Juris Galvanovskis

The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate–Dependent Nucleic Acid Demethylase

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate–dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ∼80 fF that plateaued within ∼200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K d values were 6 and 29 μM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1−/− mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1−/− mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl− into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets

1 The perforated patch whole‐cell configuration of the patch‐clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin‐secreting B‐cells and 65 % non‐B‐cells (A‐ and D‐cells). 2 Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mm glucose and had the electrophysiological characteristics of isolated B‐cells maintained in tissue culture. 3 The Ca2+ current recorded from B‐cells in situ was 80 % larger than that of isolated B‐cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4 Voltage‐dependent K+ currents were observed during depolarisations to membrane potentials above −20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between −90 and −30 mV. 5 The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP‐regulated K+ (KATP) channels, amounted to ≈4 nS. Glucose produced a concentration‐dependent reduction of KATP channel conductance with half‐maximal inhibition observed with 5 mm glucose. 6 Combining voltage‐ and current‐clamp recording allowed the estimation of the gap junction conductance between different B‐cells. These experiments indicated that the input conductance of the B‐cell at stimulatory glucose concentrations (≈1 nS) is almost entirely accounted for by coupling to neighbouring B‐cells.

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.

Selective nucleotide-release from dense-core granules in insulin-secreting cells.

Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by ∼0.1 and ∼2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.

Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells.

We have explored whether γ-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic β-cells. To this end, β-cells were engineered to express GABAA-receptor Cl−-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every β-cell contains ∼3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl−-currents, was observed during membrane depolarizations from −70 mV to voltages beyond −40 mV or when Ca2+ was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca2+ entry was inhibited using Cd2+. Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes ∼1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic β-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.

Capacitance measurements of exocytosis in mouse pancreatic α-, β- and δ-cells within intact islets of Langerhans

Capacitance measurements of exocytosis were applied to functionally identified α‐, β‐ and δ‐cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in β‐cells during a depolarization to 0 mV was equivalent to 14 granules s−1, <5% of that observed in isolated β‐cells. β‐Cell secretion exhibited bell‐shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to ∼−20 mV) the maximum rate of release was ∼4 granules s−1. Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L‐type Ca2+ channel blocker nifedipine (25 μm) but only marginally (<20%) affected by the R‐type Ca2+ channel blocker SNX482 (100 nm). Exocytosis in the glucagon‐producing α‐cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s−1) and a sustained late component (30 granules s−1). Whereas addition of the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L‐type Ca2+ channel blocker nifedipine (25 μm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca2+‐influx through N‐type rather than L‐type Ca2+ channels. Exocytosis in the somatostatin‐secreting δ‐cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s−1) component followed by a sustained slower (60 granules s−1) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in β‐cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes.

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Capacitance measurements of exocytosis in mouse pancreatic alpha-, beta- and delta-cells within intact islets of Langerhans.

Capacitance measurements of exocytosis were applied to functionally identified α‐, β‐ and δ‐cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in β‐cells during a depolarization to 0 mV was equivalent to 14 granules s−1, <5% of that observed in isolated β‐cells. β‐Cell secretion exhibited bell‐shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to ∼−20 mV) the maximum rate of release was ∼4 granules s−1. Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L‐type Ca2+ channel blocker nifedipine (25 μm) but only marginally (<20%) affected by the R‐type Ca2+ channel blocker SNX482 (100 nm). Exocytosis in the glucagon‐producing α‐cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s−1) and a sustained late component (30 granules s−1). Whereas addition of the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L‐type Ca2+ channel blocker nifedipine (25 μm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca2+‐influx through N‐type rather than L‐type Ca2+ channels. Exocytosis in the somatostatin‐secreting δ‐cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s−1) component followed by a sustained slower (60 granules s−1) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in β‐cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.