Guy A Rutter — Page 4 | PubMed Health

PubMed2002

Dynamic imaging of endoplasmic reticulum Ca2+ concentration in insulin-secreting MIN6 Cells using recombinant targeted cameleons: roles of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 and ryanodine receptors.

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](cyt), and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse beta-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.

Boron Compounds↗

PubMed2002

Stimulation of acetyl-CoA carboxylase gene expression by glucose requires insulin release and sterol regulatory element binding protein 1c in pancreatic MIN6 beta-cells.

Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet beta-cells may contribute to glucolipotoxicity. Here, we provide evidence that enhanced insulin release plays an important role in the activation of this gene by glucose. Glucose (30 vs. 3 mmol/l) increased ACCI mRNA levels approximately 4-fold and stimulated ACCI (pII) promoter activity >30-fold in MIN6 cells. The latter effect was completely suppressed by blockade of insulin release or of insulin receptor signaling. However, added insulin substantially, but not completely, mimicked the effects of glucose, suggesting that intracellular metabolites of glucose may also contribute to transcriptional stimulation. Mutational analysis of the ACCI promoter, and antibody microinjection, revealed that the effect of glucose required sterol response element binding protein (SREBP)-1c. Moreover, adenoviral transduction with dominant-negative-acting SREBP1c blocked ACCI gene induction, whereas constitutively active SREBP1c increased ACCI mRNA levels. Finally, glucose also stimulated SREBP1c transcription, although this effect was independent of insulin release. These data suggest that glucose regulates ACCI gene expression in the beta-cell by complex mechanisms that may involve the covalent modification of SREBP1c. However, overexpression of SREBP1c also decreased glucose-stimulated insulin release, implicating SREBP1c induction in beta-cell lipotoxicity in some forms of type 2 diabetes.

Acetyl-CoA Carboxylase↗