GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of mouse and human pancreatic islet glucagon secretion.

AIMS/HYPOTHESIS: Diabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7-36) to GLP-1(9-36). We hypothesised that the metabolite GLP-1(9-36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes. METHODS: We used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9-36). RESULTS: GLP-1(7-36) inhibited glucagon secretion in isolated islets with an IC50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9-36) shared this capacity. GLP-1(9-36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9-36) also potently inhibited glucagon secretion evoked by ß-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9-36) led to inhibition of Ca2+ entry via voltage-gated Ca2+ channels sensitive to ω-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9-36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9-36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content. CONCLUSIONS/INTERPRETATION: We conclude that the GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9-36)'s glucagonostatic action.

Loss of tetraspanin-7 expression reduces pancreatic ß-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

BACKGROUND: Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate ß-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic ß-cell function is not yet fully understood. METHODS: Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. ß-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. RESULTS: Tspan7 is expressed in insulin-containing granules of pancreatic ß-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- ß-cells, but ß-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent ß-cells, exocytotic Ca2+ sensitivity was reduced in a human ß-cell line (EndoC-ßH1) following Tspan7 KD. CONCLUSION: Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in ß-cells. Its function is more significant in human ß-cells than their rodent counterparts.

Perlecan: An Islet Basement Membrane Protein with Protective Anti-Inflammatory Characteristics.

Throughout the isolation process, human islets are subjected to destruction of the islet basement membrane (BM) and reduced oxygen supply. Reconstruction of the BM represents an option to improve islet function and survival post-transplant and may particularly be relevant for islet encapsulation devices and scaffolds. In the present study, we assessed whether Perlecan, used alone or combined with the BM proteins (BMPs) Collagen-IV and Laminin-521, has the ability to protect isolated human islets from hypoxia-induced damage. Islets isolated from the pancreas of seven different organ donors were cultured for 4-5 days at 2% oxygen in plain CMRL (sham-treated controls) or in CMRL supplemented with BMPs used either alone or in combination. Postculture, islets were characterized regarding survival, in vitro function and production of chemokines and reactive oxygen species (ROS). Individually added BMPs significantly doubled islet survival and increased in vitro function. Combining BMPs did not provide a synergistic effect. Among the tested BMPs, Perlecan demonstrated the significantly strongest inhibitory effect on chemokine and ROS production when compared with sham-treatment (p < 0.001). Perlecan may be useful to improve islet survival prior to and after transplantation. Its anti-inflammatory potency should be considered to optimise encapsulation and scaffolds to protect isolated human islets post-transplant.

The endoplasmic reticulum plays a key role in α-cell intracellular Ca2+ dynamics and glucose-regulated glucagon secretion in mouse islets.

Glucagon is secreted by pancreatic α-cells to counteract hypoglycaemia. How glucose regulates glucagon secretion remains unclear. Here, using mouse islets, we studied the role of transmembrane and endoplasmic reticulum (ER) Ca2+ on intrinsic α-cell glucagon secretion. Blocking isradipine-sensitive L-type voltage-gated Ca2+ (Cav) channels abolished α-cell electrical activity but had little impact on its cytosolic Ca2+ oscillations or low-glucose-stimulated glucagon secretion. In contrast, depleting ER Ca2+ with cyclopiazonic acid or blocking ER Ca2+-releasing ryanodine receptors abolished α-cell glucose sensitivity and low-glucose-stimulated glucagon secretion. ER Ca2+ mobilization in α-cells is regulated by intracellular ATP and likely to be coupled to Ca2+ influx through P/Q-type Cav channels. ω-Agatoxin IVA blocked α-cell ER Ca2+ release and cell exocytosis, but had no additive effect on glucagon secretion when combined with ryanodine. We conclude that glucose regulates glucagon secretion through the control of ER Ca2+ mobilization, a mechanism that can be independent of α-cell electrical activity.

Loss of electrical ß-cell to δ-cell coupling underlies impaired hypoglycaemia-induced glucagon secretion in type-1 diabetes.

Diabetes mellitus involves both insufficient insulin secretion and dysregulation of glucagon secretion1. In healthy people, a fall in plasma glucose stimulates glucagon release and thereby increases counter-regulatory hepatic glucose production. This response is absent in many patients with type-1 diabetes (T1D)2, which predisposes to severe hypoglycaemia that may be fatal and accounts for up to 10% of the mortality in patients with T1D3. In rats with chemically induced or autoimmune diabetes, counter-regulatory glucagon secretion can be restored by SSTR antagonists4-7 but both the underlying cellular mechanism and whether it can be extended to humans remain unestablished. Here, we show that glucagon secretion is not stimulated by low glucose in isolated human islets from donors with T1D, a defect recapitulated in non-obese diabetic mice with T1D. This occurs because of hypersecretion of somatostatin, leading to aberrant paracrine inhibition of glucagon secretion. Normally, KATP channel-dependent hyperpolarization of ß-cells at low glucose extends into the δ-cells through gap junctions, culminating in suppression of action potential firing and inhibition of somatostatin secretion. This 'electric brake' is lost following autoimmune destruction of the ß-cells, resulting in impaired counter-regulation. This scenario accounts for the clinical observation that residual ß-cell function correlates with reduced hypoglycaemia risk8.

α-cell electrophysiology and the regulation of glucagon secretion.

Glucagon is the principal glucose-elevating hormone that forms the first-line defence against hypoglycaemia. Along with insulin, glucagon also plays a key role in maintaining systemic glucose homeostasis. The cells that secrete glucagon, pancreatic α-cells, are electrically excitable cells and use electrical activity to couple its hormone secretion to changes in ambient glucose levels. Exactly how glucose regulates α-cells has been a topic of debate for decades but it is clear that electrical signals generated by the cells play an important role in glucagon secretory response. Decades of studies have already revealed the key players involved in the generation of these electrical signals and possible mechanisms controlling them to tune glucagon release. This has offered the opportunity to fully understand the enigmatic α-cell physiology. In this review, we describe the current knowledge on cellular electrophysiology and factors regulating excitability, glucose sensing, and glucagon secretion. We also discuss α-cell pathophysiology and the perspective of addressing glucagon secretory defects in diabetes for developing better diabetes treatment, which bears the hope of eliminating hypoglycaemia as a clinical problem in diabetes care.

Regulation of α-cell glucagon secretion: The role of second messengers.

Glucagon is a potent glucose-elevating hormone that is secreted by pancreatic α-cells. While well-controlled glucagon secretion plays an important role in maintaining systemic glucose homeostasis and preventing hypoglycaemia, it is increasingly apparent that defects in the regulation of glucagon secretion contribute to impaired counter-regulation and hyperglycaemia in diabetes. It has therefore been proposed that pharmacological interventions targeting glucagon secretion/signalling can have great potential in improving glycaemic control of patients with diabetes. However, despite decades of research, a consensus on the precise mechanisms of glucose regulation of glucagon secretion is yet to be reached. Second messengers are a group of small intracellular molecules that relay extracellular signals to the intracellular signalling cascade, modulating cellular functions. There is a growing body of evidence that second messengers, such as cAMP and Ca2+, play critical roles in α-cell glucose-sensing and glucagon secretion. In this review, we discuss the impact of second messengers on α-cell electrical activity, intracellular Ca2+ dynamics and cell exocytosis. We highlight the possibility that the interaction between different second messengers may play a key role in the glucose-regulation of glucagon secretion.

The ischaemic preconditioning paradox and its implications for islet isolation from heart-beating and non heart-beating donors.

The impact of ischaemia can severely damage procured donor organs for transplantation. The pancreas, and pancreatic islets in particular, is one of the most sensitive tissues towards hypoxia. The present study was aimed to assess the effect of hypoxic preconditioning (HP) performed ex-vivo in islets isolated from heart-beating donor (HBD) and non heart-beating donor (NHBD) rats. After HP purified islets were cultured for 24 h in hypoxia followed by islet characterisation. Post-culture islet yields were significantly lower in sham-treated NHBD than in HBD. This difference was reduced when NHBD islets were preconditioned. Similar results were observed regarding viability, apoptosis and in vitro function. Reactive oxygen species generation after hypoxic culture was significantly enhanced in sham-treated NHBD than in HBD islets. Again, this difference could be diminished through HP. qRT-PCR revealed that HP decreases pro-apoptotic genes but increases HIF-1 and VEGF. However, the extent of reduction and augmentation was always substantially higher in preconditioned NHBD than in HBD islets. Our findings indicate a lower benefit of HBD islets from HP than NHBD islets. The ischaemic preconditioning paradox suggests that HP should be primarily applied to islets from marginal donors. This observation needs evaluation in human islets.

Basement membrane proteins improve human islet survival in hypoxia: Implications for islet inflammation.

Enzymatic digestion of the pancreas during islet isolation is associated with disintegration of the islet basement membrane (IBM) that can cause reduction of functional and morphological islet integrity. Attempts to re-establish IBM by coating the surface of culture vessels with various IBM proteins (IBMP) have resulted in loss of islet phenotype and function. This study investigated the capability of Collagen-IV, Laminin-521 and Nidogen-1, utilised as single or combined media supplements, to protect human islets cultured in hypoxia. When individually supplemented to media, all IBMP significantly improved islet survival and in-vitro function, finally resulting in as much as a two-fold increase of islet overall survival. In contrast, combining IBMP enhanced the production of chemokines and reactive oxygen species diminishing all positive effects of individually added IBMP. This impact was concentration-dependent and concerned nearly all parameters of islet integrity. Predictive extrapolation of these findings to data from 116 processed human pancreases suggests that more than 90% of suboptimal pancreases could be rescued for clinical islet transplantation increasing the number of transplantable preparations from actual 25 to 40 when adding Nidogen-1 to pretransplant culture. This study suggests that media supplementation with essential IBMP protects human islets from hypoxia. Amongst those, certain IBMP may be incompatible when combined or applied at higher concentrations. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally-invasive treatment that can reverse type 1 diabetes in certain patients. It involves infusing of insulin-producing cell-clusters (islets) from donor pancreases. Unfortunately, islet extraction is associated with damage of the islet basement membrane (IBM) causing reduced islet function and cell death. Attempts to re-establish the IBM by coating the surface of culture vessels with IBM proteins (IBMP) have been unsuccessful. Instead, we dissolved the most relevant IBM components Collagen-IV, Laminin-521 and Nidogen-1 in media routinely used for clinical islet culture and transplantation. We found human islet survival and function was substantially improved by IBMP, particularly Nidogen-1, when exposed to a hypoxic environment as found in vivo. We also investigated IBMP combinations. Our present findings have important clinical implications.

High Concentrations of Etanercept Reduce Human Islet Function and Integrity.

BACKGROUND: Most islet transplant groups worldwide routinely use the TNFα inhibitor Etanercept in their peri-transplant protocols. Surprisingly, there have been no published dose-response studies on the effects of Etanercept on human islets. Our study aimed to address this by treating cultured human islets with increasing concentrations of Etanercept. MATERIALS AND METHODS: Isolated human islets were cultured for 3-4 days in normoxic (21% oxygen) or in hypoxic (2% oxygen) atmosphere using Etanercept dissolved in a range of 2.5-40 µg/mL prior to islet characterisation. RESULTS: In normoxic atmosphere, it was found that 5 µg/mL is the most efficient dose to preserve islet morphological and functional integrity during culture. Increasing the dose to 10 µg/mL or more resulted in detrimental effects with respect to viability and glucose-stimulated insulin release. When human islets were cultured for 3 to 4 days in clinically relevant hypoxia and treated with 5 µg/mL Etanercept, post-culture islet survival (P < 0.001) and in vitro function (P < 0.01) were significantly improved. This correlated with a substantially reduced cytokine production (P < 0.05), improved mitochondrial function (P < 0.01), and reduced production of reactive oxygen species (P < 0.001) in hypoxia-exposed islets. CONCLUSION: These findings suggest that the therapeutic window of Etanercept is very narrow and that this should be considered when optimising the dosage and route of Etanercept administration in islet-transplant recipients or when designing novel drug-delivering islet scaffolds.

Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets.

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6-8 or 3-4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.

High Seeding Density Induces Local Hypoxia and Triggers a Proinflammatory Response in Isolated Human Islets.

Hypoxia is the main threat to morphological and functional integrity of isolated pancreatic islets. Lack of oxygen seems to be of particular importance for functionality of encapsulated islets. The present study was initiated as an experimental model for the environment experienced by human islets in a confined space present during culture, shipment, and in an implanted macrodevice. Quadruplicate aliquots of isolated human islets (n = 12) were cultured for 24 h at 37°C under normoxic conditions using 24-well plates equipped with 8-µm pore size filter inserts and filled with islet aliquots adjusted to obtain a seeding density of 75, 150, 300, or 600 IEQ/cm(2). After culture viability, glucose-stimulated insulin release, DNA content as well as Bax and Bcl-2 gene expression were measured. Culture supernatants were collected to determine production of VEGF and MCP-1. Viability correlated inversely with IEQ seeding density (r = -0.71, p < 0.001), while the correlation of VEGF and MCP-1 secretion with seeding density was positive (r = 0.78, p < 0.001; r = 0.54, p < 0.001). Decreased viability corresponded with a significant increase in the Bax/Bcl-2 mRNA ratio at 300 and 600 IEQ/cm(2) and with a sigificantly reduced glucose-stimulated insulin secretion and insulin content compared to 75 or 150 IEQ/cm(2) (p < 0.01). The present study demonstrates that the seeding density is inversely correlated with islet viability and in vitro function. This is associated with a significant increase in VEGF and MCP-1 release suggesting a hypoxic and proinflammatory islet microenvironment.