Acceleration by fructose of the ATP-sensitive K(+) channel-independent pathway of glucose-induced insulin secretion.

The mechanism with which fructose augments glucose-induced insulin secretion is still unclear. The present study was aimed at examining whether the ketohexose potentiates the ATP-sensitive K(+) channel-independent pathway of glucose-induced insulin secretion and, if so, how this happens. When isolated rat islets were depolarized by incubating them with 50 mM KCl in the presence of 150 microM diazoxide (an opener of ATP-sensitive K(+) channels), 10 mM glucose plus 20 mM fructose elicited significantly higher insulin secretion than 10 mM glucose alone, whereas 20 mM fructose alone did not stimulate insulin secretion. The fructose 1,6-bisphosphate and inositol trisphosphate contents were markedly higher in islets incubated with glucose plus fructose than in islets incubated with glucose alone. The results demonstrate that fructose has the ability to potentiate the ATP-sensitive K(+) channel-independent pathway of glucose-induced insulin secretion. The increase in fructose 1,6-bisphosphate content induced by the co-presence of fructose with glucose, resulting in the rise in inositol trisphosphate content, is likely to be one of the signals involved in the fructose potentiation of glucose-induced insulin secretion.     

Dual effect of formycin a upon the hydrolysis of phosphoinositides in perifused pancreatic islets

Formycin A (1.0 mM) caused a rapid, sustained and rapidly reversible inhibition of effluent radioactivity in rat pancreatic islets prelabelled with myo-[2-³H]inositol and perifused in the presence of 8.3 mM -glucose. This coincided with a progressive decrease in islet ATP content and transient inhibition of insulin release. Theraafter, however, formycin A increased glucose-induced insulin release. Moreover, in islets that were preincubated with myo-[2-³H]inositol and then exposed during perifusion to a rise in -glucose concentration from 2.8 to 16.7 mM, the release of insulin and ³H fractional outflow rate at both the low and high hexose concentrations were much higher when both the preincubation and perifusion were conducted in the presence, rather than absence, of formycin A. It is concluded that formycin A first inhibits and later enhances both the hydrolysis of phosphoinositides and release of insulin, these effects being possibly related to changes in the islet cell content of adenosine and/or formycin A triphosphates.     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion.

The influence of down-regulation of protein kinase C on glucose-induced insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM) in RPMI 1640 culture medium (8.3 mM-glucose, 0.43 mM-Ca2+) abolished TPA (0.16 microM)-induced insulin secretion and led to a potentiation of phase 1 and a decrease in phase 2 of glucose-induced insulin secretion. Thus, although the total insulin release during 40 min of perfusion with glucose (16.7 mM) (45-85 min) was unaffected, the percentage released during phase 1 (45-55 min) was increased from 12.9 +/- 1.5 (4)% in controls to 35.8 +/- 3.9 (4)% in TPA-treated islets (P less than 0.01), and the percentage released during phase 2 (65-85 min) was decreased from 63.2 +/- 3.9 (4)% to 35.3 +/- 1.4 (4)% (P less than 0.005). In contrast, TPA exposure in TCM 199 medium (5.5 mM-glucose, 1.26 mM-Ca2+) caused a total abolition of both phases 1 and 2 of glucose-induced secretion. However, inclusion of the alpha 2-adrenergic agonists adrenaline (10 microM) or clonidine (10 microM), or lowering of the Ca2+ concentration in TCM 199 during down-regulation, preserved and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1 and inhibited phase 2 of carbamoylcholine (100 microM)-induced insulin secretion at 3.3 mM-glucose, and abolished the potentiating effect of carbamoylcholine (100 microM) at 16.7 mM-glucose. These results substantiate a role for protein kinase C in insulin secretion, and suggest that protein kinase C inhibits phase 1 and stimulates phase 2 of both glucose-induced and carbamoylcholine-induced insulin secretion.     

The conditions under which rat islets are labelled with [3H]inositol alter the subsequent responses of these islets to a high glucose concentration.

Isolated rat islets were incubated with myo-[2-3H]inositol for 2 h to label their phosphoinositide (PI) pools. Labelling was carried out under three separate conditions: in media containing low (2.75 mM) glucose, high (13.75 mM) glucose, or low (2.75 mM) glucose plus sulphated cholecystokinin (CCK-8S; 200 nM). After labelling, the islets were perifused and the insulin-secretory response to 20 mM-glucose was measured. PI hydrolysis in these same islets was assessed by measurements of both [3H]inositol efflux and the accumulation of labelled inositol phosphates. The following major observations were made. After prelabelling for 2 h in low glucose, perifusion with 20 mM-glucose resulted in a biphasic insulin-secretory response, an increase in [3H]inositol efflux and a parallel increase in the accumulation of labelled inositol phosphates. After prelabelling in high (13.75 mM) glucose, peak first-phase insulin secretion induced by 20 mM-glucose increased 2-2.5-fold, whereas the second phase of insulin release, as well as [3H]inositol efflux and inositol phosphate accumulation, were significantly decreased. The simultaneous infusion of the diacylglycerol kinase inhibitor 1-mono-oleoylglycerol (50 microM), along with 20 mM-glucose, restored the second-phase insulin-secretory response from these islets. After labelling in low (2.75 mM) glucose plus CCK-8S, the initial phases of the insulin-secretory and [3H]inositol-efflux responses to 20 mM-glucose were blunted and the sustained phases of both responses were markedly decreased. Inositol phosphate accumulation was also impaired. Labelling islets in high (13.75 mM) glucose or low (2.75 mM) glucose plus CCK-8S suppresses, in a parallel fashion, glucose-induced increases in PI hydrolysis and in second-phase insulin release. These findings suggest that desensitization of the insulin-secretory response is a consequence of impaired information flow in the inositol lipid cycle.     

Comparative study on the effects of a hypoglycemic 2-substituted-2-imidazoline derivative (DG-5128) and tolbutamide on insulin secretion from and insulin synthesis in the isolated rat pancreatic islets

2[2-(4,5-Dihydro-1H-imidazol-2-yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate (DG-5128) was found to stimulate the glucose-primed insulin secretion from the isolated rat pancreatic islets throughout the incubation period, unlike tolbutamide which stimulated it only in the initial phase of incubation. The effect of DG-5128 was more pronounced at a higher glucose concentration (5 mg/ml). In the islet perifusion study, DG-5128 was also found to stimulate the glucose-induced insulin secretion in both the first and the second phases of the reaction, in contrast to tolbutamide which stimulated only the first phase of insulin secretion from the perifused islets. DG-5128 gave no significant effect on the glucose-stimulated increase in incorporation of [³H]leucine into the pro-insulin and insulin fractions, while tolbutamide significantly inhibited the incorporation especially at a low glucose concentration (1 mg/ml). These and the previous findings indicate that DG-5128 is a new class of hypoglycemic agent with a unique mode of action different from the known hypoglycemics ever reported.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.     

Are 5-hydroxytryptamine-preloaded beta-cells an appropriate physiologic model system for establishing that insulin stimulates insulin secretion?

The release and oxidation of 5-hydroxytryptamine from 5-hydroxytryptamine-preloaded beta-cells has been used as a surrogate marker for insulin secretion. Findings made using this methodology have been used to support the concept that insulin stimulates its own release. In the present studies, the effects of 5-hydroxytryptamine on stimulated insulin secretion from isolated perifused rat islets was determined. When added together with stimulatory glucose, 5-hydroxytryptamine (0.5 mm) significantly reduced both phases of 8 mm glucose-induced secretion and reduced the first phase of 15 mm glucose-induced release by 60% without any effect on sustained insulin release rates. Preloading of beta-cells with 0.5 mm 5-hydroxytryptamine for 3 h resulted in a more severe impairment of 15 mm glucose-induced secretion. First and second phase release rates were reduced by 70 and 55%, respectively. In addition, this pretreatment protocol also abolished 200 microm tolbutamide-induced insulin secretion from perifused islets. These findings confirm that 5-hydroxytryptamine is a powerful inhibitor of stimulated insulin secretion. The responses of 5-hydroxytryptamine-preloaded beta-cells may not accurately reflect the biochemical events occurring during the physiologic regulation of insulin secretion. The suggestion that insulin stimulates its own secretion based exclusively on amperometric measurements should be reconsidered.     

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets.

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K0.5 of 10 microM) and maximum activation was observed at 50 microM arachidonate. Arachidonic acid did not affect the Ca2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 microM) inhibited (greater than 90%) calmodulin-dependent protein kinase activity (K0.5 = 50-100 microM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K0.5 = 24 microM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50-100 microM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 microM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 microM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.     

A microperifusion system with environmental control for studying insulin secretion by pancreatic tissue.

A continuous flow reactor (perifusion system) was fabricated and tested for measuring the kinetics of insulin secretion from isolated pancreatic islets of Langerhans in response to step changes in the glucose concentration and oxygen partial pressure in the perfusate flowing around the islets. The system was capable of making rapid changes in perfusate glucose concentration and pO2, had rapid dynamic response for measuring the change in insulin secretion rate as a result of these changes in perfusate, and was suitable for studying very small volumes of tissue. Initial experiments with this system demonstrated that (1) the response of isolated rat islets to glucose stimulation was very fast, with the first phase peak occurring in as little as about 10 s, (2) bulk perfusate oxygen partial pressure levels of 30 mmHg or less reduced the second-phase insulin secretion rate in graded fashion, (3) the reduction in secretion rate began within 1 min following an oxygen partial pressure decrease, and (4) the reduction in secretion rate was reversible, with a burst of insulin secretion occurring during the first minute after partial pressure restoration.     

Insulin secretion from isolated pancreatic islets in the female rat. Short and long term oestradiol influence.

An increase in the two phases of glucose-induced insulin response is observed after an oestradiol (E2) treatment or after a direct E2 action on the female rat perfused pancreas. To study such effects, experiments were carried out with isolated islets of Langerhans, and especially with a dynamic method : perifusion of the islets. The same long term E2 permissive effect was obtained with isolated islets submitted to a glucose stimulation. On the contrary, the direct E2 action seen in glucose-stimulated perfused pancreas was found neither with incubation tests, nor with perifusion studies, whatever the experimental conditions (E2 or glucose doses, exposure times, oestrous or spayed rats, 100 instead of 25 islets per perifusion chamber). The differences between isolated islets of Langerhans and the whole perfused pancreas preparation are discussed.     

Viability criteria of islets of Langerhans isolated and purified using Ficoll

Rejection of islet allografts is generally explained by immunologic problems, due to both cellular and antibody mechanisms. But another great problem is in the isolation of intact and viable islets of Langerhans: it is necessary to use a good method of pancreas distention, to determine the optimal concentration of collagenase for digestion, to select an effective technique for purifying the islets. This study correlates the morphology of isolated pancreatic islets of rats and dogs with secretion of insulin. The islets are incubated in a perifusion system and are tested during four periods; the glucose concentrations of the perifusion fluid are: 5.5 mM during the initial 70 min. period, 16.5 mM during the second 60 min. period, 5.5 mM during the third 60 min. period and 16.5 during the fourth 50 min. period. This "double glucose stimulation" is a good test of islet viability. The intact, viable isolated islets showed a significant increase of insulin secretion during the two 16.5 mM glucose periods. Damaged islets with some little morphologic alterations after showed a good insulin release during the first glucose stimulation, but a very poor insulin response to glucose during the second stimulation period.     

Characteristics of the biotin enhancement of glucose-induced insulin release in pancreatic islets of the rat

Perifused isolated rat islets were used to show that biotin plus 16.5 mM glucose evoked more insulin secretion than 16.5 mM glucose alone. Whether or not this reinforcement of glucose-induced insulin secretion by biotin is unique was studied by using perifused islets stimulated with 16.5 mM glucose plus 100 microM of one of various components of the vitamin B group. No effect of any of these vitamins was found on glucose-induced insulin secretion. These results indicate that biotin is unique among the members of the vitamin B group in enhancing glucose-induced insulin secretion. Static incubation experiments showed that biotin did not potentiate insulin release when the islets were incubated with an experimental solution containing either no or 2.8 mM glucose. The addition of biotin to 27.7 mM glucose, which is the maximal concentration for stimulating insulin release, did not significantly enhance the effect of the glucose on insulin release (although it did at 16.5 mM glucose). These findings indicate that biotin, by itself, does not stimulate insulin secretion, and does not enhance glucose-induced insulin secretion beyond the ability of glucose itself to stimulate insulin secretion.     

Hexamminecobalt(III) chloride inhibits glucose-induced insulin secretion at the exocytotic process

Hexamminecobalt(III) (HAC) chloride was found to have a potent inhibitory effect on glucose-induced insulin secretion from pancreatic islets. HAC at 2 mm inhibited the secretion in response to 22.2 mm glucose by 90% in mouse islets. Perifusion experiments revealed that the first phase of insulin secretion was severely suppressed and that the second phase of secretion was completely abrogated. Removal of HAC from the perifusate immediately restored insulin secretion with a transient overshooting above the normal level. However, HAC failed to affect glucose-induced changes in d-[6-(14)C]glucose oxidation, levels of reduced forms of NAD and NADP, mitochondrial membrane potential, ATP content, cytosolic calcium concentration, or calcium influx into mitochondria. Furthermore, HAC inhibited 50 mm potassium-stimulated insulin secretion by 77% and 10 microm mastoparan-stimulated insulin secretion in the absence of extracellular Ca(2+) by 80%. The results of a co-immunoprecipitation study of lysates from insulin-secreting betaHC9 cells using anti-syntaxin and anti-vesicle-associated membrane protein antibodies for immunoprecipitation or Western blotting suggested that HAC inhibited disruption of the SNARE complex, which is normally observed upon glucose challenge. These results suggest that the inhibitory effect of HAC on glucose-induced insulin secretion is exerted at a site(s) distal to the elevation of cytosolic [Ca(2+)], possibly in the exocytotic machinery per se; and thus, HAC may serve as a useful tool for dissecting the molecular mechanism of insulin exocytotic processes.     

Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide

The cryoprotectants dimethyl sulfoxide (Me2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at -196 degrees C after slow cooling (0.3 degrees C/min) to -70 degrees C in the presence of multimolar concentrations of either Me2SO or glycerol. Samples were rewarmed slowly (approximately 10 degrees C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 degrees C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me2SO; and in one of three animals receiving islets cryopreserved with 3 M Me2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.     

Differential effects of propofol and isoflurane on glucose utilization and insulin secretion

AimsVolatile anesthetics, such as isoflurane, reverse glucose-induced inhibition of pancreatic adenosine triphosphate-sensitive potassium (K_ATP) channel activity, resulting in reduced insulin secretion and impaired glucose tolerance. No previous studies have investigated the effects of intravenous anesthetics, such as propofol, on pancreatic K_ATP channels. We investigated the cellular mechanisms underlying the effects of isoflurane and propofol on pancreatic K_ATP channels and insulin secretion.Main methodsIntravenous glucose tolerance tests (IVGTT) were performed on male rabbits. Pancreatic islets were isolated from male rats and used for a perifusion study, measurement of intracellular ATP concentration ([ATP]_i), and patch clamp experiments.Key findingsGlucose stimulus significantly increased insulin secretion during propofol anesthesia, but not isoflurane anesthesia, in IVGTT study. In perifusion experiments, both islets exposed to propofol and control islets not exposed to anesthetic had a biphasic insulin secretory response to a high dose of glucose. However, isoflurane markedly inhibited glucose-induced insulin secretion. In a patch clamp study, the relationship between ATP concentration and channel activity could be fitted by the Hill equation with a half-maximal inhibition of 22.4, 15.8, and 218.8 μM in the absence of anesthetic, and with propofol, and isoflurane, respectively. [ATP]_i and single K_ATP channel conductance did not differ in islets exposed to isoflurane or propofol.SignificanceOur results indicate that isoflurane, but not propofol, decreases the ATP sensitivity of K_ATP channels and impairs glucose-stimulated insulin release. These differential actions of isoflurane and propofol on ATP sensitivity may explain the differential effects of isoflurane and propofol on insulin release.     

Direct effect of corticosterone upon insulin secretion studied by three different techniques.

The immediate effect of corticosterone upon insulin secretion rates estimated by three different techniques (perfusior of isolated rat pancreas and perifusion or incubation of isolated islets of Langerhans) was studied for one hour. Three corticosterone concentrations were used: 0.02, 0.2 or 20 mg/l. With 4.2 mmol/l glucose, corticosterone did not affect insulin secretion, whereas, with a stimulating glucose concentration (16.7 mmol/l), insulin secretion was inhibited by the three corticosterone concentrations tested during incubation experiments, and by only the two physiological ones (0.02 and 0.2 mg/l) during islets perifusion and pancreas perfusion experiments. Moreover the inhibitory effect appeared more rapid with perifused islets than perfused pancreas, where only the second insulin secretory phase was disturbed.     

Biotin enhances ATP synthesis in pancreatic islets of the rat, resulting in reinforcement of glucose-induced insulin secretion

Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.     

Glucose‐stimulated insulin release: Parallel perifusion studies of free and hydrogel encapsulated human pancreatic islets

To explore the effects immune‐isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose‐stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi‐channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first‐phase peak. While small microcapsules (d = 700 µm) caused only a slightly delayed and blunted first‐phase insulin response compared to unencapsulated islets, larger capsules (d = 1,800 µm) completely blunted the first‐phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time‐profiles were fitted with our complex insulin secretion computational model. This allowed further fine‐tuning of the hormone‐release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first‐phase insulin response and to a sustained‐release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long‐term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design.