Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]triphosphate

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O

Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion

Secretagogues Modulate the Calcium Concentration in the Endoplasmic Reticulum of Insulin-secreting Cells STUDIES IN AEQUORIN-EXPRESSING INTACT AND PERMEABILIZED INS-1 CELLS

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+]er was 267 +/- 9 microM. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+]er by 20-25%. Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+]er in the potency order K+ >> glucose > leucine, paralleling their actions in the cytosolic compartment. Glucose, which augmented [Ca2+]er by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+]er, but only the former generated whereas the latter consumed ATP. Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+]er, emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+]er, invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+]er to ensure beta-cell homeostasis

Long-term results in pancreatic transplantation with special emphasis on the use of prolamine

Our pancreatic transplantation programme was initiated in 1979. Since then a total of 102 pancreas transplantations have been performed, blocking exocrine secretion using the duct occlusion technique with prolamine. Early non-immunological complications are frequent. The long-term results (9 years) in combined pancreas and kidney transplanted patients are satisfying: the survival rate for pancreas is 38% and 54% for kidney. Patient survival rate in this period is 85%. Beyond the first year post-transplant the exocrine activity disappears whereas the endocrine function remains well preserved

Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

Abstract Increases in the concentration of free ATP within the islet β-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+(KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary β-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar (∼1 mm). Elevations in extracellular glucose concentration (3–30 mm) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration

Stimulus-Section Coupling in Endocrine Cell Models

Detailed understanding of biological systems governing specific mechanisms and pathways is essential in the development of novel disease therapies. Stimulus-secretion coupling in hormone secreting cells is a complex system of pathways that link activation of cellular processes by i.e. nutrients to the release of hormone. Stimulus-secretion coupling in the insulin secreting beta-cell is intensely researched to improve our understanding of type 2 diabetes (T2D), a perpetually growing global pandemic. Such research requires availability of model systems that are metabolically and functionally faithful to the cell type they represent. In my research I have characterized, evaluated and applied cell models for metabolic research in endocrine cells. In studies I and II cellular function in response to nutrient stimuli in human and murine beta cell models and isolated islets was evaluated. In these studies I investigated functional aspects such as insulin secretion as well as metabolic changes such as changes in intracellular metabolite levels, oxygen consumption rates and energy production. I found that the response was qualitatively similar in human and rat beta cell models. The same was found when comparing a clonal rat beta cell model to isolated rat islets. Hence, I concluded that the similarities outweigh the differences and as such the in vitro models investigated lend themselves useful in metabolic studies, but with the recommend use of primary material for confirmation of key findings. Stimulus-secretion coupling has been widely studied in the beta cell, providing extensive knowledge on the mechanisms governing insulin secretion elicited by various nutrients. In study III and IV, I used established alpha, beta and L-cell models to investigate similarities and differences in stimulus-secretion coupling in different endocrine cell types. In study III, an important difference in mitochondrial shuttles was found between alpha and beta cells, highlighting the malate-aspartate shuttle to be critical for glucagon secretion. These findings were subsequently verified in mouse islets. In study IV, a striking difference in the activity of glutamate dehydrogenase was found between L and beta cells. Accumulation of glycogen in the beta cell has been associated with T2D. In study V, I investigated the potential role of glycogen metabolism in beta cells and its potential role in regulation of insulin secretion. This study revealed glycogen metabolism to be active and accumulation of glycogen to occurs in response to elevated glucose levels both in a clonal cell line and human islets. Moreover, perturbation of glycogen metabolism was shown to decrease insulin secretion in vitro. Stimulus-secretion coupling is highly complex as is the pathogenesis of T2D. These studies highlight how well-characterized metabolic models may be used to further the understanding of stimulus secretion coupling in endocrine cells

Cyclic AMP signalling in pancreatic islets

Cyclic 3'5'AMP (cAMP) is an important physiological amplifier of glucose-induced insulin secretion by the pancreatic islet β-cell, where it is formed by the activity of adenylyl cyclases, which are stimulated by glucose, through elevation in intracellular calcium concentrations, and by the incretin hormones (GLP-1 and GIP). cAMP is rapidly degraded in the pancreatic islet β-cell by various cyclic nucleotide phosphodiesterase (PDE) enzymes. Many steps involved in glucose-induced insulin secretion are modulated by cAMP, which is also important in regulating pancreatic islet β-cell differentiation, growth and survival. This chapter discusses the formation, destruction and actions of cAMP in the islets with particular emphasis on the β-cell

MAFA controls genes implicated in insulin biosynthesis and secretion

Aims/hypothesis: Effects of the transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA) on the regulation of beta cell gene expression and function were investigated. Materials and methods: INS-1 stable cell lines permitting inducible up- or downregulation of this transcription factor were established. Results: MAFA overproduction enhanced and its dominant-negative mutant (DN-MAFA) diminished binding of the factor to the insulin promoter, correlating with insulin mRNA levels and cellular protein content. Glucose-stimulated insulin secretion was facilitated by MAFA and blunted by DN-MAFA. This is partly due to alterations in glucokinase production, the glucose sensor of beta cells. In addition, the expression of important beta cell genes, e.g. those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (formerly known as GLUT2), pancreatic and duodenal homeobox factor 1 (PDX1), NK6 transcription factor-related, locus 1 (NKX6-1), glucagon-like peptide 1 receptor (GLP1R), prohormone convertase 1/3 (PCSK1) and pyruvate carboxylase (PC), was regulated positively by MAFA and negatively by DN-MAFA. Conclusions/interpretation: The data suggest that MAFA is not only a key activator of insulin transcription, but also a master regulator of genes implicated in maintaining beta cell function, in particular metabolism-secretion coupling, proinsulin processing and GLP1R signalling. Our in vitro study provides molecular targets that explain the phenotype of recently reported Mafa-null mice. We also demonstrate that MAFA is produced specifically in beta cells of human islets. Glucose influenced DNA-binding activity of MAFA in rat islets in a bell-shaped manner. MAFA thus qualifies as a master regulator of beta-cell-specific gene expression and functio