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.

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.

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 Ca2+influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca2+influx eliminates the inhibitory effect of PGE1on exocytosis and allows specific characterization of the inhibitory effect of PGE1on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gzinto the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1inhibits endocytosis and Gzmediates the inhibition.

Background: The affinity of soluble adenylyl cyclase (sAC) for its substrate ATP suggested that it might be sensitive to fluctuations in ATP. Results: In sAC-overexpressing and glucose-responsive cells, sAC-generated cAMP reflects intracellular ATP levels. Conclusion: sAC can be an ATP sensor inside cells. Significance: sAC serves as a metabolic sensor via regulation by three cellular metabolites: ATP, bicarbonate, and calcium.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 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.

Insulin secretion is coupled with changes in β-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 β-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 β-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 β-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 β-cell stimulus-secretion coupling.

Norepinephrine has for many years been known to have three major effects on the pancreatic β-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 βγ 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 β-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 β-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 β-cells can cause type 2 diabetes.

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 Gz 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 Gz in insulin-secreting cells and possibly other cell types.

Aims: To determine whether protein acylation plays a role in the effects of glucose on the insulin secreting beta-cell.Main methods: The measurement of H-3-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 H-3-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 H-3-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 76 kD were identified.Significance: The data suggest that protein acylation plays multiple roles in beta-cell function. (C) 2010 Elsevier Inc. All rights reserved.

The molecular mechanisms responsible for the ‘distal’ effect by which noradrenaline (NA) blocks exocytosis in the β‐cell were examined by whole‐cell and cell‐attached patch clamp capacitance measurements in INS 832/13 β‐cells. NA inhibited Ca2+‐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 Ca2+ influx, by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ‐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 [Ca2+]i and is mediated by an interaction between Gβγ 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 Gαi1/2 blocking peptide demonstrating that the effect is mediated via activation of Gαi1 and/or Gαi2.

Norepinephrine (NE) is a well-known physiological inhibitor of insulin secretion in pancreatic β cells. We investigated modulation of exocytosis and endocytosis by NE in INS 832/13 cells using whole-cell capacitance measurements. Exocytosis was stimulated by depolarizing pulses from −70mV to +10mV of variable duration and was followed by compensatory endocytosis. Inhibition of Ca2+-evoked exocytosis by NE was overcome by increasing Ca2+ influx, either by increasing the depolarizing pulse duration (up to 500ms) or by increasing the extracellular Ca2+ concentration up to 10mM. When stimulated by a short train of 500ms pulses in the presence of NE (5μM), robust exocytosis was observed but endocytosis was markedly inhibited. The NE inhibition of endocytosis was abolished by the α2-adrenergic receptor antagonist yohimbine (10μM) and was not affected by PTX-treatment (150ng/ml), demonstrating that NE inhibition of endocytosis is mediated via the α2-adrenergic receptor and not via Gi and/or Go proteins. When a synthetic peptide that mimicked the last 13 c-terminal amino acids of the Gzα subunit was dialyzed into the cells via the whole-cell patch pipette, NE inhibition of endocytosis was fully blocked, suggesting that Gz may be mediating the inhibition. Single vesicle recordings by cell attached capacitance measurements indicate that inhibition of endocytosis by NE is due to a decreased number of endocytic events without a significant change in endocytic vesicle size. Further analysis of fission pore kinetics revealed that NE selectively inhibited the rapid fission events. Our findings establish a novel action for NE and suggest the possibility that NE may modulate endocytosis in the central nervous system and elsewhere.

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.

As it has been suggested that protein acylation plays a role in nutrient stimulus–secretion coupling in the pancreatic β-cell, we examined the insulin-secreting INS 832/13 β-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 Ca2+ currents measured in the presence and absence of the protein acylation inhibitors cerulenin and tunicamycin. Both inhibitors enhanced the peak amplitude of ICa,L. Both increased the peak inward current in the range between −40 and +30 mV and shifted the apparent maximum current by 10 mV in the hyperpolarizing direction without affecting the activation threshold of −40 mV. 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 Ca2+ charge influx was increased by the drugs at all potentials tested. In contrast to these effects on the L-type Ca2+ 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 Ca2+ channel function in the insulin-secreting β-cell.

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 pm) inhibited insulin secretion in response to glucose, and to the non-hydrolys able 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. Copyright (c) 2006 John Wiley & Sons, Ltd.

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.