Basal hypersecretion of glucagon and insulin from palmitate-exposed human islets depends on FFAR1 but not decreased somatostatin secretion.

In obesity fasting levels of both glucagon and insulin are elevated. In these subjects fasting levels of the free fatty acid palmitate are raised. We have demonstrated that palmitate enhances glucose-stimulated insulin secretion from isolated human islets via free fatty acid receptor 1 (FFAR1/GPR40). Since FFAR1 is also present on glucagon-secreting alpha-cells, we hypothesized that palmitate simultaneously stimulates secretion of glucagon and insulin at fasting glucose concentrations. In addition, we hypothesized that concomitant hypersecretion of glucagon and insulin was also contributed by reduced somatostatin secretion. We found basal glucagon, insulin and somatostatin secretion and respiration from human islets, to be enhanced during palmitate treatment at normoglycemia. Secretion of all hormones and mitochondrial respiration were lowered when FFAR1 or fatty acid ß-oxidation was inhibited. The findings were confirmed in the human beta-cell line EndoC-ßH1. We conclude that fatty acids enhance both glucagon and insulin secretion at fasting glucose concentrations and that FFAR1 and enhanced mitochondrial metabolism but not lowered somatostatin secretion are crucial in this effect. The ability of chronically elevated palmitate levels to simultaneously increase basal secretion of glucagon and insulin positions elevated levels of fatty acids as potential triggering factors for the development of obesity and impaired glucose control.

Glucose-dependent regulation of rhythmic action potential firing in pancreatic beta-cells by K(ATP)-channel modulation.

The regulation of a K(+) current activating during oscillatory electrical activity (I(K,slow)) in an insulin-releasing beta-cell was studied by applying the perforated patch whole-cell technique to intact mouse pancreatic islets. The resting whole-cell conductance in the presence of 10 mM glucose amounted to 1.3 nS, which rose by 50 % during a series of 26 simulated action potentials. Application of the K(ATP)-channel blocker tolbutamide produced uninterrupted action potential firing and reduced I(K,slow) by approximately 50 %. Increasing glucose from 15 to 30 mM, which likewise converted oscillatory electrical activity into continuous action potential firing, reduced I(K,slow) by approximately 30 % whilst not affecting the resting conductance. Action potential firing may culminate in opening of K(ATP) channels by activation of ATP-dependent Ca(2+) pumping as suggested by the observation that the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (4 microM) inhibited I(K,slow) by 25 % and abolished bursting electrical activity. We conclude that oscillatory glucose-induced electrical activity in the beta-cell involves the opening of K(ATP)-channel activity and that these channels, in addition to constituting the glucose-regulated K(+) conductance, also play a role in the graded response to supra-threshold glucose concentrations.

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.

Patch-clamp characterisation of somatostatin-secreting -cells in intact mouse pancreatic islets.

The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact mouse pancreatic islets. Three types of electrical activity were observed corresponding to alpha-, beta- and delta-cells. The delta-cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the beta-cells. By contrast, the alpha-cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. Both alpha- and beta-cells contained transient voltage-activated K+ currents. In the delta-cells, the K+ currents activated above -20 mV and were completely blocked by TEA (20 mM). The alpha-cells differed from the delta-cells in possessing a TEA-resistant K+ current activating already at -40 mV. Immunocytochemistry revealed the presence of Kv3.4 channels in delta-cells and TEA-resistant Kv4.3 channels in alpha-cells. Thus the presence of a transient TEA-resistant current can be used to functionally separate the delta- and alpha-cells. A TTX-sensitive Na+ current developed in delta-cells during depolarisations beyond -30 mV and reached a peak amplitude of 350 pA. Steady-state inactivation of this current was half-maximal at -28 mV. The delta-cells were also equipped with a sustained Ca2+ current that activated above -30 mV and reached a peak of 60 pA when measured at 2.6 mM extracellular Ca2+. A tolbutamide-sensitive KATP channel conductance was observed in delta-cells exposed to glucose-free medium. Addition of tolbutamide (0.1 mM) depolarised the delta-cell and evoked electrical activity. We propose that the KATP channels in delta-cells serve the same function as in the beta-cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.

Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels.

The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial glucagon-secreting alpha-cells in intact mouse pancreatic islets. alpha-cells were distinguished from the beta- and delta-cells by the presence of a large TTX-blockable Na+ current, a TEA-resistant transient K+ current sensitive to 4-AP (A-current) and the presence of two kinetically separable Ca2+ current components corresponding to low- (T-type) and high-threshold (L-type) Ca2+ channels. The T-type Ca2+, Na+ and A-currents were subject to steady-state voltage-dependent inactivation, which was half-maximal at -45, -47 and -68 mV, respectively. Pancreatic alpha-cells were equipped with tolbutamide-sensitive, ATP-regulated K+ (KATP) channels. Addition of tolbutamide (0.1 mM) evoked a brief period of electrical activity followed by a depolarisation to a plateau of -30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mM glucose, concentrations up to 2 microM stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the alpha-cells is dependent on the generation of Na+-dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage-dependent inactivation of voltage-gated membrane currents. Glucose may inhibit glucagon release by depolarising the alpha-cell with resultant inactivation of the ion channels participating in action potential generation.

Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

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 approximately 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 ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells.

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells.

1. Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells. 2. ACh (250 microM) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (tau) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 microM) for much longer periods in the presence than in the absence of the agonist. 3. The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in tau for the refilling of the RRP from 31 to 19 s. 4. Collectively, the effects of ACh on the RRP and tau result in a > 10-fold stimulation of the rate at which granules are supplied for release.

Specificity and target proteins of arginine-specific mono-ADP-ribosylation in T-tubules of rabbit skeletal muscle.

In order to specify that protein labeling is the result of mono-ADP ribosylation, a careful evaluation of the reaction conditions and products is necessary. To investigate the specificity and target proteins of the arginine-specific mono-ADP-ribosyltransferase (mADP-RT) in rabbit skeletal muscle T-tubules (TT) biotin- or digoxigenin-coupled NAD-derivatives were synthesized. They were used for the nonradioactive labeling of proteins and compared with radioactive mono-ADP-ribosylation. According to the results of our studies, they cannot be used as substrates to detect arginine-specific or pertussis toxin-dependent mono-ADP-ribosylation of target proteins in skeletal muscle. In contrast, radioactive NAD can be used to monitor these reactions. Under the appropriate reaction conditions, the radioactive [adenylate-14C]NAD and [32P]NAD were found to be solely consumed by the arginine-specific mADP-RT of skeletal muscle TT. The incorporation studies confirmed earlier data on the localization of the mADP-RT and its targets in TT. The T-tubular targets were purified in a single-step procedure using phenylboronate affinity chromatography. Of 18 target proteins delineated by autoradiography of electrophoretically separated T-tubular proteins, a 42-kDa protein was suggested to be the stimulatory G protein (Gsalpha). Mono-ADP-ribosylation of Gsalpha resulted in an inhibition of the T-tubular adenylate cyclase activity as proven by the suppression of this inhibition using novobiocin as a specific inhibitor of mADP-RT.

Early functional and biochemical adaptations to low-frequency stimulation of rabbit fast-twitch muscle.

To examine mechanisms underlying force reduction after the onset of chronic low-frequency (10 Hz) stimulation (CLFS), we exposed rabbit tibialis anterior muscles to various durations of CLFS. To follow changes in isometric contractile properties and electromyographic (EMG) activity, we studied stimulated and contralateral muscles during a terminal test at 10 Hz for 10 min. In addition, activities and protein amounts of the sarcoplasmic reticulum Ca(2+)-ATPase, content of Na(+)-K(+)-ATPase, and expression patterns of triad junction components were examined. Force output and EMG amplitude declined abruptly soon after the onset of stimulation, suggesting refractoriness of a large fiber population. Although twitch force and to a lesser extent EMG activity gradually recovered after stimulation for 6 days and longer, the muscles exhibited profoundly altered properties, i.e., enhanced fatigue resistance, absence of twitch potentiation, and prolonged contraction and relaxation times. These changes were associated with significant increases in Na(+)-K(+)-ATPase concentration and significant decreases in Ca(2+)-ATPase, ryanodine receptor, dihydropyridine receptor, and triadin concentrations over the course of the 20 days of stimulation. Alterations in excitability, Ca2+ handling, and excitation-contraction coupling prior to changes in myofibrillar protein isoforms may thus be responsible for early functional alterations.