Islet-enriched gene expression and glucose-induced insulin secretion in human and mouse islets.

AIMS/HYPOTHESIS: Our understanding of the transcription factors that control the development and function of rodent islet beta cells is advancing rapidly, yet less is known of the role they play in similar processes in human islets. METHODS: To characterise the abundance and regulation of key proteins involved in glucose-regulated insulin secretion in human islets, we examined the expression of MAFA, MAFB, GLUT2 (also known as SLC2A2), ßGK (also known as GCK) and PDX1 in isolated, highly purified human islets with an intact insulin secretory pattern. We also assessed these features in islets from two different mouse strains (C57BL/6J and FVB). RESULTS: Compared with mouse islets, human islets secreted more insulin at baseline glucose (5.6 mmol/l), but less upon stimulation with high glucose (16.7 mmol/l) or high glucose plus 3-isobutyl-1-methyl-xanthine. Human islets had relatively more MAFB than PDX1 mRNA, while mouse islets had relatively more Pdx1 than Mafb mRNA. However, v-maf musculoaponeurotic fibrosarcoma oncogene homologue (MAF) B protein was found in human islet alpha and beta cells. This is unusual as this regulator is only produced in islet alpha cells in adult mice. The expression of insulin, MAFA, ßGK and PDX1 was not glucose-regulated in human islets with an intact insulin secretory pattern. CONCLUSIONS/INTERPRETATION: Our results suggest that human islets have a distinctive distribution and function of key regulators of the glucose-stimulated insulin secretion pathway, emphasising the urgent need to understand the processes that regulate human islet beta cell function.

Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets.

AIMS/HYPOTHESIS: Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. METHODS: We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2](-/-)) and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As (-/-) mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. RESULTS: Fasting glucose concentrations were reduced in As (-/-) mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As (-/-) than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As (-/-) mice even during high sodium intake. There was no difference in insulin sensitivity between As (-/-) and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose- and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. CONCLUSIONS/INTERPRETATION: We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess.

New capsule with tailored properties for the encapsulation of living cells.

A new capsule for the encapsulation and transplantation of pancreatic islets has been developed. Five active ingredients are involved in the capsule formation process: high viscosity sodium alginate (SA-HV), cellulose sulfate (CS), poly(methylene-co-guanidine) hydrochloride (PMCG), calcium chloride, and sodium chloride. Complexation reaction exhibits several unique features: (1) solution of SA-HV with CS represents a physical mixture of two entangled polyanions that provide both pH-sensitive (carboxylic) and permanently charged (sulfate) groups; (2) presence of CaCl2 in the cation solution ensures formation of the gelled bead after the drop of polyanion solution is immersed in the cation solution; (3) character of the polycation (PMCG), i.e., low molecular weight and unusually high charge density, combines both high mobility and reactivity; (4) presence of PMCG in cation solution, together with CaCl2, gives rise to the competitive binding of these two cations based on their diffusion and affinity towards the anion groups; and (5) NaCl provides the anti-gelling sodium ions that significantly affect the reaction of CaCl2 with the polyanion matrix, thus altering the final properties of the capsule surface, shape, and permeability. The capsule size, mechanical strength, membrane thickness, and permeability can be precisely adjusted and quantified. Detailed information on the permeability aspects is given in another paper by Brissová et al. [J. Biomed. Mater. Sci., 39, 61 (1998)]. The new features concerning capsule processing and testing are presented. We believe that the capsule characteristics can be optimized in the next step to meet the biological criteria. The initial transplantation results suggest that this capsule is biocompatible and noncytotoxic and is a promising candidate for the immunoisolation of cells such as pancreatic islets.

Control and measurement of permeability for design of microcapsule cell delivery system.

Transplantation of immunoisolated islets of Langerhans has been proposed as a promising approach to treating insulin-dependent diabetes mellitus. Recently, a cell delivery system based on a multicomponent microcapsule has been designed for the immunoisolation of insulin-secreting pancreatic islets. The capsule, formed by polyelectrolyte complexation of sodium alginate and cellulose sulfate with poly(methylene-co-guanidine), markedly has improved mechanical strength compared with the widely used alginate/poly(L-lysine) capsules. It also provides a flexibility for readily adjusting membrane thickness and capsule size, and, more important, the membrane permeability can be altered over a wide range of molecular sizes. To rigorously test the capsule diffusion properties, we have improved capsule permeability measurement by using two complementary methods: (1) size exclusion chromatography with dextran standards; and (2) newly developed methodology for assessing permeability to a series of biologically relevant proteins. Viability and function of rat pancreatic islets enclosed in the capsules with different permeability were tested in vitro. The insulin secretion of encapsulated islets was well preserved even though slightly delayed in comparison with a control group of free islets. We believe that the unique features of this encapsulation system together with the precise characterization of its physical parameters will enable us to find the optimal range of capsule permeability for in vitro and in vivo survival and function of encapsulated pancreatic islets.

An encapsulation system for the immunoisolation of pancreatic islets.

Over a thousand combinations of polyanions and polycations were tested to search for new polymer candidates that would be suitable for encapsulation of living cells. The combination of sodium alginate, cellulose sulfate, poly (methylene-co-guanidine) hydrochloride, calcium chloride, and sodium chloride was most promising. In parallel, a novel multiloop chamber reactor was developed to control the time of complex formation and to negate gravitational effects such as pancreatic islet sedimentation and droplet deformation during the encapsulation process. Encapsulated rat islets demonstrated glucose-stimulated insulin secretion in vitro, and reversed diabetes in mice. This new capsule formulation and encapsulation system allows independent adjustments of capsule size, wall thickness, mechanical strength, and permeability, which may offer distinct advantages for immunoisolating cells.

Permeability assessment of capsules for islet transplantation.

Despite considerable progress in the development of immunoisolation devices, the optimal permeability of such devices is not known. This limitation stems partly from deficits in knowledge about which molecules should be allowed to traverse the semipermeable membrane and which molecules should be excluded, and also partly from experimental obstacles that have prevented a systematic study of permeability. To determine the optimal permeability of immunoisolation devices, we have created a series of microcapsules (800 microM diameter) that span a broad range of molecular exclusion limits yet are identical in wall thickness and chemical composition. Capsule permeability was precisely defined by two complementary methods--size exclusion chromatography (SEC) and a newly developed methodology to assess permeability of biologically relevant proteins. The entry of interleukin-1 beta-125I was significantly delayed, but not prevented, when the capsule exclusion limit was decreased from 230 kD to 3.2 kD, as determined by SEC with dextran standards. The influx of IgG was as predicted, based on the viscosity radius R eta of IgG and the capsule exclusion limit defined by SEC. Glucose-stimulated insulin secretion by encapsulated pancreatic islets did not differ as capsule permeability was decreased from a molecular exclusion limit of 230 kD to 120 kD. These studies should assist in the design of immunoisolation devices by defining the permeability optimal for cell function and also should be applicable to any cell type or immunoisolation device.

Evaluation of microcapsule permeability via inverse size exclusion chromatography.

Inverse aqueous size exclusion chromatography (SEC) was adopted to measure the permeability of microcapsules (hollow hydrogel spheres with diameter < 1 mm) using dextran molecular weight standards. Alginate/poly(L-lysine)/alginate microcapsules were chosen as a column substrate. Data from column SEC experiments were verified by kinetic studies of solute size exclusion. The permeability of tested microcapsules was modified by the reaction time with 0.05wt.% poly(L-lysine) (PLL). The exclusion limit of the microcapsules prepared at 5-min reaction time was found to be 100,000, while the microcapsules that were allowed to react with PLL for 20 min became less permeable and their exclusion limit was approximately 50,000. Based on relationships between solute size and molecular weight, the exclusion limits determined with dextrans were converted to the size and approximate molecular weight of protein presumably excluded by the capsular membrane at "ideal" conditions. The results from both column SEC and batch experiments suggest that the standard alginate/PLL/alginate capsules are permeable to immunoglobulins of IgG class. Unlike other techniques which utilize only a limited number of solutes, inverse SEC enables one to examine the capsule permeability to a homologous series of molecular weight standards. Inverse SEC also provides an opportunity to evaluate the properties of a large series of capsules directly by comparing their calibration curves. In addition, undesirable enthalpic effects in permeability studies with globular proteins as test solutes can be minimized or eliminated by using the inert molecular weight standards such as polysaccharides.

Study on the thermal stability of alpha-amylase modified by maleic anhydride copolymer.

The thermal inactivation of mesophilic Bacillus subtilis alpha-amylase modified by maleic anhydride/vinyl acetate copolymer has been studied at different polymer/enzyme ratios in the pH range of relevance to enzymatic catalysis. Enzymatic activity measurements combined with circular dichroism measurements were used to determine the enzyme thermostability. The apparent first-order rate constants and activation energies of thermo-inactivation affected by addition of Ca2+ ions as well as by modification have been calculated. The modified alpha-amylase exhibited sufficiently high catalytic activity with enhanced resistance to the thermal unfolding process.