Pierre Maechler

Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis

The hormone insulin is stored in secretory granules and released from the pancreatic β-cells by exocytosis. In the consensus model of glucose-stimulated insulin secretion, ATP is generated by mitochondrial metabolism, promoting closure of ATP-sensitive potassium (KATP) channels, which depolarizes the plasma membrane. Subsequently, opening of voltage-sensitive Ca2+ channels increases the cytosolic Ca2+ concentration ([Ca2+]c) which constitutes the main trigger initiating insulin exocytosis. Nevertheless, the Ca2+ signal alone is not sufficient for sustained secretion. Furthermore, glucose elicits a secretory response under conditions of clamped, elevated [Ca2+]c (refs 5, 6). A mitochondrial messenger must therefore exist which is distinct from ATP. We have now identified this as glutamate. We show that glucose generates glutamate from β-cell mitochondria. A membrane-permeant glutamate analogue sensitizes the glucose-evoked secretory response, acting downstream of mitochondrial metabolism. In permeabilized cells, under conditions of fixed [Ca2+]c, added glutamate directly stimulates insulin exocytosis, independently of mitochondrial function. Glutamate uptake by the secretory granules is likely to be involved, as inhibitors of vesicular glutamate transport suppress the glutamate-evoked exocytosis. These results demonstrate that glutamate acts as an intracellular messenger that couples glucose metabolism to insulin secretion.

Mitochondrial function in normal and diabetic |[beta]|-cells

The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic β-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on β-cell function reflects the importance of mitochondria in the control of insulin secretion. The β-cell mitochondria serve as fuel sensors, generating factors that couple nutrient metabolism to the exocytosis of insulin-containing vesicles. The latter process requires an increase in cytosolic Ca2+, which depends on ATP synthesized by the mitochondria. This organelle also generates other factors, of which glutamate has been proposed as a potential intracellular messenger.

Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.

Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from β-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from α-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet α- and β-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although α-cells, like β-cells, possess the inherent capacity to respond to nutrients, secretion from α-cells is normally suppressed by the simultaneous activation of β-cells. Zinc released from β-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.

Hydrogen Peroxide Alters Mitochondrial Activation and Insulin Secretion in Pancreatic Beta Cells

The effects of a transient exposure to hydrogen peroxide (10 min at 200 μmH2O2) on pancreatic beta cell signal transduction and insulin secretion have been evaluated. In rat islets, insulin secretion evoked by glucose (16.7 mm) or by the mitochondrial substrate methyl succinate (5 mm) was markedly blunted following exposure to H2O2. In contrast, the secretory response induced by plasma membrane depolarization (20 mm KCl) was not significantly affected. Similar results were obtained in insulinoma INS-1 cells using glucose (12.8 mm) as secretagogue. After H2O2 treatment, glucose no longer depolarized the membrane potential (ΔΨ) of INS-1 cells or increased cytosolic Ca2+. Both ΔΨ and Ca2+ responses were still observed with 30 mm KCl despite an elevated baseline of cytosolic Ca2+ appearing ∼10 min after exposure to H2O2. The mitochondrial ΔΨ of INS-1 cells was depolarized by H2O2 abolishing the hyperpolarizing action of glucose. These ΔΨ changes correlated with altered mitochondrial morphology; the latter was not preserved by the overexpression of the antiapoptotic protein Bcl-2. Mitochondrial Ca2+ was increased following exposure to H2O2 up to the micromolar range. No further augmentation occurred after glucose addition, which normally raises this parameter. Nevertheless, KCl was still efficient in enhancing mitochondrial Ca2+. Cytosolic ATP was markedly reduced by H2O2 treatment, probably explaining the decreased endoplasmic reticulum Ca2+. Taken together, these data point to the mitochondria as primary targets for H2O2 damage, which will eventually interrupt the transduction of signals normally coupling glucose metabolism to insulin secretion.

Preventing Mitochondrial Fission Impairs Mitochondrial Function and Leads to Loss of Mitochondrial DNA

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.

Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase

Sirtuins are homologues of the yeast transcriptional repressor Sir2p and are conserved from bacteria to humans. We report that human SIRT4 is localized to the mitochondria. SIRT4 is a matrix protein and becomes cleaved at amino acid 28 after import into mitochondria. Mass spectrometry analysis of proteins that coimmunoprecipitate with SIRT4 identified insulindegrading enzyme and the ADP/ATP carrier proteins, ANT2 and ANT3. SIRT4 exhibits no histone deacetylase activity but functions as an efficient ADP-ribosyltransferase on histones and bovine serum albumin. SIRT4 is expressed in islets of Langerhans and colocalizes with insulin-expressing β cells. Depletion of SIRT4 from insulin-producing INS-1E cells results in increased insulin secretion in response to glucose. These observations define a new role for mitochondrial SIRT4 in the regulation of insulin secretion.

New insights into amino acid metabolism, β-cell function and diabetes

Specific amino acids are now known to acutely and chronically regulate insulin secretion from pancreatic beta-cells in vivo and in vitro. Understanding the molecular mechanisms by which amino acids regulate insulin secretion may identify novel targets for future diabetes therapies. Mitochondrial metabolism is crucial for the coupling of amino acid and glucose recognition to the exocytosis of the insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP, which is the main coupling factor in insulin secretion; however, the subsequent Ca2+ signal in the cytosol is necessary, but not sufficient, for full development of sustained insulin secretion. Hence mitochondria generate ATP and other coupling factors serving as fuel sensors for the control of the exocytotic process. Numerous studies have sought to identify the factors that mediate the amplifying pathway over the Ca2+ signal in nutrient-stimulated insulin secretion. Predominantly, these factors are nucleotides (GTP, ATP, cAMP and NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and the key amino acid glutamate. This scenario highlights further the importance of the key enzymes or transporters, glutamate dehydrogenase, the aspartate and alanine aminotransferases and the malate/aspartate shuttle, in the control of insulin secretion. Therefore amino acids may play a direct or indirect (via generation of putative messengers of mitochondrial origin) role in insulin secretion.

Hepatocyte nuclear factor 4alpha regulates the expression of pancreatic beta -cell genes implicated in glucose metabolism and nutrient-induced insulin secretion.

Mutations in the HNF4α gene are associated with the subtype 1 of maturity-onset diabetes of the young (MODY1), which is characterized by impaired insulin secretory response to glucose in pancreatic β-cells. Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor critical for liver development and hepatocyte-specific gene expression. However, the role of HNF4α in the regulation of pancreatic β-cell gene expression and its correlation with metabolism secretion coupling have not been previously investigated. The tetracycline-inducible system was employed to achieve tightly controlled expression of both wild type (WT) and dominant-negative mutant (DN) of HNF4α in INS-1 cells. The induction of WT-HNF4α resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4α selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4α also caused defective mitochondrial function substantiated by reduced [14C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4α-regulated pancreatic β-cell gene expression revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase, aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling protein-2. The patterns of HNF4α-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1α. Indeed, HNF4α changed the HNF1α mRNA levels and HNF1α promoter luciferase activity through altered HNF4α binding. These results demonstrate the importance of HNF4α in β-cell metabolism-secretion coupling.

Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta-cells.

In the pancreatic β‐cell, insulin secretion is stimulated by glucose metabolism resulting in membrane potential‐dependent elevation of cytosolic Ca2+ ([Ca2+]c). This cascade involves the mitochondrial membrane potential (Δψm) hyperpolarization and elevation of mitochondrial Ca2+ ([Ca2+]m) which activates the Ca2+‐sensitive NADH‐generating dehydrogenases. Metabolism‐secretion coupling requires unidentified signals, other than [Ca2+]c, possibly generated by the mitochondria through the rise in [Ca2+]m. To test this paradigm, we have established an α‐toxin permeabilized cell preparation permitting the simultaneous monitoring of [Ca2+] with mitochondrially targeted aequorin and insulin secretion under conditions of saturating [ATP] (10 mM) and of clamped [Ca2+]c at substimulatory levels (500 nM). The tricarboxylic acid (TCA) cycle intermediate succinate hyperpolarized Δψm, raised [Ca2+]m up to 1.5 μM and stimulated insulin secretion 20‐fold, without changing [Ca2+]c. Blockade of the uniporter‐mediated Ca2+ influx into the mitochondria abolished the secretory response. Moreover, glycerophosphate, which raises [Ca2+]m by hyperpolarizing Δψm without supplying carbons to the TCA cycle, failed to stimulate exocytosis. Activation of the TCA cycle with citrate evoked secretion only when combined with glycerophosphate. Thus, mitochondrially driven insulin secretion at permissive [Ca2+]c requires both a substrate for the TCA cycle and a rise in [Ca2+]m. Therefore, mitochondrial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis

Dopamine D2-like Receptors Are Expressed in Pancreatic Beta Cells and Mediate Inhibition of Insulin Secretion

Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 μm) and the D2-like receptor agonist quinpirole (5 μm) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.