Fasting inhibits excitatory synaptic input on paraventricular oxytocin neurons via neuropeptide Y and Y1 receptor, inducing rebound hyperphagia, and weight gain.

Fasting with varying intensities is used to treat obesity-related diseases. Re-feeding after fasting exhibits hyperphagia and often rebound weight gain. However, the mechanisms underlying the hyperphagia and rebound remain elusive. Here we show that 24 h food restriction (24 h FR) and milder 50% FR, both depress synaptic transmission in the hypothalamic paraventricular nucleus (PVN) and induce acute hyperphagia in rats. 24 h FR is followed by weight rebound but 50% FR is not. Orexigenic neuropeptide Y (NPY) via the Y1 receptor (Y1R) inhibited the miniature excitatory postsynaptic current (mEPSC) on anorexigenic oxytocin neurons in the PVN. 24 h FR and 50% FR activated this neuronal pathway to induce acute hyperphagia on Days 1-3 and Days 1-2 after FR, respectively. 24 h FR induced large mEPSC depression, recurrent hyperphagia on Days 9-12 and rebound weight gain on Days 12-17, whereas 50% FR induced moderate mEPSC depression and sustained weight reduction. Transverse data analysis on Day 1 after 24 h FR and 50% FR demonstrated saturation kinetics for the mEPSC depression-hyperphagiacurve, implying hysteresis. The results reveal FR-driven synaptic plasticity in the NPY-Y1R-oxytocin neurocircuit that drives acute hyperphagia. FR with the intensity that regulates the synapse-feeding relay without hysteresis is the key for successful dieting.

Onion component, isoalliin, stimulates feeding and activates the arcuate nucleus neuropeptide Y, ghrelin- and Ninjin'yoeito-responsive neurons.

Appetite loss or anorexia substantially decreases the quality of life in patients with cancer, depression and gastrointestinal disorders, and can lead to sarcopenia and frailty. Foods that restore appetite have been sought-for but are not currently available. Historically, onion intake was adopted to treat a variety of diseases with reduced appetite including cancer and gastrointestinal disturbances. While isoalliin is a core component of onion, the effects of isoalliin on feeding behavior and feeding centers remain unknown. Neuropeptide Y (NPY) and ghrelin are the most potent central and peripheral inducers of appetite. A Japanese kampo medicine Ninjin'yoeito activates ghrelin-responsive NPY neurons in the hypothalamic arcuate nucleus (ARC) and counteracts anorexia induced by an anti-cancer drug cisplatin. This study explored the effects of isoalliin on feeding behavior and activities of ARC neurons in mice. Isoalliin, injected intraperitoneally, dose-dependently increased food intake during dark phase (DP) and daily without altering light phase (LP) food intake. We measured cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons including NPY neurons identified by GFP fluorescence. Isoalliin increased [Ca2+]i in 10 of 18 (55.6%) NPY neurons, a majority of which also responded to ghrelin with [Ca2+]i increases, indicating that the ARC ghrelin-responsive NPY neuron is the major target of isoalliin. Isoalliin also increased [Ca2+]i in the ARC neurons that responded to Ninjin'yoeito. These results indicate that isoalliin enhances feeding at the active period and activates ARC ghrelin-responsive NPY neurons and Ninjin'yoeito-responsive neurons. These abilities of isoalliin to stimulate DP feeding and activate ARC orexigenic neurons provide scientific evidence for the health beneficial effects of onion experienced historically and globally.

Prostaglandin E1 inhibits endocytosis in the ß-cell endocytosis.

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca(2+) influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca(2+) influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In ß cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

Time-resolved metabolomics analysis of ß-cells implicates the pentose phosphate pathway in the control of insulin release.

Insulin secretion is coupled with changes in ß-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 ß-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the ß-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 ß-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in ß-cell stimulus-secretion coupling.

Evolving insights regarding mechanisms for the inhibition of insulin release by norepinephrine and heterotrimeric G proteins.

Norepinephrine has for many years been known to have three major effects on the pancreatic ß-cell which lead to the inhibition of insulin release. These are activation of K(+) channels to hyperpolarize the cell and prevent the gating of voltage-dependent Ca(2+) channels that increase intracellular Ca(2+) concentration ([Ca(2+)](i)) and trigger release; inhibition of adenylyl cyclases, thus preventing the augmentation of stimulated insulin release by cyclic AMP; and a "distal" effect that occurs downstream of increased [Ca(2+)](i) to inhibit exocytosis. All three are mediated by the pertussis toxin (PTX)-sensitive heterotrimeric Gi and Go proteins. The distal inhibitory effect on exocytosis is now known to be due to the binding of G protein ßγ subunits to the synaptosomal-associated protein of 25 kDa (SNAP-25) on the soluble NSF attachment protein receptor (SNARE) complex. Recent studies have uncovered two more actions of norepinephrine on the ß-cell: 1) retardation of the refilling of the readily releasable granule pool after it has been discharged, an action that is mediated by Gαi(1) and/or Gαi(2); and 2) inhibition of endocytosis that is mediated by Gz. Of importance also are new findings that Gαo regulates the number of docked granules in the ß-cell, and that Gαo(2) maintains a tonic inhibitory influence on secretion. The latter provides another explanation as to why PTX, which blocks the effect of Gαo(2), was initially called "islet activating protein." Finally, there is clear evidence that overexpression of α(2A)-adrenergic receptors in ß-cells can cause type 2 diabetes.

Glucose stimulation of protein acylation in the pancreatic ß-cell.

AIMS: To determine whether protein acylation plays a role in the effects of glucose on the insulin secreting ß-cell. MAIN METHODS: The measurement of (3)H-palmitate incorporation into protein in the INS 832/13 cell that has a robust and well-characterized biphasic insulin secretory response to stimulation with glucose. KEY FINDINGS: Stimulating the cells with glucose increased the incorporation of (3)H-palmitic acid into protein by up to 90%. Similarly, 2-aminobicyclo [2.2.1] heptane-2-carboxylic acid (BCH) the non-metabolizable analog of leucine that mimics the stimulatory effect of glucose on insulin secretion also increased the incorporation of (3)H-palmitic acid into protein. Treatment of cell lysates with hydroxylamine substantially reduced the incorporation indicating that most of the incorporation was due to enzymatic palmitoylation of proteins. Cerulenin, a classical inhibitor of protein acylation also substantially reduced the incorporation. Using PAGE and autoradiography a glucose-induced increase in protein palmitoylation and specific glucose-induced increases in the palmitoylation of proteins of 30, 44, 48 and 76kD were identified. SIGNIFICANCE: The data suggest that protein acylation plays multiple roles in ß-cell function.

Hormonal inhibition of endocytosis: novel roles for noradrenaline and G protein G(z).

The modulation of endocytosis following exocytosis by noradrenaline (NA), a physiological inhibitor of insulin secretion, was investigated in INS 832/13 cells using patch-clamp capacitance measurements. Endocytosis was inhibited by NA in a pertussis toxin-insensitive manner. Dialysing a synthetic peptide mimicking the C-terminus of the α-subunit of G(z) into the cells blocked the inhibition of endocytosis by NA. Cell-attached capacitance measurements indicated that inhibition by NA was due to a decreased number of endocytotic events without a change in vesicle size. Analysis of fission pore closure kinetics revealed two distinct fission modes, with NA selectively inhibiting the rapid fission pore closure events. Comparison of the actions of NA and deltamethrin, a calcineurin antagonist and potent inhibitor of endocytosis, demonstrated that they inhibit endocytosis by different mechanisms. These findings establish novel actions for NA and G(z) in insulin-secreting cells and possibly other cell types.

Noradrenaline inhibits exocytosis via the G protein ßγ subunit and refilling of the readily releasable granule pool via the α(i1/2) subunit.

The molecular mechanisms responsible for the 'distal' effect by which noradrenaline (NA) blocks exocytosis in the ß-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 ß-cells. NA inhibited Ca(2+)-evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA-induced inhibition of exocytosis was abolished by a high level of Ca(2+) influx, by intracellular application of antibodies against the G protein subunit Gß and was mimicked by the myristoylated ßγ-binding/activating peptide mSIRK. NA-induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C-terminal nine amino acids of SNAP-25, and also by a SNAP-25 C-terminal-blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca(2+)](i) and is mediated by an interaction between Gßγ and the C-terminus of SNAP-25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a Gα(i1/2) blocking peptide demonstrating that the effect is mediated via activation of Gα(i1) and/or Gα(i2).

Both G i and G o heterotrimeric G proteins are required to exert the full effect of norepinephrine on the beta-cell K ATP channel.

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (K ATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on K ATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the K ATP channels approximately 2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the K ATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric G i/G o proteins. To identify the G proteins involved, antisera raised against alpha and beta subunits (anti-G alpha common, anti-G beta, anti-G alpha i1/2/3, and anti-G alpha o) were used. Anti-G alpha common totally blocked the effects of NE on membrane potential and K ATP channels. Individually, anti-G alpha i1/2/3 and anti-G alpha o only partially inhibited the action of NE on K ATP channels. However, the combination of both completely eliminated the action. Antibodies against G beta had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the alpha subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G alpha i3 and G alpha o1 were not involved. In conclusion, both G i and G o proteins are required for the full effect of norepinephrine to activate the K ATP channel.

The inhibitors of protein acylation, cerulenin and tunicamycin, increase voltage-dependent Ca(2+) currents in the insulin-secreting INS 832/13 cell.

As it has been suggested that protein acylation plays a role in nutrient stimulus-secretion coupling in the pancreatic beta-cell, we examined the insulin-secreting INS 832/13 beta-cell line for evidence that protein acylation was involved. The perforated whole-cell configuration was employed to voltage-clamp INS 832/13 cells. Voltage pulses were applied and Ca(2+) currents measured in the presence and absence of the protein acylation inhibitors cerulenin and tunicamycin. Both inhibitors enhanced the peak amplitude of I(Ca,L). Both increased the peak inward current in the range between -40 and +30mV and shifted the apparent maximum current by 10mV in the hyperpolarizing direction without affecting the activation threshold of -40mV. The two drugs had qualitatively and quantitatively similar effects. Steady-state activation curves revealed that cerulenin and tunicamycin shifted the activation curves in the hyperpolarization direction. Activation time constants were significantly reduced in the presence of both drugs. The Ca(2+) charge influx was increased by the drugs at all potentials tested. In contrast to these effects on the L-type Ca(2+) channel, the two inhibitors of protein acylation had no effect on the ATP-sensitive K(+) channel. The results suggest that protein acylation exerts a tonic inhibitory effect on L-type Ca(2+) channel function in the insulin-secreting beta-cell.

Inhibitory role of Src family tyrosine kinases on Ca2+-dependent insulin release.

Both neurotransmitter release and insulin secretion occur via regulated exocytosis and share a variety of similar regulatory mechanisms. It has been suggested that Src family tyrosine kinases inhibit neurotransmitter release from neuronal cells (H. Ohnishi, S. Yamamori, K. Ono, K. Aoyagi, S. Kondo, and M. Takahashi. Proc Natl Acad Sci USA 98: 10930-10935, 2001). Thus the potential role of Src family kinases in the regulation of insulin secretion was investigated in this study. Two structurally different inhibitors of Src family kinases, SU-6656 and PP2, but not the inactive compound, PP3, enhanced Ca2+-induced insulin secretion in both rat pancreatic islets and INS-1 cells in a concentration-dependent and time-dependent manner. Furthermore, Src family kinase-mediated insulin secretion appears to be dependent on elevated intracellular Ca2+ and independent of glucose metabolism, the ATP-dependent K+ channel, adenylyl cyclase, classical PKC isoforms, extracellular signal-regulated kinase 1/2, and insulin synthesis. The sites of action for Src family kinases seem to be distal to the elevation of intracellular Ca2+ level. These results indicate that one or more Src family tyrosine kinases exert a tonic inhibitory role on Ca2+-dependent insulin secretion.

Inhibition of insulin secretion by cerulenin might be due to impaired glucose metabolism.

BACKGROUND: Cerulenin, an inhibitor of protein acylation, has been used as a tool to study the potential role of protein acylation in a variety of activities in different cells, and in stimulus-secretion coupling in pancreatic islets and clonal beta-cells. METHODS: In the present study we investigated its effects on stimulated insulin secretion, glucose metabolism and utilization, oxygen consumption and ATP levels. RESULTS: In isolated rat pancreatic islets, cerulenin pre-treatment (100 microM) inhibited insulin secretion in response to glucose, and to the non-hydrolysable analogue of leucine, aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH). These data are in accord with the hypothesis that protein acylation could be involved in the stimulation of insulin secretion. However, we also found that cerulenin profoundly decreased glucose oxidation, glucose utilization, oxygen consumption and ATP levels. Consequently, decreased metabolism provides an alternative mechanism to inhibition of protein acylation that could explain the inhibition of insulin secretion by cerulenin. CONCLUSIONS: Inhibition of insulin secretion by cerulenin can no longer be taken as evidence in favour of a role for protein acylation in the control of insulin release. As protein acylation is known to be involved in the normal functioning of proteins in stimulus-secretion coupling and exocytosis, more direct approaches to understand its role(s) are required.

Massive augmentation of stimulated insulin secretion induced by fatty acid-free BSA in rat pancreatic islets.

Incubation of rat pancreatic islets for 4-6 h with 100 micromol/l fatty acid-free BSA induced a 3- to 10-fold enhancement of insulin release to a subsequent challenge with 16.7 mmol/l glucose, without changing the typical biphasic pattern of the response. A similar enhancement was observed with other stimuli, such as leucine, depolarizing concentrations of KCl and tolbutamide, pointing to a general phenomenon and common mechanism for the augmentation. Norepinephrine completely blocked the stimulated response. The protein kinase C (PKC) inhibitor Ro 31-8220, which acts at the ATP-binding site and inhibits all PKC isoforms, strongly inhibited the enhancement of a subsequent glucose challenge when present during the BSA pretreatment period. In contrast, Go 6976, an inhibitor of conventional PKC isoforms, was without effect, even at the high concentration of 1 micromol/l. Preincubation with calphostin C, which competes for the diacylglycerol (DAG)-binding site, therefore inhibiting conventional, novel, and PKC isoforms of the PKD type, completely abolished the enhancing effect of the BSA but did not affect secretion in islets treated with 10 micromol/l fatty acid-free BSA. We conclude that the remarkable enhancement of insulin release is due to a change in glucose signaling and activation of a novel PKC isoform or a DAG-binding protein.

Stimulation of insulin release by glucose is associated with an increase in the number of docked granules in the beta-cells of rat pancreatic islets.

Electron microscopy and quantitative stereological techniques were used to study the dynamics of the docked granule pool in the rat pancreatic beta-cell. The mean number of granules per beta-cell was 11,136. After equilibration in RPMI containing 5.6 mmol/l glucose, 6.4% of the granules (approximately 700) were docked at the plasma membrane (also measured as [means +/- SE] 4.3 +/- 0.6 docked granules per 10 microm of plasma membrane at the perimeter of the cell sections). After a 40-min exposure to 16.7 mmol/l glucose, 10.2% of the granules (approximately 1,060) were docked (6.4 +/- 0.8 granules per 10 microm of plasma membrane). Thus, the docked pool increased by 50% during stimulation with glucose. Islets were also exposed to 16.7 mmol/l glucose in the absence or presence of 10 micromol/l nitrendipine. In the absence and presence of nitrendipine, there were 6.1 +/- 0.7 and 6.3 +/- 0.6 granules per 10 microm of membrane, respectively. Thus, glucose increased granule docking independently of increased [Ca2+]i and exocytosis. The data suggest a limit to the number of docking sites. As the rate of docking exceeded the rate of exocytosis, docking is not rate limiting for insulin release. Only with extremely high release rates, glucose stimulation after a 4-h incubation with a high concentration of fatty acid-free BSA, was the docked granule pool reduced in size.

Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets.

Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the beta-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca(2+) concentration ([Ca(2+)](i)). The second phase requires increased [Ca(2+)](i) and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca(2+)-stimulated release via the K(ATP) channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K(+) channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased alpha-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of alpha-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP.

Hypothesis: one rate-limiting step controls the magnitude of both phases of glucose-stimulated insulin secretion.

The biphasic secretory response of pancreatic beta-cells to abrupt and sustained exposure to glucose is well documented. Some of the ATP-sensitive K(+) (K(ATP)) channel-dependent mechanisms underlying the first phase of insulin release are known; the mechanisms underlying the second phase are less well known. The hypothesis we propose is that one rate-limiting step, controlling the conversion of granules in a readily releasable (RR) docked granule pool to an immediately releasable (IR) pool, is responsible for the magnitude of both phases of release. Furthermore, we propose that the K(ATP) channel-independent signaling pathway regulates this rate-limiting step. The size of the IR pool of granules that constitutes the first phase is determined under resting conditions by the forward and reverse rates of conversion of granules in the RR and IR pools. The resulting equilibrium position determines the maximum number of beta-cell granules available for release during the first phase upon exposure to glucose. At the nadir between the two phases, the IR pool has been depleted so that the rate of granule release is equal to the low forward rate for the conversion of RR to IR granules. After the nadir, the forward rate is accelerated during the rising portion of the second phase until it reaches a maximum rate at the plateau.

Y-26763: ATP-sensitive K+ channel activation and the inhibition of insulin release from human pancreatic beta-cells.

The effect of Y-26763 [(-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol], a novel ATP-sensitive K(+) (K(ATP)) channel activator, was tested on insulin secretion from human pancreatic islets in vitro. Y-26763 was able to inhibit both glucose- and tolbutamide-induced insulin secretion from islets as assessed by radioimmunoassay. The mechanism for inhibition of insulin secretion was characterised using patch clamp electrophysiology on dispersed human pancreatic beta-cells which express K(ATP) channels comprised of Kir6.2 and SUR1, and the NES2Y human beta-cell line, transfected with Kir6.2DeltaC26. Y-26763 activated K(ATP) channels in a reversible manner with a similar activity to diazoxide. This required the presence of hydrolysable nucleotides and appeared to be mediated by interaction of Y-26763 with SUR1 since: (a) tolbutamide was able to reverse the actions of Y-26763 and (b) Y-26763 failed to activate Kir6.2DeltaC26 in the absence of SUR1. We conclude that Y-26763-induced inhibition of insulin release is dependent upon the activation of K(ATP) channels in human beta-cells.

Protein acylation in the inhibition of insulin secretion by norepinephrine, somatostatin, galanin, and PGE2.

The major physiological inhibitors of insulin secretion, norepinephrine, somatostatin, galanin, and prostaglandin E2, act via specific receptors that activate pertussis toxin (PTX)-sensitive G proteins. Four inhibitory mechanisms are known: 1) activation of ATP-sensitive K channels and repolarization of the beta-cell; 2) inhibition of L-type Ca2+ channels; 3) decreased activity of adenylyl cyclase; and 4) inhibition of exocytosis at a "distal" site in stimulus-secretion coupling. We have examined the underlying mechanisms of inhibition at this distal site. In rat pancreatic islets, 2-bromopalmitate, cerulenin, and polyunsaturated fatty acids, all of which suppress protein acyltransferase activity, blocked the distal inhibitory effects of norepinephrine in a concentration-dependent manner. In contrast, control compounds such as palmitate, 16-hydroxypalmitate, and etomoxir, which do not block protein acylation, had no effect. Furthermore, 2-bromopalmitate also blocked the distal inhibitory actions of somatostatin, galanin, and prostaglandin E2. Importantly, neither 2-bromopalmitate nor cerulenin affected the action of norepinephrine to decrease cAMP production. We also examined the effects of norepinephrine, 2-bromopalmitate, and cerulenin on palmitate metabolism. Palmitate oxidation and its incorporation into lipids seemed not to contribute to the effects of 2-bromopalmitate and cerulenin on norepinephrine action. These data suggest that protein acylation mediates the distal inhibitory effect on insulin secretion. We propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyltransferase, causing the acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of the essential and precise interactions involved in core complex formation would block exocytosis.

Activation of the KATP channel-independent signaling pathway by the nonhydrolyzable analog of leucine, BCH.

Leucine and glutamine were used to elicit biphasic insulin release in rat pancreatic islets. Leucine did not mimic the full biphasic response of glucose. Glutamine was without effect. However, the combination of the two did mimic the biphasic response. When the ATP-sensitive K+ (KATP) channel-independent pathway was studied in the presence of diazoxide and KCl, leucine and its nonmetabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) both stimulated insulin secretion to a greater extent than glucose. Glutamine and dimethyl glutamate had no effect. Because the only known action of BCH is stimulation of glutamate dehydrogenase, this is sufficient to develop the full effect of the KATP channel-independent pathway. Glucose, leucine, and BCH had no effect on intracellular citrate levels. Leucine and BCH both decreased glutamate levels, whereas glucose was without effect. Glucose and leucine decreased palmitate oxidation and increased esterification. Strikingly, BCH had no effect on palmitate oxidation or esterification. Thus BCH activates the KATP channel-independent pathway of glucose signaling without raising citrate levels, without decreasing fatty acid oxidation, and without mimicking the effects of glucose and leucine on esterification. The results indicate that increased flux through the TCA cycle is sufficient to activate the KATP channel-independent pathway.