Aniko Varadi

Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression.

AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet beta-cells [da Silva Xavier, Leclerc, Salt, Doiron, Hardie, Kahn and Rutter (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4023-4028]. Using pharmacological and molecular strategies to regulate AMPK activity in rat islets and clonal MIN6 beta-cells, we show here that the effects of AMPK are exerted largely upstream of insulin release. Thus forced increases in AMPK activity achieved pharmacologically with 5-amino-4-imidazolecarboxamide riboside (AICAR), or by adenoviral overexpression of a truncated, constitutively active form of the enzyme (AMPK alpha 1.T(172)D), blocked glucose-stimulated insulin secretion. In MIN6 cells, activation of AMPK suppressed glucose metabolism, as assessed by changes in total, cytosolic or mitochondrial [ATP] and NAD(P)H, and reduced increases in intracellular [Ca(2+)] caused by either glucose or tolbutamide. By contrast, inactivation of AMPK by expression of a dominant-negative form of the enzyme mutated in the catalytic site (AMPK alpha 1.D(157)A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca(2+)], but led to the unregulated release of insulin. These results indicate that inhibition of AMPK by glucose is essential for the activation of insulin secretion by the sugar, and may contribute to the transcriptional stimulation of the preproinsulin gene. Modulation of AMPK activity in the beta-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus.

Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca2+ concentrations ([Ca2+]c) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2–synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet β-cells: (a) increases in [Ca2+]c cause a prompt increase in intravesicular-free Ca2+ concentration ([Ca2+]SV), which is mediated by a P-type Ca2+-ATPase distinct from the sarco(endo) plasmic reticulum Ca2+-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2+ pumps; (b) steady state Ca2+ concentrations are 3–5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca2+; (c) inositol (1,4,5) trisphosphate has no impact on [Ca2+]SV in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca2+]SV. Thus, secretory vesicles represent a dynamic Ca2+ store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca2+-induced Ca2+ release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

Regulation of Gene Expression by Glucose in Pancreatic β-Cells (MIN6) via Insulin Secretion and Activation of Phosphatidylinositol 3′-Kinase

Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet β-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998)Mol. Cell 1, 933–938), the role of insulin in the control of other β-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 β-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1–20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca2+ channel blocker verapamil, the ATP-sensitive K+ channel opener diazoxide, or the α2-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mmglucose failed to induce the promoter after inhibition of phosphatidylinositol 3′-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3′-kinase (Δp85) or the phosphoinositide 3′-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5′-AMP-activated protein kinase with anti-5′-AMP-activated protein kinase α2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 μm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 β-cells, stimulation of insulin secretion is important for the activation by glucose of L-PK as well as the PPI promoter, but does not cause increases in glucokinase gene expression or glucose metabolism.

Functional metabotropic glutamate receptors are expressed in oligodendrocyte progenitor cells

We investigated the expression of metabotropic glutamate receptor (mGluR) isoforms in CG‐4 rodent oligodendroglial progenitor cells (OPC) and rat brain oligodendrocytes. Our RT‐PCR analysis detected mRNAs for mGluR3 and mGluR5 isoforms in OPCs. Although neurons express both mGluR5a and mGluR5b splice variants, only mGluR5a was identified in OPCs. Antibodies to mGluR2/3 and mGluR5 detected the corresponding receptor proteins in immunoblots of OPC membrane fractions. Furthermore, immunocytochemical analysis identified mGluR5 in oligodendrocyte marker O4‐positive OPCs. The expression of mGluR5 was also demonstrated in oligodendrocyte marker (O4 and O1) positive cells in white matter of postnatal 4‐ and 7‐day‐old rat brain sections using immunofluorescent double labelling and confocal microscopy. The mGluR5 receptor function was assessed in CG‐4 OPCs with fura‐2 microfluorometry. Application of the mGluR1/5 specific agonist (S)‐3,5‐dihydroxyphenylglycine (DHPG) induced calcium oscillations, which were inhibited by the selective mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl) pyridine hydrochloride (MPEP). The DHPG induced calcium oscillations required Ca2+ release from intracellular stores. In OPCs the group II mGluR agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV) decreased forskolin‐stimulated cAMP synthesis, indicating the presence of functional mGluR3. The newly identified mGluR3 and mGluR5a may be involved in the differentiation of oligodendrocytes, myelination and the development of white matter damage.