Glucose Dependence of the Regulated Secretory Pathway in αTC1-6 Cells

Abstract

We have investigated the effects of chronically elevated glucose concentrations on the pancreatic alpha-cell line alphaTC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2. The secretion of GLP-1, a proglucagon-derived peptide produced by cleavage of proglucagon by PC1/3, was also increased in response to high glucose. Gene expression profiling experiments showed that a number of components of the regulated secretory pathway were up-regulated at high glucose concentrations, including processing enzymes and exocytotic proteins. Immunoblot analysis showed that the expression of the exocytotic SNARE proteins, as well as that of PC1/3, chromogranin A, and 7B2, were all increased after chronic exposure to high glucose levels. Immunocytochemistry showed no changes in the expression of the mature alpha-cell markers glucagon and brn-4 and no induction of the immature alpha-cell marker pdx-1. We conclude that chronically elevated glucose concentrations up-regulate the regulated secretory response of the alpha-cell.

Upregulation of alpha cell glucagon-like peptide 1 (GLP-1) in Psammomys obesus—an adaptive response to hyperglycaemia?

“…One possibility is increased production or activity of PC1/3. Indeed it has been shown that PC1/3 is upregulated by high glucose in alpha cells [34] and in hyperglycaemic db/db mice [17]. Therefore, it is possible that this is also the mechanism in our studies.…”

Physiology of the pancreatic α-cell and glucagon secretion: role in glucose homeostasis and diabetes

“…It was concluded that insulin, unlike glucose, modulates glucagon expression. The lack of response to glucose was further confirmed in isolated rat islets (Gremlich et al 1997, Dumonteil et al 2000, contrasting with the up-regulation of glucagon expression observed in clonal a-cells (Dumonteil et al 1999, McGirr et al 2005. The effect of amino acids on glucagon gene regulation has also been studied.…”

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes

“…Lys 19 (mtt) was selectively deprotected using 2% trifluoroacetic acid, 5% triisopropylsilane in dichloromethane, followed by coupling to Cy5 succinimidyl ester (0.025 mmol, 19 mg) in the presence of N,N-diisopropylethylamine (0.6 mmol, 100 L). Once fully functionalized, Dpr 3 (octanoyl), Lys 19 (Cy5) ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) was cleaved from the resin using 95% trifluoroacetic acid, 2.5% triisopropylsilane and 2.5% water (2 mL). The crude peptide was precipitated using ice cold tert-butylmethylether (20 mL) and the dark blue crude solid was then isolated by centrifugation.…”

“…HEK 293 cells were transiently transfected with the cDNA encoding GHS-R1a in order to visualize the binding of Cy5-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) in situ. Both wt HEK and HEK/GHS-R1a cells were plated at a density of 1 × 10 6 cells/well in a 6-well plate, and serum starved overnight.…”

“…These results indicate that GHS-R signaling is altered in cardiovascular disease, and point toward the use of ghrelin in pharmacologic therapy of heart disease. Therefore, given the importance of GHS-R signaling and expression in the heart, we are developing ghrelin analogs for the purpose of generating agents for in situ and in vivo imaging of cardiac GHS-R. We have previously designed a fluorescent ghrelin analog consisting of a fluorescein moiety attached through a lysine to the N-terminal 18 amino acids of ghrelin (fluorescein-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)) [20] and have shown that it specifically binds to GHS-R1a in mouse heart tissue.…”