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Interstitial GABA activates tonic GABA A receptor currents in rat pancreatic islets. A. A representative trace showing GABAactivated tonic current, recorded from a cell in an intact rat islet (Vp = 290 mV). The current was activated by interstitial GABA and inhibited by application of SR-95531, causing a clear outward shift in the holding current (line 1 to line 2). B. Gaussian fits to all-points histogram derived from 20 s current recordings in (A) before (1) and after (2) application of SR-95531. SR-95531 insensitive current was subtracted for analysis. The difference between the peaks of the Gaussian fits denotes the mean tonic current (5.3 pA). C. Whole-cell current-voltage (IV) relationship of GABA-activated current in rat islet cells (n = 3). The current activated by the interstitial GABA (I-I SR-95531 = I GABA ) is plotted. The IV relation shows outward rectification. D. A representative current showing GABA-activated tonic current (21 pA, Vp = 290 mV) that is significantly larger than the current shown in (A). E. Gaussian fits to all-points histogram derived from 20 s current recordings in (D) before (1) and after (2) application of SR-95531. SR-95331 insensitive current was subtracted for analysis. F. Whole-cell IV relationship of GABA-activated current in rat islet cells (n = 3). The IV relation shows outward rectification. doi:10.1371/journal.pone.0067228.g003
Source publication
In the rat islets γ-aminobutyric acid (GABA) is produced by the β-cells and, at least, the α-cells express the GABAA receptors (GABAA channels). In this study, we examined in intact islets if the interstitial GABA activated the GABAA receptors. We used the patch-clamp technique to record whole-cell and single-channel currents and single-cell RT-PCR...
Contexts in source publication
Context 1
... b-cells and thereby maximize the interstitial GABA concentration in the islets during our experi- ments. The highest concentration is expected to be around the b- cells release sites from where it then diffuses to the rest of the islet. We examined if the interstitial GABA activated GABA A receptors in the islet cells and the results are shown in Fig. 3. Although no GABA was added experimentally, whole-cell currents were reduced by SR-95531, a GABA A receptor specific antagonist. SR-95531 reduced the baseline current but increased again when SR-95531 was withdrawn. The difference in the whole-cell current level in the presence and absence of SR-95531 revealed the level of the ...
Context 2
... added experimentally, whole-cell currents were reduced by SR-95531, a GABA A receptor specific antagonist. SR-95531 reduced the baseline current but increased again when SR-95531 was withdrawn. The difference in the whole-cell current level in the presence and absence of SR-95531 revealed the level of the GABA-activated tonic current in the cell (Fig. 3A, B Gaussian fits to all-points histogram derived from 20 s current recordings in (A) before (2) and after application of (1) pentobarbital and (3) SR-95531 SR-95531 insensitive current was subtracted for analysis. The difference between the peaks of the Gaussian fits shows the evoked (5 pA, (2) - (3)) and the enhanced (17 pA, (1) - (3)) ...
Context 3
... on the gel contain amplified RT-PCR products: insulin (primer: Ins), glucagon (primer: Gcg2) or somatostatin (primer: Sst2). The cell-type was identified as a b-cell. Positive controls were purified rat islets RNA samples and the negative control was water. doi:10.1371/journal.pone.0067228.g004 GABA-activated current was outwardly rectifying (Fig. 3C) demonstrating that more current passes through the GABA A receptors at depolarized as compared to hyperpolarized potentials in rat pancreatic islets. In another four cells the tonic current was significantly larger (Fig. 3D and E, 21 pA, Vp = 290 mV; Fig 3F). Whether the larger current level is due to more GABA A receptors in the ...
Context 4
... current was outwardly rectifying (Fig. 3C) demonstrating that more current passes through the GABA A receptors at depolarized as compared to hyperpolarized potentials in rat pancreatic islets. In another four cells the tonic current was significantly larger (Fig. 3D and E, 21 pA, Vp = 290 mV; Fig 3F). Whether the larger current level is due to more GABA A receptors in the plasma membrane or different subpopulations of GABA A receptors expressed in the cells is not known. ...
Citations
... These subunits suggest mouse beta cells are attuned to tonic GABA (a4b3d). Expression of GABA A R receptor subunits has also been studied in rat islets with many different subunits detected that could comprise both tonic and phasic receptor subtypes (11,102). ...
... Measurements of rat and human beta cell native GABA A R responses to interstitial GABA (no external GABA applied) showed clear tonic conductances (11,45), so it reasonably clear that beta cells have tonic GABA responses. Information on phasic GABA responses is more elusive as the best data available are from human beta cells transduced to overexpress the synaptic a1b1 GABA A R which showed both phasic and tonic responses (62,76,83). ...
... Experiments to measure the average open probability and mean current of the native beta cell GABA A Rs at different membrane potentials showed beta cell GABA A Rs are outwardly rectifying (10,11). This means that the beta cell GABA A R carries a low Clcurrent at hyperpolarized potentials but current increases as the membrane becomes more depolarized. ...
Gamma aminobutyric acid (GABA) is a non-proteinogenic amino acid and neurotransmitter that is produced in the islet at levels as high as in the brain. GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), of which the 65 kDa isoform (GAD65) is a major autoantigen in type 1 diabetes. Originally described to be released via synaptic-like microvesicles or from insulin secretory vesicles, beta cells are now understood to release substantial quantities of GABA directly from the cytosol via volume-regulated anion channels (VRAC). Once released, GABA influences the activity of multiple islet cell types through ionotropic GABAA receptors and metabotropic GABAB
receptors. GABA also interfaces with cellular metabolism and ATP production via the GABA shunt pathway. Beta cells become depleted of GABA in type 1 diabetes (in remaining beta cells) and type 2 diabetes, suggesting that loss or reduction of islet GABA correlates with diabetes pathogenesis and may contribute to dysfunction of alpha, beta, and delta cells in diabetic individuals. While the function of GABA in the nervous system is wellunderstood, the description of the islet GABA system is clouded by differing reports describing multiple secretion pathways and effector functions. This review will discuss and attempt to unify the major experimental results from over 40 years of literature characterizing the role of GABA in the islet.
... In the pancreatic islets, GABA is concentrated in synaptic-like microvesicles, which are different from insulin-containing vesicles in β cells (Fig. 5) [6,7,35]. It is generally accepted that GABA A receptors are expressed in α cells [8,17,49,50]. Our current findings clearly demonstrate that the α cells express KCC2, which is consistent with previous physiological studies showing that GABA is released from β cells, causes membrane hyperpolarization, and reduces glucagon release (Fig. 5) [39,49,50]. ...
Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the mature brain; however, it acts excitatory during development. This difference in action depends on the intracellular chloride ion concentration, primarily regulated by potassium chloride co-transporter2 (KCC2). Sufficient KCC2 expression results in its inhibitory action. GABA is also abundant in pancreatic islets, where it acts differentially on the islet cells, and is involved in carbohydrate metabolism. However, the mechanisms underlying the differential action remain unknown. We performed immunohistochemistry for glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and KCC2 in normal adult islets. GAD was co-localized with insulin in β cells, whereas KCC2 was expressed in glucagon-positive α cells. These results are in line with previous observations that GABA decreases glucagon release but increases insulin release, and suggest that GABA and insulin may work together in reducing blood glucose levels under hyperglycemia. Next, we examined the streptozotocin-induced type1 diabetes mellitus mouse model. GAD and insulin expression levels were markedly decreased. KCC2 was expressed in glucagon-positive cells, whereas insulin- and somatostatin-positive cells were KCC2-negative. These findings suggest that in diabetes model, reduced GABA release may cause disinhibition of glucagon release, resulting in increased blood sugar levels and the maintenance of hyperglycemic state.
... 70 estradiol inhibits the Ca 2+ influx oscillations and subsequently glucagon secretion in pancreatic α-cells (Nadal et al. 2004). However, the β-cells produce significant amounts of aminobutyric acid (GABA), resulting in GABA receptor type A activation (GABAAR) and subsequent inhibition of glucagon secretion in pancreatic α-cells (Jin et al. 2013). Meanwhile, insulin negatively modulates GABAAR via an ERK-dependent negative feedback loop mechanism (Bansal et al. 2011). ...
As gestational diabetes mellitus (GDM) is both a frequent and serious complication, steroid levels in pregnancy are extremely elevated and their role in pregnancy is crucial, this review focuses on the role of steroids and related substances in the GDM pathophysiology. Low SHBG levels are associated with insulin resistance and hyperinsulinemia, while also predicting a predisposition to GDM. Other relevant agents are placental hormones such as kisspeptin and CRH, playing also an important role beyond pregnancy, but which are synthesized here in smaller amounts in the hypothalamus. These hormones affect both the course of pregnancy as well as the synthesis of pregnancy steroids and may also be involved in the GDM pathophysiology. Steroids, whose biosynthesis is mainly provided by the fetal adrenal glands, placenta, maternal adrenal glands, and both maternal and fetal livers, are also synthesized in limited amounts directly in the pancreas and may influence the development of GDM. These substances involve the sulfated Δ5 steroids primarily acting via modulating different ion channels and influencing the development of GDM in different directions, mostly diabetogenic progesterone and predominantly anti-diabetic estradiol acting both in genomic and non-genomic way, androgens associated with IR and hyperinsulinemia, neuroactive steroids affecting the pituitary functioning, and cortisol whose production is stimulated by CRH but which suppresses its pro-inflammatory effects. Due to the complex actions of steroids, studies assessing their predominant effect and studies assessing their predictive values for estimating predisposition to GDM are needed.
... However, the β-cells produce significant amounts of aminobutyric acid (GABA), resulting in GABA receptor type A activation Vol. 70 (GABA A R) and subsequent inhibition of glucagon secretion in pancreatic α-cells (Jin et al. 2013). Meanwhile, insulin negatively modulates GABA A R via an ERK-dependent negative feedback loop mechanism (Bansal et al. 2011). ...
As gestational diabetes mellitus (GDM) is both a frequent and serious complication, steroid levels in pregnancy are extremely elevated and their role in pregnancy is crucial, this review focuses on the role of steroids and related substances in the GDM pathophysiology. Low SHBG levels are associated with insulin resistance and hyperinsulinemia, while also predicting a predisposition to GDM. Other relevant agents are placental hormones such as kisspeptin and CRH, playing also an important role beyond pregnancy, but which are synthesized here in smaller amounts in the hypothalamus. These hormones affect both the course of pregnancy as well as the synthesis of pregnancy steroids and may also be involved in the GDM pathophysiology. Steroids, whose biosynthesis is mainly provided by the fetal adrenal glands, placenta, maternal adrenal glands, and both maternal and fetal livers, are also synthesized in limited amounts directly in the pancreas and may influence the development of GDM. These substances involve the sulfated Δ5 steroids primarily acting via modulating different ion channels and influencing the development of GDM in different directions, mostly diabetogenic progesterone and predominantly anti-diabetic estradiol acting both in genomic and non-genomic way, androgens associated with IR and hyperinsulinemia, neuroactive steroids affecting the pituitary functioning, and cortisol whose production is stimulated by CRH but which suppresses its pro-inflammatory effects. Due to the complex actions of steroids, studies assessing their predominant effect and studies assessing their predictive values for estimating predisposition to GDM are needed.
... The newly produced cDNA was directly applied as a pattern for RT-PCR by the succeeding reaction: 2 μl of cDNA template was amplified in 25 μl of total reaction volume containing 0.5 μl of each specific primer ß actin (housekeeping gene) and γ2 (target genes). We used two sets of primers as designed by Briner et al (19). Primer sequences for ß actin were 5´-cctggcacccagcacaat-3´ (sense) and 5´-gggccggactcgtcatact-3´ (anti-sense). ...
Objectives:
Stress during pregnancy is able to bring extensive effects on neurobehavioral development in offspring. The GABAergic system plays a pivotal role in neuronal excitability, which can be affected by prenatal stress (PS). This study aimed to evaluate impact of the PS on γ2 subunit of gamma-aminobutyric acid A (GABAA) receptor gene expression in the hippocampus and seizure induced by pentylenetetrazol (PTZ) in developing rats.
Materials and methods:
In this experimental study, female Wistar rats were exposed to restraint stress during gestation and their offspring were studied on postnatal days 14 and 21 (P14 and P21, respectively) for epileptic behaviors and γ2 GABAA receptor subunit gene expression. Quantitative real-time PCR was used for evaluating the γ2 GABAA receptor subunit gene expression in rat pups. Meanwhile, PTZ was injected into the pups, and seizure behaviors were recorded for 60 min.
Results:
The results showed that γ2 subunit mRNA expression significantly increased in the hippocampus of the stressed pups. The expression level of γ2 subunit was higher on P21 compared to that on P14 in both groups. Number of seizures with tonic-clonic features increased in pups of stressed group compared to the control group. Prenatal stress significantly caused an increase in the total score of seizure on P21.
Conclusion:
The effect of PS on seizure susceptibility is age-specific; the increased γ2 subunit level in the hippocampus might be, at least in part, the underlying mechanism for PS-induced augmentation of seizures in immature rats.
... As the data from type 2 diabetic donors were limited and overlapped in values of the analysed parameters with the data from the non-diabetic donors, we combined the results from the two groups when examining iGABA A R single-channel properties and effects of days in culture on the channel properties (Figures 2 and 3). In rodent islets, the cell size normally correlates with the major cell-types [21,22], but the situation is somewhat different for human islet cells where we did not detect any difference in cell size among α, β and δ cells in intact islets [17]. However, it is possible that alterations in size reflect transdifferentiation of one cell-type to another. ...
... The cytosome harvesting procedure and single-cell RT-PCR were previously described [17,21]. Briefly, after completing the patch-clamp experiment in the whole-cell configuration, the negative pressure was applied to the back of the pipette and was relieved at the moment of the whole-cell configuration destroying. ...
... The 20 µL of RT-reaction was exposed to 42 • C for 30 min and then incubated at 95 • C for 2 min. PCR was accomplished according to a standard procedure [17,21]. In brief, the amplification of the PCR products was done with the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA) according to the following procedure: initial denaturation step at 95 • C for 5 min; followed by 45 cycles of 95 • C for 15 s, 60 • C for 30 s and 72 • C for 1min; followed by one melting curve step. ...
In pancreatic islets, the major cell-types are α, β and δ cells. The γ-aminobutyric acid (GABA) signalling system is expressed in human pancreatic islets. In single hormone transcript-expressing cells, we have previously characterized the functional properties of islet GABAA receptors (iGABAARs). Here, we extended these studies to islet cells expressing mRNAs for more than one hormone and sought for correlation between iGABAAR activity level and relative mRNA expression ratio. The single-cell RT-PCR in combination with the patch-clamp current recordings was used to examine functional properties of iGABAARs in the multiple hormone mRNA-expressing cells. We detected cells expressing double (α/β, α/δ, β/δ cell-types) and triple (α/β/δ cell-type) hormone transcripts. The most common mixed-identity cell-type was the α/β group where the cells could be grouped into β- and α-like subgroups. The β-like cells had low GCG/INS expression ratio (<0.6) and significantly higher frequency of iGABAAR single-channel openings than the α-like cells where the GCG/INS expression ratio was high (>1.2). The hormone expression levels and iGABAAR single-channel characteristics varied in the α/β/δ cell-type. Clearly, multiple hormone transcripts can be expressed in islet cells whereas iGABAAR single-channel functional properties appear to be α or β cell specific.
... Glutamic acid decarboxylase 1 (GAD1), the enzyme that synthesizes GABA, is highly enriched in beta cells and is a major type 1 diabetes autoantigen 32,33 . In rodents, GABA inhibits alpha cells through ionotropic GABA A receptors [123][124][125] (Fig. 3b), although metabotropic GABA B1 receptor expression is also detectable. GABA from beta cells has been proposed to be the reason why alpha cells are silenced under high glucose concentrations 124 , but GABA treatment by itself does not fully suppress rodent glucagon secretion 126 . ...
The pancreatic islet is a complex mini organ composed of a variety of endocrine cells and their support cells, which together tightly control blood glucose homeostasis. Changes in glucose concentration are commonly regarded as the chief signal controlling insulin-secreting beta cells, glucagon-secreting alpha cells and somatostatin-secreting delta cells. However, each of these cell types is highly responsive to a multitude of endocrine, paracrine, nutritional and neural inputs, which collectively shape the final endocrine output of the islet. Here, we review the principal inputs for each islet-cell type and the physiological circumstances in which these signals arise, through the prism of the insights generated by the transcriptomes of each of the major endocrine-cell types. A comprehensive integration of the factors that influence blood glucose homeostasis is essential to successfully improve therapeutic strategies for better diabetes management. Pancreatic islets are heterogeneous clusters of endocrine cells responsible for glucose homeostasis. Here Noguchi and Huising review the main stimuli for each islet-cell type and their response, guided by insights from islet-cell transcriptomes.
... The synthesized cDNA was used directly as a template for RT-PCR by the following reaction in total volume of 25 μ1: 2 μl of cDNA template was amplified in 25 μl of total reaction volume containing 0.5 μl of each specific primer of ß actin (housekeeping gene) and α5 (target gene). Primer sequences were 5´-cctggcacccagcacaat-3´(sense) and 5´-gggccggactcgtcatact-3´(anti-sense) for ß actin and 5´-ttattcttactgggaatggacaatgg-3´(sense) and 5´-ttaaaccgcagcctttcatctttc-3´(antisense) for α5 (Jin et al., 2013). PCR amplification was carried out using a Gene Amp 9600 PCR system (Perkin Elmer, Norwalk, CT, USA) under the following conditions: initial denaturation at 94•C for 3 min followed by 30 amplification cycles, each consisting of denaturation at 94•C for 30 s, annealing at 59•C for 30 s and extension at 72•C for 50 s, with an additional extension step at the end of the procedure at 72•C for 5 min. ...
The GABAergic synapses go through structural and functional maturation during early brain development. Maternal stress alters GABAergic synapses in developing brain, which are associated with the pathophysiology of neuropsychiatric disorders in adults. The present study aimed to investigate the effect of prenatal restraint stress (PS) on pilocarpine-induced seizure and ɑ5 subunit of γ-amino butyric acid type A (GABAA) receptor expression in hippocampus. Pregnant Wistar rats were subjected to PS at gestational days 15-17 and the pups were examined for susceptibility to seizure and ɑ5 subunit of GABAA receptor expression in hippocampus at postnatal days 14 and 21 (P14 and PND 21). Quantitative real-time PCR was used for evaluating the gene expression in the pups. Pilocarpine was injected intraperitoneally into the pups and seizure behaviors were recorded. The results showed that ɑ5 subunit mRNA expression significantly increased in hippocampus at both the P14 and P21 in the stressed rats. However, ɑ5 subunit level was greater at the P21 than at the P14 in both the groups. Latency of first tonic-clonic seizure significantly decreased in the PS group compared to the control pups. Number and duration of tonic-clonic seizures increased in the PS rats compared to the controls. PS led to an increase in total score of seizure at the P14 and P21. It can be concluded that PS increases the seizure susceptibility and GABAA receptor ɑ5 subunit gene expression in offspring; it is likely that the mechanism of increased seizure susceptibility by PS, at least in part, can increase the GABAA receptor ɑ5 subunit gene expression in hippocampus.
... This renders the human islet GABA signaling distinct from e.g. signaling in rat and guinea pig islets where functional GABA A receptors are not expressed in the β cells (Gilon et al. 1991;Jin et al. 2013;Rorsman et al. 1989;Wendt et al. 2004). The GABA A receptor activity in the brain is enhanced by a number of medicines such as the benzodiazepines, anesthetics and even by the metabolic hormone GLP-1 and its analogue exendin-4 (Korol et al. 2015;Olsen and Sieghart 2008). ...
... The 20 μl reverse transcription reaction was incubated at 42°C for 30 min followed by a second incubation at 95°C for 2 min. PCR was performed in a 10 μl reaction mixture containing 3 μl cDNA, 5× SYBR Green I (Life Technologies), 1× PCR reaction buffer, MgCl2 (3 mM), dNTP (0.3 mM), 1× ROX reference dye, 0.8 U JumpStart Taq DNA polymerase (Sigma-Adrich; Jin et al. 2013) and hormone genespecific primers. The PCR amplification was performed using the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) with an initial denaturation step of 5 min at 95°C, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 1 min, and one melting curve step. ...
Highlights
• In human islets GABA (≤μM) activates β cell-specific GABAA receptors that become supersensitive to GABA in type 2 diabetes.
• GABAA receptors activity in β cells is enhanced by diazepam, anesthetics, the incretin GLP-1 but not the hypnotic zolpidem.
• GABA modulates rate of insulin granule exocytosis and glucose-stimulated insulin secretion.
GABA is a signal molecule in the brain but is also secreted by the insulin-producing β cells in pancreatic islets. GABA has many roles in human islets that most aim at optimizing function and survival of β cells. In the report by Korol, Jin et al. the authors identify and characterize the molecular unit that GABA binds to in human β cells, the GABAA receptors. These receptors normally sensitive become super-sensitive to GABA in type 2 diabetes. The GABAA receptors regulate insulin secretion and can themselves be regulated by the anxiolytic diazepam, anesthetics, the incretin GLP-1 but not the hypnotic zolpidem. Targeting GABA signaling in human islets in diabetes mellitus is likely to be a part of the solution when curing diabetes.
... Importantly, using both X-Gal staining and immunohistochemical analyses ( Figures 6G-6J), the vast majority of newly formed insulin-producing cells were found to be labeled with b-galactosidase (permanently marking cells that once expressed glucagon), demonstrating that most of the regenerated b-like cells passed through a glucagon-expressing transitional phase. Accordingly, the repetition of this b cell ablation procedure in presence of GABA and of an antagonist of the a cell-specific GABA A receptor, SR-95531 (Jin et al., 2013), prevented the recovery from the lethal hyperglycemia, indicating that GABA mainly acts on a cells via the GABA A receptor ( Figure S6B compared to 6A). It is important to mention that mouse GABA A receptors are indeed expressed in mouse a cells (Bailey et al., 2007) (A) Two-month-old WT mice were subjected to high dose streptozotocin (STZ) treatment to ablate b cells and then treated with GABA (or saline) once they were overtly diabetic (glycemia R300 mg/dL). ...
The recent discovery that genetically modified α cells can regenerate and convert into β-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-β-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into β-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated β-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in β-like cell counts, suggestive of α-to-β-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated β-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.
