AMP-activated protein kinase (AMPK) has recently been implicated in the control of preproinsulin gene expression in pancreatic islet β-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 β-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 (AMPKα1.T172D), 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 [Ca2+] 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 (AMPKα1.D157A) did not affect glucose-stimulated increases in [ATP], NAD(P)H or intracellular [Ca2+], 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 β-cell may thus represent a novel therapeutic strategy for the treatment of type 2 diabetes mellitus.

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.

While the subcellular organisation of mitochondria is likely to influence many aspects of cell physiology, its molecular control is poorly understood. Here, we have investigated the role of the retrograde motor protein complex, dynein-dynactin, in mitochondrial localisation and morphology. Disruption of dynein function, achieved in HeLa cells either by over-expressing the dynactin subunit, dynamitin (p50), or by microinjection of an anti-dynein intermediate chain antibody, resulted in (a) the redistribution of mitochondria to the nuclear periphery, and (b) the formation of long and highly branched mitochondrial structures. Suggesting that an alteration in the balance between mitochondrial fission and fusion may be involved in both of these changes, overexpression of p50 induced the translocation of the fission factor dynamin-related protein (Drp1) from mitochondrial membranes to the cytosol and microsomes. Moreover, a dominant-negative-acting form of Drp1 mimicked the effects of p50 on mitochondrial morphology, while wild-type Drp1 almost completely restored normal mitochondrial distribution in p50 over-expressing cells. Thus, the dynein/dynactin complex plays an unexpected role in the regulation of mitochondrial morphology in living cells, by controlling the recruitment of Drp1 to these organelles.

Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal β-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein(phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHCmut) blocked all vesicular movements with velocity>0.4 μm second-1, which demonstrates that kinesin activity was essential for vesicle motility in live β-cells. Moreover, expression of KHCmut strongly inhibited the sustained, but not acute,stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.

The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic beta-cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative-acting globular tail domain of MyoVa decreased by approximately 50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in beta-cells.

These data suggest that mGluRs and GABA(B)Rs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca(2+)-channels, cAMP generation and G-protein-mediated modulation of K(ATP) channels.

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca 2؉ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na ؉ -Ca 2؉ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca 2؉ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca 2؉ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca 2؉ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation (OXPHOS) 1 system, comprising 39 nuclear encoded and seven mitochondrially encoded subunits. Malfunction of this complex is associated with a wide variety of clinical syndromes ranging from often early lethal disorders, of which Leigh disease, a progressive encephalopathy, is the most frequent, to neurodegenerative disorders in adulthood, including Leber's hereditary optic neuropathy and Parkinson disease. In recent years, all human nuclear structural complex I genes have been characterized, which allowed us to elucidate the genetic defect in 40% of a cohort of complex I-deficient patients in which the enzyme defect was present in at least skeletal muscle and cultured skin fibroblasts (1-7). To enhance our understanding of the pathophysiological consequences of these diseases, with the final aim of developing new treatment strategies to stabilize or even cure these conditions, we study genetically characterized human complex I-deficient fibroblast cell lines as a model for OXPHOS system disease, knowing that these cells are glycolytic (8). Several hypotheses concerning the pathophysiology of OXPHOS diseases have been investigated of which the most consistent are (a) increased production of reactive oxygen species (9), (b) decreased potential across the mitochondrial inner membrane (10), (c) decreased intracellular ATP levels (11-13), and (d) altered Ca 2ϩ homeostasis (10, 12). In agreement with the reactive oxygen species hypothesis (a), we found that metallothioneins were up-regulated in all of the genetically characterized complex I-deficient cell lines (14). Pilot experiments w...

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-4OPCs 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 Ca 2+ release from intracellular stores. InOPCs 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. Glutamate is the major excitatory neurotransmitter in the CNS and its responses are mediated by ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptors. The iGluRs mediate fast synaptic transmission and the mGluRs are involved in a variety of modulatory events associated with neuronal activity. The iGluRs are multimeric, cation-specific ion channels that are classified into three families on the basis of their pharmacology, electrophysiology and sequence homology: namely the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors (Dingledine et al. 1999). The mGluRs are G-protein coupled receptors, which mediate relatively slow responses to glutamate. There are eight mGluR genes (mGluR1-8) and these have been divided into three groups based on pharmacological, signal transduction and sequence similarities. Group I comprises mGluR1 and mGluR5, which are involved in the mobilization of intracellular calcium by stimulating phospholipase C and are activated by (S)-3,5-dihydroxyphenylglycine (DHPG). In group II, the mGluR2 and mGluR3 are typically linked to inhibition of adenylyl cyclase (AC) and react effectively with (2S,1¢S,2¢S)-2-(carboxycyclopropyl)glycine (DCG-IV). In group III, the mGluR4, mGluR6, mGluR7 and mGluR8 are linked to inhibition of cAMP formation by AC and respond effectively to L-2-amino-4-phosphonobutyrate (Conn and Pin 1997;Schoepp et al. 1999). Oligodendrocytes are responsible for axon myelination and they a...