron diameter pores of Millipore filters. The membrane-derived activators were nondialyzable and destroyed by heat and trypsin in the same manner as adenylate cyclase activators detectable in cytosol. Stimulation by factors from membranes and cytosol was not additive. The amount of the activators which could be freed from membranes by sonication was 12-15% of that contained in cytosol previously separated from the membranes. Soluble activators from the two sources had limited ability to restore adenylate cyclase activity to membranes from the cyclone of S49 mouse lymphoma cells which are deficient in the enzymes guanine nucleotide-binding stimulatory protein, Ns. Cytosol did not contain a substrate for ADP-ribosylation by cholera toxin that corresponded electrophoretically to Ns. Furthermore, purified Ns did not affect adenylate cyclase activity in preparations stimulated by the soluble activators. These findings suggest that the activating factors found in cytosol may be released from membranes during tissue homogenization. Because these protein activators can be obtained from membranes without use of detergents and can neither substitute for nor be substituted for by Ns in functional assays, they are distinct from Ns.

Iodate promotes guanosine triphosphate stimulation of human fat-cell adenylate cyclase.

Abstract Iodate promoted GTP activation of human fat-cell adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and reversed the usual inhibitory effect which GTP presumably exerts through interaction with a distinct regulatory subunit of the enzyme. GTP (0.1 m m ) inhibited adenylate cyclase by 52%, while NaIO3 (0.1 to 5 m m ) was minimally stimulatory. GTP stimulation in the presence of IO3− was dependent on the concentrations of both salt and nucleotide with maximal stimulation (up to 2.5-fold times basal) occurring at 0.5–1.0 m m IO3− and 0.1 m m GTP. Iodate salts of Li+, Na+, K+, and Rb+ all enhanced GTP action with similar salt concentration dependence, although the cation species did affect the magnitude of GTP activation. The action of IO3− in promoting GTP stimulation differed in several respects from that of Na+ cation which allows activation by the nucleotide relatively independent of the accompanying anion ( M. S. Katz, J. S. Partilla, C. R. Schneyer, M. A. Pineyro, R. I. Gregerman, 1981, Proc. Nat. Acad. Sci. USA78, 7417–7421 ). Effective IO3− concentrations were 100-fold lower than those for Na+.IO3−-induced GTP stimulation was not increased by raising the temperature from 30 to 37 °C, in contrast to the temperature dependence of the Na+ effect. Furthermore, unlike Na+, IO3− did not allow stimulation of adenylate cyclase by the nonhydrolyzable GTP analog, 5′-guanylyl-β-γ-imidodiphosphate. Iodate did, however, eliminate inhibition of enzyme by the analog, thereby suggesting that IO3− did not act by inhibiting GTPase activity. Time courses showed that basal adenylate cyclase activity decreased with time but that in the presence of IO3− the initial basal rate of cyclic AMP formation was maintained for at least 10 min. Iodate, when added with GTP, reduced the lag time caused by the nucleotide and produced an increase in enzyme acivity over basal. Vanadate, like IO3−, stimulated adenylate cyclase at millimolar concentrations but had only a minimal effect on the interaction of GTP with the fat-cell enzyme, while molybdates modulation of the nucleotide effect appeared to be entirely attributable to the accompanying Na+ cation. Our results show for the first time modulation of GTP effect by an anion and suggest that IO3− may be a useful probe of the GTP-binding regulatory protein and its interaction with the adenylate cyclase complex.