Michael B. Hoppa

Novel aspects of the molecular mechanisms controlling insulin secretion.

Pancreatic β‐cells secrete insulin by Ca2+‐dependent exocytosis of secretory granules. β‐cell exocytosis involves SNARE (soluble NSF‐attachment protein receptor) proteins similar to those controlling neurotransmitter release and depends on the close association of L‐type Ca2+ channels and granules. In most cases, the secretory granules fuse individually but there is ultrastructural and biophysical evidence of multivesicular exocytosis. Estimates of the secretory rate in β‐cells in intact islets indicate a release rate of ∼15 granules per β‐cell per second, 100‐fold higher than that observed in biochemical assays. Single‐vesicle capacitance measurements reveal that the diameter of the fusion pore connecting the granule lumen with the exterior is ∼1.4 nm. This is considerably smaller than the size of insulin and membrane fusion is therefore not obligatorily associated with release of the cargo, a feature that may contribute to the different rates of secretion detected by the biochemical and biophysical measurements. However, small molecules like ATP and GABA, which are stored together with insulin in the granules, are small enough to be released via the narrow fusion pore, which accordingly functions as a molecular sieve. We finally consider the possibility that defective fusion pore expansion accounts for the decrease in insulin secretion observed in pathophysiological states including long‐term exposure to lipids.

Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca2+ Channels From Secretory Vesicles

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca2+]i) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca2+]i. The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and β-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20–50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca2+]i signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca2+ entry in HFD β-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca2+ entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.

Insulin granule recruitment and exocytosis is dependent on p110γ in insulinoma and human β-cells

OBJECTIVE Phosphatidylinositol 3-OH kinase (PI3K) has a long-recognized role in β-cell mass regulation and gene transcription and is implicated in the modulation of insulin secretion. The role of nontyrosine kinase receptor–activated PI3K isoforms is largely unexplored. We therefore investigated the role of the G-protein–coupled PI3Kγ and its catalytic subunit p110γ in the regulation of insulin granule recruitment and exocytosis. RESEARCH DESIGN AND METHODS The expression of p110γ was knocked down by small-interfering RNA, and p110γ activity was selectively inhibited with AS605240 (40 nmol/l). Exocytosis and granule recruitment was monitored by islet perifusion, whole-cell capacitance, total internal reflection fluorescence microscopy, and electron microscopy in INS-1 and human β-cells. Cortical F-actin was examined in INS-1 cells and human islets and in mouse β-cells lacking the phosphatase and tensin homolog (PTEN). RESULTS Knockdown or inhibition of p110γ markedly blunted depolarization-induced insulin secretion and exocytosis and ablated the exocytotic response to direct Ca2+ infusion. This resulted from reduced granule localization to the plasma membrane and was associated with increased cortical F-actin. Inhibition of p110γ had no effect on F-actin in β-cells lacking PTEN. Finally, the effect of p110γ inhibition on granule localization and exocytosis could be rapidly reversed by agents that promote actin depolymerization. CONCLUSIONS The G-protein–coupled PI3Kγ is an important determinant of secretory granule trafficking to the plasma membrane, at least in part through the negative regulation of cortical F-actin. Thus, p110γ activity plays an important role in maintaining a membrane-docked, readily releasable pool of secretory granules in insulinoma and human β-cells.

Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3.

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons

The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment.