Insulin secretion from perifused rat pancreatic pseudoislets

Summary Isolated adult rat pancreatic islets were dispersed into single cells and cultured free-floating for 3 to 4 d, during which time islet cells reaggregated spontaneously into spherical clusters or pseudoislets. The gross morphology of these tissues resembled nondissociated islets. Electron microscopy revealed well-preserved cell ultrastructure and intercellular membrane connections. Immunofluorescent localization of islet cell types showed that A cells tended to be peripherally distributed around a B cell core, with D cells scattered throughtout the aggregate, mass. The dynamics of insulin release from pseudoislets were evaluated in vitro by perifusion techniques. Pseudoislets exhibited clear biphasic dose-dependent insulin responses to 30 min glucose stimulation over the range 5.5 to 30 mM. Repeated 2-min pulses with 22 mM glucose elicited brief monophasic spikes of insulin release of, consistent magnitude.l-Arginine (5 to 20 mM) evoked biphasic insulin release but these responses were not dose-dependent. These data indicate that islet cells reaggregate into structures with close morphologic similarities to intact islets, and that pseudoislet B cells continue to secrete insulin in response to nutrient secretagogues, comparable to that seen with islets in vitro and in situ. This work was supported by grants from the Medical Research Council of New Zealand. D. W. H. was the recipient of a Novo Diabetes Research Scholarship.     

Effect of calcitonin gene-related peptide on glucose and gastric inhibitory polypeptide-stimulated insulin release from cultured newborn and adult rat islet cells

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide that is present in peripheral cells of islets and in nerves around and within islets. CGRP can inhibit insulin secretion in vitro and in vivo. Whether the inhibitory action of CGRP is mediated by somatostatin or by nerve terminals is, however, not known. The objective of this study was to examine the effect of CGRP on insulin secretion, using cultured newborn and adult rat islet cells which did not contain nerve terminals. In adult rat islet cells, CGRP (10(-10) to 10(-8) M) significantly inhibited glucose-stimulated and gastric inhibitory polypeptide (GIP)-potentiated insulin secretion, but in newborn rat islet cells, CGRP did not inhibit glucose-stimulated insulin secretion. Inhibition of glucose-stimulated and GIP-potentiated insulin release was dependent on the glucose concentration during the prestimulation period. CGRP did not stimulate release of somatostatin. These findings suggest that rat CGRP can act directly on beta-cells through a specific receptor that is absent in newborn rat beta-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.     

Glucose dependent stimulation by prostaglandin D2 of glucagon and insulin in perfused rat pancreas

Effects of prostaglandin D2 on pancreatic islet function in perfused rat pancreas were examined in comparison with those of prostaglandin E2, which has hitherto been suggested to be a modifier of pancreatic hormone release. In the presence of 2.8 mM glucose, only glucagon release was strongly stimulated by 14 microM of prostaglandin D2, while release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2. When the glucose concentration was elevated to 11.2 mM, insulin release was accelerated by 14 microM of prostaglandin D2 but there was no effect upon glucagon release. Again, release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2 in the presence of 11.2 mM of glucose. The regulation of glucagon and insulin release through prostaglandin D2 is apparently adapted to glycemic changes, and may be a physiological modulator of pancreatic islet function.     

INGAP-related pentadecapeptide: its modulatory effect upon insulin secretion

We examined the effects of a pentadecapeptide having the 104-118 aminoacid sequence of islet neogenesis-associated protein (INGAP-PP) on insulin secretion, and the morphological characteristics of adult and neonatal pancreatic rat islets cultured in RPMI and 10 mM glucose for 4 days, with or without different INGAP-PP concentrations (0.1-100 mug/ml). A scrambled 15 aminoacid peptide was used as control for the specificity of INGAP-PP effect. Cultured neonatal and adult islets released insulin in response to glucose (2.8-16.7 mM) in a dose-dependent manner, and to leucine and arginine (10 mM). In all cases, the response was greater in adult islets. INGAP-PP added to the culture medium significantly enhanced glucose- and aminoacid-induced insulin release in both adult and newborn rats; however, no changes were observed with the scrambled peptide. Similar results were obtained incubating freshly isolated adult rat islets with INGAP-PP. Whereas INGAP-PP did not induce significant changes in islet survival rate or proportion/number of islet cells, it increased significantly beta-cell size. This first demonstration of the enhancing effect of INGAP-PP on the beta-cell secretory response of adult and newborn islets opens a new avenue to study its production mechanism and potential use to increase the secretory capacity of endogenous islets in intact animals or of islets preserved for future transplants.     

Effect of glucose and K+ depolarization on cytosolic free Ca2+ in cultured neonatal islets.

In cultured neonatal islet cells, glucose (16.7 mM) and K+ (50 mM) increased cytosolic free Ca2+ ([Ca2+]i). The increments in [Ca2+]i induced by either glucose or K+ were similar to those obtained in cultured adult islet cells but only half of that recorded in freshly isolated adult islet cells. These data indicate that, in neonatal islet cells, the reduced insulin release in response to glucose is associated with a diminished increase in [Ca2+]i. This reduced insulin response may not solely be due to an impaired regulation of the ATP-sensitive K+ channels as previously suggested. It may also result from some alteration in the process of Ca2+ inflow through voltage-sensitive Ca2+ channels.     

Formation of islet cell monolayers from fetal human and neonatal rat pancreatic islets: protein biosynthesis, insulin secretion, and binding of islet cell antibodies

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) has been used to promote monolayer formation in cultured islets isolated from fetal human or neonatal rat pancreas. Immunofluorescence with specific antiserum to insulin revealed B-cells in the outgrown monolayers. The rat islet cells were further characterized by their secretory and biosynthetic response to the action of IBMX. Both glucose-stimulated insulin release and recoverable insulin, i.e. intracellular insulin plus insulin secreted, were increased by the addition of IBMX (0.1 mmol/l) to the medium containing 10 mmol/l glucose. 3H-leucine incorporation into (pro)insulin was significantly higher following culture in 10 mmol/l glucose plus IBMX (0.1 and 1.0 mmol/l) than after cultivation with glucose alone. However, the percentage of (pro)insulin synthesized in relation to total protein synthesis was increased to a lesser extent after an acute incubation for 3 h at 1.5 mmol/l or in the absence of glucose. Moreover, the culture system employed in the present study proved to be useful for detecting islet cell antibodies bound to human as well as rat islet cells after exposure to islet cell antibody-positive sera.     

Effects of glucose and acetylcholine on islet tissue NADH and insulin release.

The relationship between islet tissue NADH and insulin release resulting from glucose or acetylcholine was investigated with the isolated perfused rat pancreas. Switching the perfusate from 4 to 16 mM glucose or adding 1 μM acetylcholine to 4.4 mM basal glucose elicited biphasic insulin release and rapidly elevated the NADH content of islet tissue, suggesting that intermediary metabolism was stimulated. The biochemical basis for this NADH increase and its significance in islet physiology are discussed.     

Participation of intestinal hormones in the development of the pancreatic B-cell reactivity of rat fetuses to the action of glucose

The involvement of intestinal hormones in the development of insulin release from rat fetal pancreas was investigated. B-cell responses were determined by changes in the concentration of immunoreactive insulin after glucose addition to the incubation medium. Coincubation of fragments of fetal pancreas and duodenum from adult and newborn rats and from 21.5-day-old fetus has shown that intestinal factors can recover the response of pancreas to glucose in fetuses with experimentally removed hypothalamus and hypophysis. Besides, the intestinal factors in the fetus were found to potentiate the effect of high glucose concentrations on B cells, but had no insulinotropic effect at physiological glucose concentration in the medium. The data obtained suggest that even in the antenatal period the intestinal, along with cephalic factors, can serve as modulators of glucose action on islet B cells.     

Microcarriers: A new approach to pancreatic islet cell culture

Summary Free islet cell suspensions were prepared from isolated fetal rat islets using a modified enzyme dispersion technique. The islet cells were dispensed into a culture flask containing microcarriers (Cytodex) suspended in culture medium RPMI 1640 by a slowly rotating bar magnet. Microscopical examination of the beads showed that the islet cells attached and then progressively proliferated on the surface of the beads as a monolayer. A highly sustained release of insulin from the beads to the medium was observed during the 7 d culture period. The functional viability of the cultured islet cells was further demonstrated by the ability of batches of the cell-coated beads to synthesize insulin and to increase the insulin release in response to an acute challenge (16.7 mmol/l glucose plus 5 mmol/l theophylline). The results suggest that bead microcarriers may provide a new approach to monolayer islet cell culture providing functional monolayers, which can easily be transferred to different test systems and further manipulated. Financial support for the study was received from the University of Uppsala, the Swedish Diabetes Association, Stiftelsen Expressens Prenatalforskningsfond, Nordisk Insulinfond, Stiftelsen Claes Groschinskys Minnesfond, the Swedish Society of Medical Sciences and the Swedish Medical Research Council (Grants No. 12X-109 and 12X-2297). This work was presented in part at the 15th Annual Meeting of the Scandinavian Society for the Study of Diabetes (June 5–7, 1980; Oslo, Norway) and the 16th Annual Meeting of the European Association for the Study of Diabetes (September 24–27th, 1980; Athens, Greece).     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Preparation of single cells from pancreatic islets of adult rat by the use of dispase.

A high yield of viable single cells was attained from isolated pancreatic islets of adult rat by the sequential treatment with EDTA and Dispase. The percentage of single cells was consistently higher with EDTA-Dispase in comparison with EDTA-trypsin treatment, being 65.8 +/- 7.9% and 36.0 +/- 5.4% respectively, when more than 90% of total islet cells were viable. Excellent preservation of free islet cells dissociated with EDTA-Dispase was demonstrated morphologically by light and electron microscopy. The secretory response of dissociated B cells to glucose was stabilized earlier with EDTA-Dispase than with EDTA-trypsin treatment. The amount of insulin released into the medium was proportional to the number of cells inoculated, thus permitting the quantitative analysis of B-cell function in vitro.     

Abnormal glucose metabolism accompanies failure of glucose to stimulate insulin release from a rat pancreatic cell line (RINm5F).

Glucose metabolism and insulin release were studied in isolated rat islets and in an insulin-producing rat cell-line (RINm5F). Intact islets displayed two components of glucose utilization, with glucose stimulation of insulin release being associated with the high-Km component (reflecting glucokinase-like activity). Glucose failed to stimulate insulin release from RINm5F cells, which only displayed a single low-Km component of glucose utilization. Only low-Km (hexokinase-like) glucose-phosphorylating activity was found for disrupted RINm5F cells. These changes in glucose metabolism may contribute towards the failure of glucose to stimulate insulin release from RINm5F cells.     

Inhibition of glucose-stimulated insulin release by pseudo-alpha-DL-glucose as a glucokinase inhibitor

Pseudo-alpha- and pseudo-beta-DL-glucose, the isomers of 5-hydroxymethyl-1,2,3,4-cyclohexanetetrol with alpha-gluco and beta-gluco configurations, were used as synthetic analogs of glucose anomers to study the mechanism of glucose-stimulated insulin release by pancreatic islets. Neither isomer was phosphorylated by liver glucokinase nor stimulated insulin release from islets. Incubation of islets with pseudo-alpha-DL-glucose resulted in a considerable accumulation of the glucose analog, probably the D form, in islets. The alpha-isomer, but not the beta-isomer, inhibited both glucose-stimulated insulin release (44% inhibition at 20 mM) and islet glucokinase activity (36% inhibition at 20 mM) in a concentration-dependent manner and to a comparable degree. These results strongly suggest that the inhibition of glucose-stimulated insulin release by pseudo-alpha-DL-glucose is due to the inhibition of islet glucokinase by the glucose analog, providing additional evidence for the essential role of islet glucokinase in glucose-stimulated insulin release.     

Metabolic and secretory effects of methylamine in pancreatic islets

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5-³H] histamine into either N,N-dimethylcasein or endogenous islet proteins. Methylamine (2 mM ) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of ¹⁴C-methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non-nutrient insulinotropic agents such as L -arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM ) was more efficiently taken up by islet cells than methylamine (2.0 mM ), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.     

Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release.

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent on extracellular Ca2+ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the beta-cell glucose-sensor mechanism.     

Effect of 2-deoxy-D-glucose on maintenance in culture of neonatal B cell of rat

The effect of 2-deoxy-D-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-D-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-D-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Effects of phloretin and dextran-linked phloretin on pancreatic islet metabolism and insulin release

The effects of phloretin on islet metabolism and insulin release have been studied in isolated pancreatic islets of the rat. At a concentration of 0.18 mM phloretin inhibited insulin release stimulated by glucose or leucine but did not affect the oxidation rates of glucose or leucine, the rate of glucose utilization and the islet content of ATP. Higher concentrations of phloretin caused inhibition of the rate of glucose metabolism, but stimulation of insulin release. Insulin release stimulated by phloretin was inhibited by mannoheptulose but was independent of extracellular Ca²⁺ and was not potentiated by caffeine. Both inhibitory and stimulatory effects of dextran-linked phloretin on insulin release were also seen; a concentration of dextran-linked phloretin that did not inhibit islet metabolism inhibited glucose-stimulated insulin release, but not release stimulated by leucine or glyceraldehyde. Higher concentrations of dextran-linked phloretin inhibited glucose oxidation but stimulated insulin release. These data are discussed in terms of current models of the β-cell glucose-sensor mechanism.     

Insulin secretion kinetics from single islets reveals distinct subpopulations

Type II diabetes progresses with inadequate insulin secretion and prolonged elevated circulating glucose levels. Also, pancreatic islets isolated for transplantation or tissue engineering can be exposed to glucose over extended timeframe. We hypothesized that isolated pancreatic islets can secrete insulin over a prolonged period of time when incubated in glucose solution and that not all islets release insulin in unison. Insulin secretion kinetics was examined and modeled from single mouse islets in response to chronic glucose exposure (2.8‐20 mM). Results with single islets were compared to those from pools of islets. Kinetic analysis of 58 single islets over 72 h in response to elevated glucose revealed distinct insulin secretion profiles: slow‐, fast‐, and constant‐rate secretors, with slow‐secretors being most prominent (ca., 50%). Variations in the temporal response to glucose therefore exist. During short‐term (<4 h) exposure to elevated glucose few islets are responding with sustained insulin release. The model allowed studying the influence of islet size, revealing no clear effect. At high‐glucose concentrations, when secretion is normalized to islet volume, the tendency is that smaller islets secrete more insulin. At high‐glucose concentrations, insulin secretion from single islets is representative of islet populations, while under low‐glucose conditions pooled islets did not behave as single ones. The characterization of insulin secretion over prolonged periods complements studies on insulin secretion performed over short timeframe. Further investigation of these differences in secretion profiles may resolve open‐ended questions on pre‐diabetic conditions and transplanted islets performance. This study deliberates the importance of size of islets in insulin secretion. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1059–1068, 2018