Incorporation of Sulfonylurea into N-Isopropylacrylamide as an Extracellular Matrix for an Artificial Pancreas

High-molecular-weight N-isopropylacrylamide copolymers with small amounts of sulfonylurea (SU, typically 2-4 mol% in the feed) were synthesized by free radical polymerization in benzene. SU-incorporated polymer solutions (5, 6, 8, and 10% w/v) in a culture medium (pH 7.4, 0.15 M ionic strength) with islet cells were mixed and poured into Millicells which supported gel formation. In order to increase the gelation temperature, the SU-incorporated copolymer gel, p(NiPAAm-co-SU), was blended with the p(NiPAAm-co-AAc) polymer at a ratio of 4 to 96. Interaction between the islet cells and the synthetic matrix of SU-incorporated copolymer gel resulted in effective cell viability and such cell functions as insulin secretion. To verify the specific interaction between the SU (K⁺ channel closer)-incorporated copolymer and islet cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K⁺ channel (K⁺ channel opener), before interaction between the polymer and islet cells. This treatment suppressed the action of SU on the islet cells. The results from this study provide evidence that the SU-incorporated copolymer stimulated insulin secretion by specific interaction between SU moieties in the polymer and the islet cells.     

Determination of the specific interaction between sulfonylurea-incorporated polymer and rat islets

A SU derivative, mimicking glibenclamide in chemical structure, was synthesized to incorporate it into a water-soluble polymeric backbone as a biospecific and stimulating polymer for insulin secretion. The ability of insulin secretion was examined with different glucose concentrations (3.3 and 11.6 mM). Although the vinylated SU did not exhibit significant activity compared to the control, the SU-incorporated copolymer could enhance insulin secretion as much as or more than glibenclamide did. In this study, a polymer fluorescence-labeled with rodamine-B isothiocyanate was used to visualize the interactions and we found that the labeled polymer was strongly absorbed to rat islets, probably due to its specific interaction mediated by SU receptors on the cell membrane. To verify the specific interaction between the SU (K+ channel closer)-incorporated copolymer and rat islets, cells were pretreated with diazoxide, an agonist of ATP-sensitive K+ channels (K+ channel opener), before adding the incorporated polymer to the cell culture medium. This treatment suppressed the action of SUs on rat islets. A confocal laser microscopic study further confirmed this interaction. The results of this study provided evidence that the SU-incorporated copolymer stimulates insulin secretion through specific interactions of SU moieties in the polymer with rat islets.     

Visualization of the specific interaction of sulfonylurea-incorporated polymer with insulinoma cell line MIN6

A derivative of sulfonylurea (SU) that mimics glibenclamide in chemical structure was synthesized and incorporated into a water-soluble polymeric backbone as a biospecific polymer for stimulating insulin secretion. In this study, a backbone polymer fluorescence-labeled with rodamine-B isothiocyanate was found to be strongly adsorbed onto MIN6 cells, probably due to its specific interaction mediated by SU receptors on the cell membrane. The intensity of fluorescence on the cells was significantly increased by increasing the incubation time and polymer concentration. To verify the specific interaction between the SU (K(+) channel closer)-incorporated copolymer and MIN6 cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K(+) channel (K(+) channel opener), before adding the polymer to the cell culture medium. This treatment suppressed the interaction between SU and MIN6 cells. A confocal laser microscopic study confirmed this effect. The results of this study provide evidence that SU-incorporated copolymer stimulates insulin secretion through the specific interactions of SU moieties in the polymer with MIN6 cells.     

Probing of specific binding of synthetic sulfonylurea with the insulinoma cell line MIN6

To overcome the limitation of conventional sulfonylurea (SU) for investigation of biological mechanisms related to KATP channels, a hypoglycemic sulfonylurea (SU) was conjugated with a non-reducing glucose bearing polystyrene (PS) derivative to provide enhanced interaction with an insulinoma cell line (MIN6). The specific interaction between the SU (K+ channel closer)-conjugated copolymer and MIN6 cells was confirmed by confocal laser microscopic images using rhodamine B isothiocyanate (RITC)-labeled SU-conjugated polymer, which revealed the specific interaction between SU-conjugated polymer and MIN6 cells. Moreover, the location of labeled polymer and the site of Ca2+ ion mobilization obtained from the same MIN6 cells were identical. Based on the specificity and insulinotropic activity, the SU-conjugated polymer is expected to be useful tool for the study of biological mechanisms of KATP channels.     

Targeted overactivity of beta cell K(ATP) channels induces profound neonatal diabetes

A paradigm for control of insulin secretion is that glucose metabolism elevates cytoplasmic [ATP]/[ADP] in beta cells, closing K(ATP) channels and causing depolarization, Ca2+ entry, and insulin release. Decreased responsiveness of K(ATP) channels to elevated [ATP]/[ADP] should therefore lead to decreased insulin secretion and diabetes. To test this critical prediction, we generated transgenic mice expressing beta cell K(ATP) channels with reduced ATP sensitivity. Animals develop severe hyperglycemia, hypoinsulinemia, and ketoacidosis within 2 days and typically die within 5. Nevertheless, islet morphology, insulin localization, and alpha and beta cell distributions were normal (before day 3), pointing to reduced insulin secretion as causal. The data indicate that normal K(ATP) channel activity is critical for maintenance of euglycemia and that overactivity can cause diabetes by inhibiting insulin secretion.     

Metabolic regulation of the K(ATP) and a maxi-K(V) channel in the insulin-secreting RINm5F cell

K channels in the cell membrane of the insulin-secreting RINm5F cell line were studied using the patch-clamp technique in cell-attached patch mode. With 140 mM K in the pipette, two channels displaying different conductive and kinetics properties were observed. A voltage-independent, inward-rectifying, 55-pS channel was active at rest (no glucose, -70 mV), but was almost completely inhibited by 5 mM glucose. A 140-pS channel was seen in the absence of glucose only after cell membrane depolarization with high (30 mM) K. This channel was voltage dependent, with a linear slope conductance between -60 and +60 mV, and was completely inhibited only by greater than 15 mM glucose. The former channel we identify as an ATP-sensitive channel previously described in excised patches and refer to it as the K(ATP) channel. The latter, because of its large conductance and voltage-dependent kinetics, will be referred to as the maxi-K(V) channel, adopting a nomenclature previously used to classify highly conductive K channels (Latorre, R., and C. Miller, 1983, Journal of Membrane Biology, 71:11-30). In addition to glucose, mannose and 2-ketoisocaproate, which also initiate insulin secretion and electrical activity in the islet beta cell, reduced the activity of both the K(ATP) and the maxi-K(V) channel. Lactate and arginine, which potentiate but do not initiate insulin secretion or beta cell electrical activity in normal islets, each caused a large reduction in maxi-K(V) channel activity, without consistently affecting the activity of K(ATP) channels. Another agonist that potentiates insulin secretion and electrical activity in normal cells, the tumor-promoting phorbol ester TPA, blocked maxi-K(V) channel activity while stimulating the activity of the K(ATP) channel, thereby implicating phosphorylation in the control of channel activity. These results indicate that metabolic substrates that initiate electrical activity and insulin secretion in normal beta cells reduce the activity of both the K(ATP) and the maxi-K(V) channel, while potentiating agents reduce only the maxi-K(V) channel. The possible role of these two channels in the processes of initiation and potentiation of the beta cell response is discussed.     

Conjugation of Arg-Gly-Asp sequence into thermo-reversible gel as an extracellular matrix for 3T3-L1 fibroblast cell culture

High molecular weight N-isopropylacrylamide copolymers with small amounts of acrylic acid (typically 2–5 mol% in feed) were synthesized by free radical polymerization in benzene and then conjugated with adhesion molecules of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides. Aqueous polymer solutions (5, 6, 8 and 10% w/v) in culture medium (pH 7.4, ionic strength; 0.15 M) with 3T3-L1 fibroblast cells were mixed and poured in Millicells, which supported the gel formation without a significant gel induction time at 36 °C (gelation temperature). The initially formed gel was translucent and became more opaque as the temperature increased. The interaction between fibroblast cells and an artificial matrix of GRGDS containing p(NiPAAm-co-AAc) copolymer gel resulted in effective cell attachment, proliferation and growth. This study supported that specific attachment is the result of the interaction between the integrin families on the fibroblast and the RGD sequence on the p(NiPAAm-co-AAc) copolymer gel.     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

The 25-kDa synaptosome-associated protein (SNAP-25) binds and inhibits delayed rectifier potassium channels in secretory cells

Delayed-rectifier K(+) channels (K(DR)) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K(+) channels results in membrane repolarization, leading to the closure of the Ca(2+) channels and cessation of insulin secretion in neuroendocrine islet beta cells. Using patch clamp techniques, we have demonstrated that the activity of the K(DR) channel subtype, K(V)1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15 beta cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the K(V)1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of K(DR). This inhibitory effect of SNAP-25 is mediated by the N terminus of K(V)1.1, likely by direct interactions with K(Valpha)1.1 and/or K(V)beta subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with K(DR) channels.     

Insulin as a surface marker on isolated cells from rat pancreatic islets

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.     

The insulin-secreting and proliferative activity of established islet cells in the presence of sulfonylurea]

Exponentially growing rat islet cells (RINr) and hamster islet cells (HIT T-15) were incubated in presence of tolbutamide (10-1000 microM), gliclazide (0.1-10 microM) or glibenclamide (0.01-10 microM) for 15 hrs. Accumulation of insulin in culture medium was estimated by RIA. Effects of sulfonylureas (SU) on cell proliferation were assessed by 3H-thymidine (3H-T) incorporation into cellular DNA. All of SUs used stimulated insulin production in RIN and HIT cell cultures (with an exception of tolbutamide, which markedly suppressed insulin secretion in HIT cells at 1000 microM). 3H-T incorporation into RIN cells was elevated only in presence of gliclazide (10 microM), whereas tolbutamide at 1000 M significantly inhibited RIN cell proliferation. Gliclazide (0.1 microM) and glibenclamide (0.01-10 microM) enhanced 3H-T incorporation into HIT cells. Further detailed investigations of mechanisms of SU effects on islet cell reproduction will be of use for designing optimal strategy of hypoglycemizing therapy of diabetes mellitus.     

Conjugation of Arg-Gly-Asp (RGD) sequence in copolymer bearing sugar moiety for insulinoma cell line (MIN6) culture

Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane.     

Conjugation of Arg-Gly-Asp (RGD) Sequence in Copolymer Bearing Sugar Moiety for Insulinoma Cell Line (MIN6) Culture

Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

Visual evidence and quantification of interaction of polymeric sulfonylurea with pancreatic islet

Using a polymeric sulfonylurea (PSU) designed from glibenclamide, we examined the interactions of sulfonylurea with pancreatic islets rather than genetically remodeled beta-cell lines to clarify the biological roles of ATP-sensitive K+ (KATP) channels to which sulfonylurea binds. PSU enhanced insulin secretion from the islets with 10 nM (SU equivalent) treatment, especially at low glucose concentration, but its activity was inhibited by 100 microM diazoxide. Confocal microscopy visualized PSU interactions with the islet and revealed that the modulation of intracellular Ca2+ occurred in the same region of an islet where PSU was also bound. In quantification method of the confocal microscopic images, competition of PSU with glibenclamide on its binding sites and glucose inhibition against PSU binding were confirmed. In this study, it was concluded that the PSU was a comparable drug with glibenclamide and offered a new standard method to study intact islets.     

Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion.

Microdissected beta-cell-rich pancreatic islets of ob/ob mice were used in studies of the relationship between intracellular pH (pHi) and 45Ca2+ uptake and insulin release. Stepwise increases in extracellular pH (pHo) from 6.80 to 8.00 resulted in a parallel, although less pronounced, elevation of pHi from 7.24 to 7.69. Experimental conditions that alkalinize the islet cell interior, i.e. addition of 5 mM-NH4+, sudden withdrawal of extracellular bicarbonate buffer or increase in pHo, induced insulin secretion in the absence of other types of secretory stimulation (1 mM-D-glucose). Intracellular acidification by lowering pHo below 7.40 or sudden addition of bicarbonate buffer did not induce insulin secretion. The removal of extracellular bicarbonate buffer, increase in pHo from 7.40 to 8.00, or the addition of 5 mM-L-5-hydroxytryptophan or 5 mM-NH4+, which all alkalinize the islet cells and induce insulin secretion, also increased the La3+-non-displaceable 45Ca2+ uptake in the presence of 1 mM-D-glucose. The results suggest that intracellular alkalinization in beta-cells can trigger insulin secretion. Taken together with the fact that D-glucose increases pHi in the islet cells, the results also point to the possibility that alkalinization may be a link in the stimulus-secretion coupling sequence in beta-cells.     

Lactate stimulates insulin secretion without blocking the K+ channels in HIT-T15 insulinoma cells.

To clarify the mechanism by which lactate affects insulin secretion, we investigated the effect of lactate on insulin secretion, cytosolic free Ca2+ ([Ca2+](i), the ATP sensitive K+ channel (K(ATP)) and the Ca2+-activated K+ channel (K(Ca)) in HIT-T15 cells, and the results were compared with those of glucose and glibenclamide. All three agents caused insulin secretion and increased [Ca2+](i), but the effects on the K+ channels were different. In cell-attached patch configurations, 10 mmol/l glucose blocked both the K(ATP) and KCa channels, while 100 nmol/l glibenclamide had no effect on KCa channels, but blocked K(ATP) channels. Lactate at a concentration of 10 mmol/l activated both the K(ATP) and KCa channels, not only in cell-attached, but also in inside-out patch configurations, indicating that the increase in [Ca2+](i) and secretion of insulin by lactate cannot be explained by the blocking of the K+ channels. Lactate, at concentrations of 10 mmol/l and 50 mmol/l decreased 45Ca2+ efflux, while glibenclamide increased the efflux. These results suggest that the lactate-induced Ca2+ increase is not due to the closing of K+ channels, but at least in part, to the suppression of Ca2+ efflux from HIT cells.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.     

PACAP is an islet neuropeptide which contributes to glucose-stimulated insulin secretion

Pituitary adenylate cyclase activating peptide (PACAP) is a ubiquitously distributed neuropeptide which also is localized to pancreatic islets and stimulates insulin secretion. We examined whether endogenous PACAP within the islets might contribute to glucose-stimulated insulin secretion by immunoneutralizing endogenous PACAP. Immunocytochemistry showed that PACAP immunoreactivity is expressed in nerve terminals within freshly isolated rat islets, but not in islets that had been cultured for 48 h. In contrast, islet endocrine cells did not display PACAP immunoreactivity. Addition of either of two specific PACAP antisera markedly inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from freshly isolated rat islets, whereas a control rabbit serum did not affect glucose-stimulated insulin secretion. In contrast, the PACAP antisera had no effect on glucose-stimulated insulin secretion in cultured islets. Based on these results we therefore suggest that PACAP is an islet neuropeptide which is required for the normal insulinotropic action of glucose.