A new paradigm for improved co-ordination and efficacy of European biomedical research: taking diabetes as a model.

Today, European biomedical and health-related research is insufficiently well funded and is fragmented, with no common vision, less-than-optimal sharing of resources, and inadequate support and training in clinical research. Improvements to the competitiveness of European biomedical research will depend on the creation of new infrastructures that must be dynamic and free of bureaucracy, involve all stakeholders and facilitate faster delivery of new discoveries from bench to bedside. Taking diabetes research as the model, a new paradigm for European biomedical research is presented, which offers improved co-ordination and common resources that will benefit both academic and industrial clinical research. This includes the creation of a European Council for Health Research, first proposed by the Alliance for Biomedical Research in Europe, which will bring together and consult with all health stakeholders to develop strategic and multidisciplinary research programmes addressing the full innovation cycle. A European Platform for Clinical Research in Diabetes is proposed by the Alliance for European Diabetes Research (EURADIA) in response to the special challenges and opportunities presented by research across the European region, with the need for common standards and shared expertise and data.

Non-muscle myosin IIA is involved in focal adhesion and actin remodelling controlling glucose-stimulated insulin secretion.

AIMS/HYPOTHESIS: Actin and focal adhesion (FA) remodelling are essential for glucose-stimulated insulin secretion (GSIS). Non-muscle myosin II (NM II) isoforms have been implicated in such remodelling in other cell types, and myosin light chain kinase (MLCK) and Rho-associated coiled-coil-containing kinase (ROCK) are upstream regulators of NM II, which is known to be involved in GSIS. The aim of this work was to elucidate the implication and regulation of NM IIA and IIB in beta cell actin and FA remodelling, granule trafficking and GSIS. METHODS: Inhibitors of MLCK, ROCK and NM II were used to study NM II activity, and knockdown of NM IIA and IIB to determine isoform specificity, using sorted primary rat beta cells. Insulin was measured by radioimmunoassay. Protein phosphorylation and subcellular distribution were determined by western blot and confocal immunofluorescence. Dynamic changes were monitored by live cell imaging and total internal reflection fluorescence microscopy using MIN6B1 cells. RESULTS: NM II and MLCK inhibition decreased GSIS, associated with shortening of peripheral actin stress fibres, and reduced numbers of FAs and insulin granules in close proximity to the basal membrane. By contrast, ROCK inhibition increased GSIS and caused disassembly of glucose-induced central actin stress fibres, resulting in large FAs without any effect on FA number. Only glucose-induced NM IIA reorganisation was blunted by MLCK inhibition. NM IIA knockdown decreased GSIS, levels of FA proteins and glucose-induced extracellular signal-regulated kinase 1/2 phosphorylation. CONCLUSIONS/INTERPRETATION: Our data indicate that MLCK-NM IIA may modulate translocation of secretory granules, resulting in enhanced insulin secretion through actin and FA remodelling, and regulation of FA protein levels.

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model.

AIMS/HYPOTHESIS: Cardiotrophin 1 (CT-1) is a recently described cytokine originally isolated from the heart where it has been shown to play an important role in apoptotic protection of cardiomyocytes and heart hypertrophy. Its beneficial properties have also been described in other organs such as liver and neuromuscular tissue. In the present study, we investigated whether CT-1 can confer protection against pro-apoptotic stimuli in pancreatic beta cells, and its role in insulin secretion and diabetes development. METHODS: The effects of CT-1 on apoptosis and function were studied using MIN6B1 cells and freshly isolated murine pancreatic islets. The impact on the development of diabetes was evaluated in Ct1-null (Ct1 (-/-)) mice (the gene Ct1 is also known as Ctf1) using two streptozotocin (STZ)-induced models of diabetes. RESULTS: CT-1 has a protective effect in MIN6B1 cells and murine islets under the pro-apoptotic stimulus of serum deprivation, which correlates with the expression of B cell lymphoma-extra large, or following exposure to a mixture of cytokines. In addition, CT-1 enhances glucose-stimulated insulin secretion in MIN6B1 cells and this was repressed by inhibitors of phospholipase C. Furthermore, Ct1 (-/-) mice were more prone to develop diabetes, and their glucose tolerance test showed impaired plasma glucose clearance which correlated with decreased pancreatic insulin secretion. CONCLUSIONS/INTERPRETATION: The results obtained from both in vitro and in vivo experiments show that CT-1 improves beta cell function and survival, and protects mice against STZ-induced diabetes.

Bimodal impact of skeletal muscle on pancreatic ß-cell function in health and disease.

Diabetes is a complex disease that affects many organs directly or indirectly. Type 2 diabetes mellitus is characterized by insulin resistance with a relative deficiency in insulin secretion. It has become apparent that inter-organ communication is of great importance in the pathophysiology of diabetes. Far from being an inert tissue in terms of inter-organ communication, it is now recognized that skeletal muscle can secrete so-called myokines that can impact on the function of distant organs/tissues both favourably and unfavourably. We have proposed that communication between insulin-resistant skeletal muscle and ß-cells occurs in diabetes. This is a novel route of communication that we further suggest is modified by the prevailing degree of insulin resistance of skeletal muscle. This review focuses on the various myokines [interleukin-6 (IL-6), tumor necrosis factor-α, CXCL10, follistatin and IL-8] which have been identified either after different types of exercise or in the secretome from control and insulin-resistant human skeletal myotubes. We will also summarize studies on the impact of several myokines on pancreatic ß-cell proliferation, survival and function.

Hypercholesterolaemia, signs of islet microangiopathy and altered angiogenesis precede onset of type 2 diabetes in the Goto-Kakizaki (GK) rat.

AIMS/HYPOTHESIS: The adult non-obese Goto-Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals. METHODS: Blood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates). RESULTS: Systemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms. CONCLUSIONS/INTERPRETATION: These data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass.

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway.

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.

SNAP-25 is expressed in islets of Langerhans and is involved in insulin release.

SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.

Cell therapy for type 2 diabetes: is it desirable and can we get it?

The functional mass of beta-cells is decreased in type 2 diabetes. Replacing missing beta-cells or triggering their regeneration may thus allow for improved treatment of type 2 diabetes, to the extent that this is combined with therapy for improved insulin sensitivity. Although progress has been made in deriving beta-cell-like cells from stem or precursor cells in vitro, these cannot yet be obtained in sufficient quantities or well enough differentiated to envisage their therapeutic use in beta-cell replacement therapy. Likewise, our very limited understanding of beta-cell regeneration in adult man does not yet allow for development of a valid strategy for kick-starting such a process in individuals with type 2 diabetes, whether by bona fide neogenesis or self-replication of existing beta-cells. Regardless of how beta-cell mass is restored in type 2 diabetes, it will be important to prevent any renewed decrease thereafter. Current understanding suggests that islet inflammation as well as signals from (insulin-resistant/inflamed) adipose tissue and skeletal muscle contribute towards decreased beta-cell mass in type 2 diabetes. It will likely be important to protect newly formed or implanted beta-cells from these negative influences to ensure their long-term survival.

Effects of aging on insulin synthesis and secretion. Differential effects on preproinsulin messenger RNA levels, proinsulin biosynthesis, and secretion of newly made and preformed insulin in the rat.

Aging in men and rodents is associated with a marked decline in glucose stimulated insulin secretion by pancreatic beta cells (B cells). Secreted insulin is the end result of a series of steps along the biosynthetic protein-secretion pathway, including insulin gene transcription, processing of transcripts to preproinsulin mRNA, translation of mRNA, segregation and processing of newly made proinsulin in secretory vesicles, proinsulin to insulin conversion, transport of vesicles to the plasma membrane, and exocytosis. We have examined the influence of age at three stages along this pathway: preproinsulin mRNA levels, proinsulin synthesis, and secretion of newly made and preformed insulin, using Fischer rats, a widely studied rodent model of aging. Pancreatic weights and total insulin contents, islet sizes, and mean insulin content per islet were the same in young adult (4-5 mo) and senescent (21-22 mo) animals. There was no effect of age on preproinsulin mRNA levels in whole pancreata of fed animals, or in isolated islets cultured for 16 h in 5.5 mM glucose. Proinsulin biosynthesis and the secretion of newly made insulin were compared in isolated islets preincubated in 5.5 mM glucose. After a pulse label at 16.7 mM glucose, proinsulin synthesis, assayed by immunoprecipitation, was decreased 16% in 7 mo islets and 39% in 21-22 mo islets, compared with 4-5 mo islets, though total protein synthesis was not reduced. When chased at 2.8 mM glucose, 4-5 month and 21-22 mo islets showed no difference in release of preformed or newly made insulin. When chased at 16.7 mM glucose, there was a significant decrease in the secretion of newly made insulin in the old islets compared with the young islets. There was preferential release of newly made insulin over preformed insulin in both young and old islets. However, since secretion of preformed insulin was decreased much more than secretion of newly made insulin in senescent islets, these displayed a two- to threefold increase in the proportion of newly made insulin relative to total immunoreactive insulin released compared with young adult islets. The differential effects of aging on these steps in the insulin synthesis-secretion pathway may be due to varying impairments in signals transducing the glucose stimulus into the wide range of B cell responses to glucose.

Tumor necrosis factor-alpha modifies adhesion properties of rat islet B cells.

The characteristic three-dimensional cell type organization of islets of Langerhans is perturbed in animal models of diabetes, suggesting that it may be important for islet function. Rat islet cells in culture are able to form aggregates with an architecture similar to native islets (pseudoislets), thus providing a good model to study the molecular basis of islet architecture and its role in islet function. Sorted islet B cells and non-B cells were permanently labeled with two different fluorescent dyes (DiO and DiI), mixed, and allowed to form aggregates during a 5-d culture in the presence or absence of TNF-alpha (100 U/ml), a cytokine suggested to be implicated in the early physiological events leading to insulin-dependent diabetes mellitus. Confocal microscopy of aggregates revealed that TNF-alpha reversibly perturbs the typical segregation between B and non-B cells. Insulin secretion, was altered in the disorganized aggregates, and returned towards normal when pseudoislets had regained their typical architecture. The homotypic adhesion properties of sorted B and non-B cells cultured for 20 h in the presence or absence of TNF-alpha were studied in a short term aggregation assay. TNF-alpha induced a significant rise in Ca(2+)-independent adhesion of B cells (from 24 +/- 1.1% to 44.3 +/- 1.2%; n = 4, P < 0.001). These findings raise the possibility that the increased expression of Ca(2+)-independent adhesion molecules on B cells leads to altered islet architecture, which might be a factor in the perturbation of islet function induced by TNF-alpha.

Functional differences between rat islets of ventral and dorsal pancreatic origin.

Do functional linkages between islet endocrine cells exist? The effect of differences in frequency and distribution of islet endocrine cells on B cell function was examined in islets from the ventral (ventral islets) and dorsal (dorsal islets) areas of the rat pancreas. Dorsal islets contained 10 times as much glucagon as ventral islets, whereas insulin and total protein contents were similar. Basal rates of insulin secretion and proinsulin biosynthesis were similar in the two types of islet, but, under conditions of glucose stimulation, both insulin secretion and proinsulin biosynthesis were significantly greater in the glucagon-rich dorsal islets. Similarly, glucose utilization rates an ATP levels were greater in dorsal islets. In contrast, the rates of processing of newly synthesized proinsulin were similar in ventral and dorsal islets. That the islet glucagon content may have affected B cell function is inferred from two independent findings. Firstly, basal and glucose-stimulated cyclic AMP contents of glucagon-rich dorsal islets were greater than those of ventral islets. Secondly, in the presence of excess exogenous glucagon (1 microgram/ml), the differences in glucose-induced insulin secretion and proinsulin biosynthesis rates between the two types of islets were eliminated. These results strongly suggest that changes in the relative proportions of the different islet endocrine cells exert marked effects on islet function. In particular, a greater A cell and glucagon content is associated with higher rates of glucose-induced insulin secretion and biosynthesis.

Increased clearance and degradation of [3H]insulin in streptozotocin diabetic rats.

The role of the insulin-receptor compartment in the pharmacokinetics of intravenously injected insulin in rats was studied. Since streptozotocin-diabetes in rats results in increased insulin binding to tissues in vitro, insulin pharmacokinetics in streptozotocin-diabetic rats were compared to controls, using semisynthetic [(3)H]insulin as the tracer. The initial distribution volume for [(3)H]insulin was elevated by 60% in diabetic rats. By contrast, no difference in initial distribution volume for [(14)C]inulin was observed, and the absolute values were lower than those found for [(3)H]insulin. The metabolic clearance rate of [(3)H]insulin was elevated by 44% in diabetic rats. That these differences were the result of increased binding of insulin to a specific receptor compartment in diabetic rats was shown by three additional experiments. The first involved receptor saturation by injection of 10 U native insulin 2 min before the tracer injection, resulting in identical [(3)H]insulin disappearance rates in the two groups of rats. The second consisted of displacing [(3)H]insulin from receptors by injecting 10 U unlabeled insulin 6 min after the tracer injection. Displacement of intact [(3)H]insulin from receptors and subsequent reappearance in the circulation occurred in both control and diabetic animals; however, such displacement was 25% greater in the diabetic rats. Finally, treatment of diabetic rats with insulin for 8 d normalized [(3)H]insulin clearance even though the tracer was injected at a time when the animals were again hyperglycemic and hypoinsulinemic. This suggests that down-regulation of insulin receptors had occurred during insulin therapy. These results confirm that a specific compartment for insulin exists (the insulin-receptor compartment) and that this compartment plays an important role in insulin clearance.

Release of incompletely processed proinsulin is the cause of the disproportionate proinsulinemia of NIDDM.

The production of insulin from proinsulin involves cleavage of intact proinsulin into proinsulin conversion intermediates by the processing of enzymes PC2 and PC3 before fully processed insulin is produced. Intact proinsulin and these conversion intermediates are measured in many immunoreactive insulin (IRI) assays, and therefore contribute to the absolute IRI measurement. The proportion of basal IRI made up of proinsulin (PI)-like molecules (PI/IRI) is increased in NIDDM. Whether stimulated IRI levels are similarly made up of disproportionately increased PI/IRI or whether the relative proportions of proinsulin and its conversion intermediates are altered has not been evaluated. An index of the efficiency of proinsulin processing within the pancreatic beta-cell can be achieved by measuring PI/IRI immediately following acute stimulation of beta-cell secretion, and then determining the proportion of intact proinsulin and proinsulin conversion intermediates contributing to circulating proinsulin-like molecules. In this study, we determined the PI/IRI levels under basal and arginine-stimulated conditions in 17 healthy and 16 NIDDM subjects; high-performance liquid chromatography (HPLC) was also performed in a subset of these subjects to measure the relative contribution of intact proinsulin and its conversion intermediates to total proinsulin-like molecules. In NIDDM subjects, levels of both basal (44.6 +/- 9.6 vs. 9.3 +/- 1.5 pmol/l; P = 0.0007) and stimulated (64.0 +/- 12.7 vs. 19.8 +/- 2.8 pmol/l; P = 0.001) proinsulin-like molecules were higher than in healthy subjects. Although IRI was higher in NIDDM than in control subjects under basal conditions (106 +/- 19 vs. 65.1 +/- 8.1 pmol/l; P = 0.05), it was lower in NIDDM than in control subjects following stimulation (increment: 257 +/- 46 vs. 416 +/- 51 pmol/l; P = 0.03). PI/IRI ratios were increased in NIDDM subjects under both basal (43.3 +/- 5.0 vs. 14.0 +/- 1.3%; P < 0.0001) and stimulated (increment: 10.1 +/- 2.1 vs. 2.5 +/- 0.2%; P = 0.0006) conditions, compatible with the release of a disproportionately increased amount of proinsulin-like products. HPLC analysis revealed that, in the stimulated state, intact proinsulin made up 40.1 +/- 6.7% of proinsulin-like molecules in NIDDM individuals (n = 9) and 30.1 +/- 5.6% in healthy subjects (n = 7; NS). The remainder of the proinsulin-like molecules comprised the des-31,32-split proinsulin conversion intermediate. The increase in PI/IRI in NIDDM under basal and especially under stimulated conditions suggests that proinsulin conversion is indeed perturbed in this disorder. Because the relative proportions of intact and des-31,32-split proinsulin are similar in both healthy and NIDDM subjects, the orderly cleavage of proinsulin at its two junctions appears preserved. However, at the time of exocytosis, the secretory granule in the islet of NIDDM subjects contains an increased proportion of incompletely processed proinsulin, presumably reflecting a slower rate of conversion or granules' reduced time of residence in beta-cells.

Influence of glucose on insulin handling by rat islets in culture. A reflection of integrated changes in insulin biosynthesis, release, and intracellular degradation.

Rat islets were maintained for 3 days in tissue culture at 2.8, 8.3, or 16.7 mM glucose, in the presence of [3H]leucine to label insulin stores. The immunoreactive insulin content of islets after 3 days at either 2.8 or 16.7 mM glucose was lower than that of islets exposed to 8.3 mM glucose. The specific radioactivity of stored labeled insulin, however, increased with increasing glucose. Islet insulin turnover was thus highest at 16.7 mM glucose. During a subsequent 1-day chase period at 2.8, 8.3, or 16.7 mM glucose for each of the three groups of islets prelabeled at these same three concentrations, all islets responded to glucose with increased insulin release. Release of immunoreactive insulin was highest at all three chase glucose concentrations for those islets preexposed for 3 days to 16.7 mM glucose. After 3-day labeling of islets at 8.3 mM glucose, there was an inverse relationship between the rate of release of labeled insulin and its intracellular degradation in response to the three glucose concentrations during the chase period. Those islets prelabeled at either 2.8 or 16.7 mM glucose showed much lower rates of intracellular degradation of labeled insulin stores during the chase period regardless of the ambient glucose or of the rate of release of the labeled hormone. Adaptive changes in islet insulin handling thus occurred during the 3-day maintenance period at different glucose concentrations. Whereas glucose concentration is, of course, thought to regulate insulin biosynthesis and release directly, regulation of degradation of stored insulin within B-cells appears to reflect both the relative activity of biosynthesis and release of the hormone and the insulin content of the islets. This last and striking relation is best interpreted as reflecting two labile, exchangeable, granule pools, one preferentially available for release and the other for intracellular degradation.

Spontaneous reassociation of dispersed adult rat pancreatic islet cells into aggregates with three-dimensional architecture typical of native islets.

Islets of Langerhans consist of four major endocrine cell types assembled in a highly organized manner critical for their function. The molecular forces governing islet cellular architecture are not understood. We determined whether adult rat islet cells carry information necessary for orderly assembly. Dispersed cells from adult rat islets were maintained in static suspension culture for 6 days. During this time the cells reassociated to form numerous aggregates. These aggregates were approximately half the size of native islets with a commensurate reduction in DNA and insulin content. However, both cellular composition and organization were remarkably similar to that of adult rat islets, in which the beta-cells form a central core surrounded by a discontinuous mantle of non-beta-cells. Thus, immunoperoxidase staining showed that in the aggregates, just as in intact islets cultured in parallel, 26% of the cells were non-beta-cells and of these, 94% were clearly peripheral. Non-beta-cells were similarly found to be peripheral, with beta-cells located centrally, even when the ratio of non-beta-cells to beta-cells had been altered. This was achieved by sorting the two cell populations by fluorescence-activated flow cytometry, resulting in aggregates with 79% non-beta-cells and 21% beta-cells. Insulin release from the aggregates was stimulated approximately ninefold by raising glucose from 50 to 300 mg/dl, which was comparable to that found for intact islets. The spontaneous formation of isletlike aggregates displaying appropriate cellular architecture indicates that the signals (molecules) needed for such organization are intrinsic to islet cells and are still expressed by them in adult life.

Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3.

Proinsulin conversion to insulin occurs in secretory granules of pancreatic beta-cells. This processing has been suggested to require both the endoproteases PC2 and PC3 with each cleaving at only one of the two sites linking the insulin A- and B-chains with C-peptide. To evaluate this in an appropriate cellular setting, conversion of human proinsulin was followed in GH3 (rat pituitary) cells normally unable to convert this prohormone but equipped with the regulated secretory pathway. For this purpose, human proinsulin was expressed in GH3 cells, alone or in combination with PC2 and/or PC3, using recombinant adenoviruses. Cells were infected with the given adenoviruses and 24 h later were pulse-chased. Kinetics of proinsulin conversion were monitored by reverse-phase high-performance liquid chromatography. It was observed that while the two endoproteases do display a preference for a single site of cleavage (PC2 at the A-chain/C-peptide junction; PC3 at the B-chain/C-peptide junction) and act in a synergistic manner to promote proinsulin conversion, either PC2 or PC3 alone can cleave at both sites to fully convert proinsulin to insulin. These results also show that a cell can be successfully infected by three different recombinant adenoviruses.

Vascularization of purified pancreatic islet-like cell aggregates (pseudoislets) after syngeneic transplantation.

To clarify whether avascular purified endocrine cell aggregates derived from islets of Langerhans (pseudoislets) revascularize similarly to what is known for intact pancreatic islet grafts, we studied the process of angiogenesis and revascularization of syngeneically transplanted pseudoislets using intravital fluorescence microscopy. Pseudoislets were composed of pure beta-cells (B) or non-beta-cells (NB), as well as of mixed beta- and non-beta-cells (B/NB; 70/30%) or nonsorted-cells (NC), and were transplanted into the dorsal skinfold of Syrian golden hamsters. Intact islet grafts served as controls. At day 6 after transplantation, microvascularization of all types of pseudoislets was found to be less than in controls, as indicated by a reduced number of transplants that contained newly formed microvessels (take-rate: B, 38.8; NB, 38.7; B/NB, 43.8; and NC, 40.3% vs. intact islet grafts, 71.9%; P < 0.01). Moreover, those pseudoislets that had developed a microvascular network revealed a significantly lower functional capillary density (145.8+/-49.5 to 241.0+/-47.5 cm(-1) vs. intact islet grafts: 459.8+/-65.6 cm(-1); P < 0.05). After 20 days, the take-rate of pseudoislets was still lower (B, 67.4; NB, 45.3; B/NB, 48.4; and NC, 64.2%) when compared with intact islet grafts (88%; P < 0.05); however, islet-like aggregates with vascularization now showed an islet-specific glomerulus-like network of capillaries with a functional capillary density (498.5+/-49.1 to 601.4+/-124.0 cm[-1]) similar to that of intact islet grafts (644.3+/-26.8 cm[-1]). We conclude that the dissociation of pancreatic islets, followed by reaggregation of the purified endocrine cells to islet-like clusters (pseudoislets), delays the process of angiogenesis and revascularization after free transplantation; however, this does not influence the capacity to form an intact islet-specific microvasculature (angio-architecture), which appears to be independent from the cellular composition of pseudoislets.

Relative hypersecretion of proinsulin in rat model of NIDDM.

The plasma ratio of proinsulin to insulin is raised in individuals with non-insulin-dependent diabetes mellitus (NIDDM). Increased secretion of proinsulin relative to insulin is thought to be the cause, although differential changes in clearance have not been ruled out. This study was conducted in a rat model of NIDDM, 90% pancreatectomized (Px) rats, to investigate the pathophysiology of this observation. Proinsulin storage and secretion were assessed with high-performance liquid chromatography separation of the insulins and the proinsulins, followed by quantification of the peaks by insulin radioimmunoassay. In Px rats, the relative proportion of proinsulin in pancreas extracts was twice that of control (sham-operated) rats (15.6 +/- 1.4 vs. 8.3 +/- 1.4%, P less than 0.01). Samples obtained from the portal vein during in vitro pancreas perfusion also had an elevated proinsulin fraction (Px, 10.3 +/- 3.0; sham, 3.0 +/- 0.6%; P less than 0.006). In summary, 90% Px rats share many pathophysiological features with NIDDM, including loss of normal proinsulin homeostasis. Our results suggest that chronic hyperglycemia causes an intrinsic change in beta-cells that is characterized by the increased storage and secretion of proinsulin.

Tris(hydroxymethyl)aminomethane inhibits the synthesis and processing of proinsulin in isolated rat pancreatic islets without affecting release of insulin stores.

Isolated rat islets of Langerhans were pulse-labeled (5 min, [3H]leucine) and then exposed to 10 or 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 during an 85-min chase period. There was a dose-related inhibition of the conversion of labeled proinsulin to insulin by Tris. At 50 mM, Tris also inhibited the release of newly synthesized (labeled) proinsulin and insulin. These inhibitory effects of Tris were almost absent if the islets were exposed to 50 mM Tris during only the last 60 min of the 85-min chase period. Both proinsulin and total islet protein synthesis (as indexed by incorporation of [3H]leucine) were inhibited acutely by 50 mM Tris (5-min exposure); after 85 min of exposure to 50 mM Tris, the inhibition of proinsulin biosynthesis was more marked than that of total islet protein. In contrast to its effects on newly synthesized products, 50 mM Tris failed to inhibit the release of immunoreactive insulin during an 85-min incubation. However, when islets were exposed to 50 mM Tris for a longer period, a partial inhibition of immunoreactive insulin release was observed as from 120 min. Insulin released from islets consists of a mixture of older stored material and of newly synthesized products, the latter being released preferentially. These results are consistent with a selective effect of 50 mM Tris on the production of newly synthesized insulin. During the first 120 min of exposure to Tris, islet reserves of newly synthesized products will be depleted thereby leading to a new, reduced, rate of release of immunoreactive material consisting only of older insulin stores.(ABSTRACT TRUNCATED AT 250 WORDS)