Glucose-induced Activity Research Articles

347-LB: MEA Technology as an Easy Electrophysiological Approach for Noninvasive Recordings of Intact Murine and Human Islets

Increase of blood glucose concentration leads to glucose-induced electrical activity of pancreatic beta-cells followed by insulin secretion. Electrical activity appears in glucose concentration-dependent oscillations easily recorded by microelectrode arrays (MEA). In comparison to other electrophysiological techniques MEAs can record non-invasively from intact islets and can be used for long term studies to investigate chronic effects of drugs. Murine islets or islets isolated from human biopsies were used for recordings. Electrical activity was quantified as fraction of plateau Phase (FOPP) or number of single spike activity. Islets were placed via suction or cultivated on MEAs. The glucose concentration response curve recorded with murine islets plotted as FOPP revealed a half-maximal activity of 12 ± 2 mM which is in line with traditional methods like e.g., with intracellular electrodes. Glucose-induced activity of cultured islets remains stable over a month. Application of well-known substances (like e.g., diazoxide, tolbutamide) matched regular results. Human islets displayed typical glucose-induced activity (10 mM). Validation studies showed the existence of functional KATP channels and TTX reduced spike activity from 1047 ± 314 to 199 ± 90 spikes/5min after the application of 300 nM TTX. The washout led to an increased spike activity of 779 ± 301 spikes/minutes. The glucose concentration response curve revealed an EC50 value of 8 ± 3 mM glucose. The glucose responsiveness recorded with the MEA Technology is in line with other reports showing that the EC50 value of human beta-cells (∼ 6 mM) is lower than in mouse beta-cells. MEA Technology allows medium throughput recordings from up to 40 intact islets simultaneously. While previous electrophysiological attempts failed MEAs offer large scale research in both academics and industry. The principle of function is not limited to rodent but is also applicable to human or stem cell derived islets of Langerhans. Disclosure S. Schönecker: None. M. Oza: Employee; Self; Multichannel Systems, Gmbh. U. Kraushaar: None.

Distinct Effects of Royal Jelly on Human Endothelial Cells Under High Glucose Condition.

To assess different effects of royal Jelly in protecting the human endothelial cells from high glucose level, human umbilical vein endothelial cells were exposed to various concentrations of royal jelly, from 0.625 to 10 mg/mL, at the presence of 5 and 30 mM glucose contents over a course of 72 h. In addition to cell viability assessment by conventional MTT assay, we also analyzed the feature of stemness by expression of Sox-2 and CD133 factors. Moreover, fatty acid profile, the expression of autophagy-related factor, namely microtubule-associated protein light chain 3 and activity of metalloproteinase 2 and 9 and were investigated. Royal jelly supplementation at the concentrations lower than 2.5 mg/mL did not influence the survival rate of cells and partially blunted the cytotoxic effects of 30 mM glucose. The expression of CD133 and Sox-2 factors were increased by royal jelly alone. Interestingly, an up-regulated expression of Sox-2 (58.8 ± 4%) coincided with a reduction in the levels of CD133 (15.1 ± 8.3%) in the combined treatment. We notified that the contents of palmitate and trans-palmitate as well as linoleate decreased by 30 mM glucose content while cis-palmitate levels increased when RJ returned them to near-normal levels (p < 0.05). The expression of autophagy marker was prominently induced in the presence of royal jelly in both conditions (p < 0.05). The glucose-induced activity of metalloproteinases was also reduced. Royal jelly is able to attenuate the abnormal status of 30 mM glucose condition in endothelial cells by different mechanisms.

CARDIOVASCULAR SAFETY OF GLIPTINS. FOCUS ON ALOGLIPTIN

Type 2 diabetes is a chronic progressive disease the prevalence of which is increasing. The development of new strategies for the treatment of diabetes, among which drugs that modulate the “incretin effect” are worth noting, continues. Dipeptidyl peptidase-4 inhibitors (DPP-4, gliptins) are a group of oral antidiabetic incretin drugs that enhance the glucose-induced activity of β-cells and suppress excessive glucagon secretion by pancreatic α-cells. This group of antidiabetic drugs is widespread due to a number of relevant benefits, such as neutral effect on body weight, low risk of hypoglycaemia, convenient administration scheme and good adherence to treatment. Efficacy and safety of treatment demonstrated by global multicenter trials allow for a wide use of gliptins in clinical practice.

Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction.

CFTR: a missing link between exocrine and endocrine pancreas?

CFTR: a missing link between exocrine and endocrine pancreas?

Extracellular factors and immunosuppressive drugs influencing insulin secretion of murine islets

Approximately 60% of transplanted islets undergo apoptosis within the first week post-transplantation into the liver attributed to poor engraftment, immune rejection and toxicity of immunosuppressive drugs. Understanding how extracellular matrix (ECM) components, immunosuppressive drugs and proinflammatory cytokines affect insulin secretion will contribute to an improved clinical outcome of islet transplantations. In this study, functional activity of isolated murine islets was measured by glucose-stimulated insulin secretion (GSIS) and by electrophysiological measurements using patch-clamp. Cultivating islets with soluble fibronectin or laminin, as opposed to with coated laminin, markedly increased GSIS. Addition of cyclosporin A reduced GSIS and suppressed glucose-induced spike activity. Tacrolimus affected neither GSIS nor spike activity, indicating a different mechanism. To evaluate the influence of proinflammatory cytokines, islets were incubated with interleukin (IL)-1β, tumour necrosis factor (TNF)-α or with supernatants from cultured Kupffer cells, the main mediators of inflammation in the hepatic sinusoids. IL-1β exerted a bimodal effect on insulin secretion, stimulating below 2 ng/ml and suppressing above 10 ng/ml. Soluble laminin in combination with a stimulatory IL-1β concentration further increased insulin secretion by 20% compared to IL-1β alone, while with high IL-1β concentrations soluble laminin slightly attenuated GSIS inhibition. TNF-α alone did not affect GSIS, but with stimulatory IL-1β concentrations completely abolished it. Similarly, supernatants derived from Kupffer cells exerted a bimodal effect on GSIS. Our data suggest that improved insulin secretion of transplanted islets could be achieved by including soluble laminin and low IL-1β concentrations in the islet cultivation medium, and by a simultaneous inhibition of cytokine secretion from Kupffer cells.

High glucose promotes intracellular lipid accumulation in vascular smooth muscle cells by impairing cholesterol influx and efflux balance

High glucose promotes macrophage-derived foam cell formation involved in increased influx or reduced efflux of lipids. The aim of this study is to investigate the influence of hyperglycaemia on foam cell transformation of vascular smooth muscle cells (VSMCs) and possible mechanisms contributing to these effects. The results showed that high glucose increased the expression of CD36, a regulator of lipid influx, and suppressed the expression and activity of the adenosine triphosphate-binding cassette (ABC) transporter ABCG1, a regulator of cholesterol efflux to high-density lipoprotein, in a dose- and time-dependent manner. However, cholesterol efflux to lipid-free apoAI was not impaired. VSMCs exposed to high glucose readily developed into lipid-loaded cells, as demonstrated by Oil Red O staining and cholesterol content analysis. In addition, high glucose-induced down-regulation of ABCG1 was reversed by nuclear factor-kappaB (NF-kappaB) inhibitors BAY 11-7085 and tosyl-phenylalanine chloromethyl ketone and by the antioxidant N-acetyl-L-cysteine (NAC). This reversal was accompanied by reduced cellular lipid content. Also, NAC and NF-kappaB inhibitors can effectively block the high glucose-induced activity of NF-kappaB binding to DNA and/or peroxide production. These results suggested that hyperglycaemia-induced foam cell formation in VSMCs was related to the imbalanced lipid flux by increasing CD36-mediated modified low-density lipoprotein uptake and reducing ABCG1-regulated cellular cholesterol efflux. Moreover, this effect was associated with increased oxidative stress and activated NF-kappaB pathway signalling.

The Effect of Epicatechin on the High Glucose-induced TSP-1 Expression and MMP-2 Activity in Rat Vascular Smooth Muscle Cells

Background: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. Methods: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 μM) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. Results: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. Conclusion: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis. (J Kor Soc Endocrinol 21:302~310, 2006)

Glucose mediates transcriptional repression of the human angiotensin type-1 receptor gene: role for a novel cis-acting element.

Human angiotensin type 1 receptor (hAT1R) gene is regulated by hormones, second messengers, and both pathophysiological and developmental states. The focus of the present study was to determine the role of glucose in the trans-repression of hAT1R gene transcription and to identify the functional cis-acting response element(s). Serial deletions of the hAT1R promoter region indicated that an area between -1717 and -1543 base pairs upstream of the 5' end of the cDNA sequence has a glucose responsive regulatory element (GluRE) to down-regulate the gene expression. Further analysis revealed a putative 29-bp (5'-AACTGATTTTTGTATATTGATCTTGTATT-3') repressor element located between -1582 and -1610 bp was necessary for transcriptional repression. Removal of this region from promoter construct abolished repression of the hAT1R gene transcription in human proximal tubule epithelial cells (hPTECs). Using mobility shift assays, we demonstrated DNA binding activity to the labeled repressor element in hPTEC nuclear extracts. Additional studies demonstrated increased DNA binding activity to the labeled repressor element in nuclear extracts treated with high glucose (25 mM). Southwestern analysis identified two GluRE binding proteins of 34 and 36 kDa in glucose-treated extracts. Glucose-induced activity of the repressor trans-acting factor(s) reached a maximum at 4 h, which correlated with decreased transcriptional activity of the hAT1R gene, suggesting that glucose can down-regulate the transcription of the hAT1R gene through the repressor element. Furthermore, insertion of the glucose response element into heterologous SV40 promoter (SV40) chloramphenicol acetyl transferase (CAT) vector showed orientation/distance-independent repression of SV40 promoter-mediated CAT activity in hPTECs. Our results show that the glucose response factor(s) acts as trans-acting factor(s) binding to the cis-acting repressor element in the hAT1R promoter, which may participate in the control of basal transcription as well as glucose-mediated transcriptional inhibition of the hAT1R gene.

Compound exocytosis in voltage-clamped mouse pancreatic beta-cells revealed by carbon fibre amperometry.

Capacitance measurements of exocytosis were combined with carbon fibre amperometry for time-resolved measurements of the properties of secretion in single, insulin-secreting, mouse pancreatic beta-cells pre-loaded with the amine serotonin (5-HT). Glucose-induced electrical activity was associated with the appearance of brief and transient amperometric currents reflecting the serotonin co-released with insulin. The integrated amperometric responses resulting from voltage-clamp depolarisations were proportional to the corresponding increase in cell capacitance. Both parameters exhibited U-shaped relationships to the membrane potential with maximums around 0 mV. There was a variable latency (40-730 ms, average 230 ms) between the onset of the depolarisation and the amperometric current. During high-frequency repetitive stimulation, a progressive decrease in the exocytotic capacity ("depression") was observed. This was paralleled by a corresponding reduction of the amperometric responses. Using the carbon fibre to map the beta-cell for release sites indicated that exocytosis was confined to the part of the cell containing the highest density of secretory granules. Two types of amperometric responses were observed. In about 50% of the cells, a smooth increase was observed with no discernible discrete events. In the remaining cells, the amperometric records contained large spikes. These were ten or more times larger than that expected for the fusion of individual secretory granules. We propose that these large spikes reflect the exocytosis of multigranular complexes formed inside the beta-cell prior to exocytosis.

Glucose-Induced Activity of Liver-Type Pyruvate Kinase Promoter in Primary Rat Hepatocytes

Glucose-Induced Activity of Liver-Type Pyruvate Kinase Promoter in Primary Rat Hepatocytes

Adenosine triphosphate-sensitive K+ channels may not be the sole regulators of glucose-induced electrical activity in pancreatic B-cells

Adenosine triphosphate-sensitive K+ channels may not be the sole regulators of glucose-induced electrical activity in pancreatic B-cells

The influence of temperature on the effects of acetylcholine and adrenaline on the membrane potential and 86Rb efflux in mouse pancreatic B-cells

Pancreatic mouse islets were used to evaluate the influence of temperature on the B-cell response to acetylcholine and adrenaline. At 20 degrees C, the rate of 86Rb efflux from islet cells was lower, the membrane potential of B-cells was slightly less negative, and glucose-induced electrical activity was characterized by longer slow waves than at 37 degrees C. At 20 degrees C, the acceleration of 86Rb efflux produced by 1 microM-ACh was only reduced by 25%, but its reversibility was slower. Acetylcholine rapidly depolarized the B-cell membrane and increased electrical activity regardless of the temperature. However, this increase was characterized by the appearance of short slow waves of high frequency at 37 degrees C and by continuous spiking at 20 degrees C. Adrenaline (1 microM) inhibited 86Rb efflux at 37 and 20 degrees C, but the amplitude of the inhibition was decreased and its time course and reversibility were altered at the lower temperature. Adrenaline repolarized the B-cell membrane and abolished glucose-induced electrical activity for a longer period at 20 degrees C than at 37 degrees C. In conclusion, no marked decrease in signal transduction occurs at 20 degrees C. This suggests that the difficulty of identifying the currents induced by acetylcholine and adrenaline in patch-clamp experiments performed at room temperature is probably due to the small magnitude of these currents.

Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells

Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved.

Membrane and intracellular effects of adenosine in mouse pancreatic beta-cells.

Mouse islets were used to study the effects of adenosine and its stable analogue L-N6-phenylisopropyladenosine (L-PIA) on pancreatic beta-cell function. At a high concentration (500 microM), adenosine augmented glucose-induced electrical activity in beta-cells and potentiated insulin release. These effects were prevented by the inhibitor of nucleoside transport nitrobenzylthioguanosine. They probably result from the metabolism of adenosine by beta-cells. At a lower concentration (50 microM), adenosine caused a small and transient inhibition of glucose-induced electrical activity and insulin release. L-PIA (10 microM) slightly and transiently inhibited insulin release, 45Ca efflux and 86Rb efflux from islet cells, and decreased electrical activity in beta-cells. When adenylate cyclase was stimulated by forskolin in the presence of 15 mM glucose, insulin release was strongly augmented. Under these conditions, L-PIA and adenosine (with nitrobenzylthioguanosine) caused a sustained inhibition. No such inhibition was observed when insulin release was potentiated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). These data are consistent with the existence of A1 purinergic receptors on mouse beta-cells. They could mainly serve to attenuate the amplification of insulin release brought about by agents acting via cAMP.

Ultracytochemical nuclear calcium distribution in pancreatic B cells: its relation to glucose-stimulated insulin secretion.

Calcium distribution in pancreatic B cells was studied, with the aid of the pyroantimonate technique, at different times of glucose-induced secretory activity in the perfused rat pancreas. Specificity of the pyroantimonate precipitates for calcium was assessed by EGTA cross-incubation. Quantitative studies for these calcium pyroantimonate precipitates were performed by a morphometric technique. Pyroantimonate precipitates within the B cell show both time and glucose dependence. At any time-point studied, in the nucleus as well as in other organelles, they were more numerous when glucose was increased in the medium from 3.3 to 16.6 mmol/l. The total number of nuclear calcium pyroantimonate precipitates rose sharply at the time corresponding to the refractory period, falling after that to almost the number found at the prestimulatory period. Otherwise, glucose significantly modifies the temporal distribution of precipitates bound to euchromatin, heterochromatin and perichromatin. These changes in nuclear calcium pyroantimonate precipitates during different periods of B cell secretory activity may indicate an active role of the cation in some nuclear regulatory function during glucose-induced release of insulin.

PH modulation of glucose-induced electrical activity in B-cells: involvement of Na/H and HCO3/Cl antiporters.

Regulation of intracellular pH is an essential function and may be especially significant in the B-cell in which the influence of glucose on electrical activity is modulated by alterations in pH. Two possible regulatory processes have been examined: Na/H and HCO3/Cl exchange, by using inhibitors, an ionophore, and changes of ionic concentrations. In the presence of 11.1 mM glucose we found that DIDS, an inhibitor of anion exchange, elicited a dose-response increase in the relative duration of the active phase with an ED50 of 99 microM. Probenecid (0.5 mM), an inhibitor of anion fluxes, also augmented the electrical activity (EA) due to glucose. Withdrawal of HCO-3 elicited constant spike activity followed by a resumption of burst activity with a greater duration of the active phase compared to control. These data are consistent with predicted cellular acidification. However, reduction of Cl-o by isethionate substitution produced no marked effect on EA. In contrast, SO-4- substitution for Cl- resulted in variable effects characterized by constant spike activity or a decrease in the duration of the active and silent phases along with silent hyperpolarization. Tributyltin, a Cl/OH, ionophore enhanced EA at 0.25 microM with 120 mM Cl-o, but reduced EA with 10 mM Cl- as would be predicted with either cellular acidification or alkalinization, respectively. Amiloride at 100 microM elicited constant spike activity perhaps due to inhibition of Na/H exchange. Reduction of Na+o from 142.8 to 40.8 mM had a similar effect and enhanced the influence of amiloride. It appears therefore that interference with putative pH regulatory mechanisms in the B-cell are consistent with the hypothesis that cell pH is involved in regulation of EA.

Influence on anion transport on glucose-induced electrical activity in the B-cell

Influence on anion transport on glucose-induced electrical activity in the B-cell