Search

In human pancreatic islets, the neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule synthesized by and released from the insulin-secreting β cells. The effective, physiological GABA concentration range within human islets is unknown. Here we use native GABAA receptors in human islet β cells as biological sensors and reveal that 100–1000 nM GABA elicit the maximal opening frequency of the single-channels. In saturating GABA, the channels desensitized and stopped working. GABA modulated insulin exocytosis and glucose-stimulated insulin secretion. GABAA receptor currents were enhanced by the benzodiazepine diazepam, the anesthetic propofol and the incretin glucagon-like peptide-1 (GLP-1) but not affected by the hypnotic zolpidem. In type 2 diabetes (T2D) islets, single-channel analysis revealed higher GABA affinity of the receptors. The findings reveal unique GABAA receptors signaling in human islets β cells that is GABA concentration-dependent, differentially regulated by drugs, modulates insulin secretion and is altered in T2D. Keywords: GABA, GABAA receptor, Pancreatic islet, Type 2 diabetes

The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100 nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100 nM, 500 nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion., Highlights • GABA regulates cytokine secretion in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. • GABA inhibits secretion of 47 cytokines in PBMCs from type 1 diabetes patients. • GABA regulates secretion of pro- and anti-inflammatory cytokines in a concentration-dependent manner. GABA is a signal molecule in the brain, blood and pancreatic islets where it is secreted by the insulin-producing β cells. GABA has many roles in human islets including optimizing function and survival of β cells. Bhandage et al. now show that GABA is a potent regulator of secretion of both pro- and anti-inflammatory cytokines in stimulated immune cells. In type 1 diabetes the β-cell mass is diminished and thus the protective effect of GABA in the islets although not in blood. Targeting GABA signaling in diabetes mellitus is likely to be a part of the solution when curing diabetes.

In the vertebrate brain, inhibition is largely mediated by raminobutyric acid (GABA). This neurotransmitter comprises a signaling machinery of GABA(A), GABA(B) receptors, transporters, glutamate decarboxylases (gads) and 4-aminobutyrate aminotransferase (abat), and associated proteins. Chloride is intimately related to GABAA receptor conductance, GABA uptake, and GADs activity. The response of target neurons to GABA stimuli is shaped by chloride-cation co-transporters (CCCs), which strictly control Cl- gradient across plasma membranes. This research profiled the expression of forty genes involved in GABA signaling in the zebrafish (Danio rerio) brain, grouped brain regions and retinas. Primer pairs were developed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The mRNA levels of the zebrafish GABA system share similarities with that of mammals, and confirm previous studies in non-mammalian species. Proposed GABAA receptors are alpha(1)beta(2)gamma(2), alpha(1)beta(2)delta, alpha(2b)beta(3), alpha(2b)beta(3)delta, alpha(4)beta(2)gamma(2), alpha(4)beta(2)gamma, alpha(6b)beta(2)gamma(2) and alpha(6b)beta(2)delta. Regional brain differences were documented. Retinal hetero- or homomeric rho-composed GABAA receptors could exist, accompanying alpha(1)beta(y)gamma(2), alpha(1)beta(y)delta, alpha(6a)beta(y)gamma(2,) alpha(6a)beta(y)delta. Expression patterns of alpha(6a) and alpha(6b) were opposite, with the former being more abundant in retinas, the latter in brains. Given the stoichiometry alpha(6w)beta(y)gamma(z), alpha(6a-) or alpha(6b)-containing receptors likely have different regulatory mechanisms. Different gene isoforms could originate after the rounds of genome duplication during teleost evolution. This research depicts that one isoform is generally more abundantly expressed than the other. Such observations also apply to GABAB receptors, GABA transporters, GABA-related enzymes, CCCs and GABAA receptor associated proteins, whose presence further strengthens the proof of a GABA system in zebrafish.

Glucagon-like peptide-1 (GLP-1) is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM), an allosteric positive modulator of GABAA receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC) amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABAA current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM) plus diazepam (1 μM), only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABAA receptors in the CA3 pyramidal neurons.

Glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion. Receptors for GLP-1 are also found in the brain, including the hippocampus, the center for memory and learning. Diabetes is a risk factor for decreased memory functions. We studied effects of GLP-1 and exendin-4, a GLP-1 receptor agonist, on γ-aminobutyric acid (GABA) signaling in hippocampal CA3 pyramidal neurons. GABA is the main inhibitory neurotransmitter and decreases neuronal excitability. GLP-1 (0.01–1 nmol/L) transiently enhanced synaptic and tonic currents, and the effects were blocked by exendin (9-39). Ten pmol/L GLP-1 increased both the spontaneous inhibitory postsynaptic current (sIPSC) amplitudes and frequency by a factor of 1.8. In 0.1, 1 nmol/L GLP-1 or 10, 50, or 100 nmol/L exendin-4, only the sIPSC frequency increased. The tonic current was enhanced by 0.01–1 nmol/L GLP-1 and by 0.5–100 nmol/L exendin-4. When action potentials were inhibited by tetrodotoxin (TTX), inhibitory postsynaptic currents decreased and currents were no longer potentiated by GLP-1 or exendin-4. In contrast, although the tonic current decreased in TTX, it was still enhanced by GLP-1 or exendin-4. The results demonstrate GLP-1 receptor regulation of hippocampal function and are consistent with GLP-1 receptor agonists enhancing GABAA signaling by pre- and postsynaptic mechanisms.

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain., This work was supported by the Swedish Research Council, the School of Pharmaceutical Sciences (FLÄK), Lund University, the UK Medical Research Council and by grants from the Spanish government (BFU2009-07835 and CONSOLIDER-INGENIO 2010 CSD2007-0023).

In the rat islets γ-aminobutyric acid (GABA) is produced by the β-cells and, at least, the α-cells express the GABA(A) receptors (GABA(A) channels). In this study, we examined in intact islets if the interstitial GABA activated the GABA(A) receptors. We used the patch-clamp technique to record whole-cell and single-channel currents and single-cell RT-PCR to identify the cell-type we recorded from, in the intact rat islets. We further identified which GABA(A) receptor subunits were expressed. We determined the cell-type of 43 cells we recorded from and of these 49%, 28% and 7% were α, β and δ-cells, respectively. In the remaining 16% of the cells, mRNA transcripts of more than one hormone gene were detected. The results show that in rat islets interstitial GABA activates tonic current in the α-cells but not in the β-cells. Seventeen different GABA(A) receptor subunits are expressed with high expression of α1, α2, α4, α6, β3, γ1, δ, ρ1, ρ2 and ρ3 subunits whereas no expression was detected for α5 or ε subunits. The abundance of the GABA(A) receptor subunits detected suggests that a number of GABA(A) receptor subtypes are formed in the islets. The single-channel and tonic currents were enhanced by pentobarbital and inhibited by the GABA(A) receptor antagonist SR-95531. The single-channel conductance ranged from 24 to 105 pS. Whether the single-channel conductance is related to subtypes of the GABA(A) receptor or variable interstitial GABA concentrations remains to be determined. Our results reveal that GABA is an extracellular signaling molecule in rat pancreatic islets and reaches concentration levels that activate GABA(A) receptors on the glucagon-releasing α-cells.