A Microfluidic Hanging-Drop-Based Islet Perifusion System for Studying Glucose-Stimulated Insulin Secretion From Multiple Individual Pancreatic Islets

Islet perifusion systems can be used to monitor the highly dynamic insulin release of pancreatic islets in glucose-stimulated insulin secretion (GSIS) assays. Here, we present a new generation of the microfluidic hanging-drop-based islet perifusion platform that was developed to study the alterations in insulin secretion dynamics from single pancreatic islet microtissues at high temporal resolution. The platform was completely redesigned to increase experimental throughput and to reduce operational complexity. The experimental throughput was increased fourfold by implementing a network of interconnected hanging drops, which allows for performing GSIS assays with four individual islet microtissues in parallel with a sampling interval of 30 s. We introduced a self-regulating drop-height mechanism that enables continuous flow and maintains a constant liquid volume in the chip, which enables simple and robust operation. Upon glucose stimulation, reproducible biphasic insulin release was simultaneously observed from all islets in the system. The measured insulin concentrations showed low sample-to-sample variation as a consequence of precise liquid handling with stable drop volumes, equal flow rates in the channels, and accurately controlled sampling volumes in all four drops. The presented device will be a valuable tool in islet and diabetes research for studying dynamic insulin secretion from individual pancreatic islets.

Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic β cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A−/−) mice. Synt1A−/− β cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets

Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the β-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca2+ concentration ([Ca2+]i). The second phase requires increased [Ca2+]i and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca2+-stimulated release via the KATP channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K+ channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased α-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of α-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP.

Activation of the KATP channel-independent signaling pathway by the nonhydrolyzable analog of leucine, BCH

Leucine and glutamine were used to elicit biphasic insulin release in rat pancreatic islets. Leucine did not mimic the full biphasic response of glucose. Glutamine was without effect. However, the combination of the two did mimic the biphasic response. When the ATP-sensitive K+ (KATP) channel-independent pathway was studied in the presence of diazoxide and KCl, leucine and its nonmetabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) both stimulated insulin secretion to a greater extent than glucose. Glutamine and dimethyl glutamate had no effect. Because the only known action of BCH is stimulation of glutamate dehydrogenase, this is sufficient to develop the full effect of the KATP channel-independent pathway. Glucose, leucine, and BCH had no effect on intracellular citrate levels. Leucine and BCH both decreased glutamate levels, whereas glucose was without effect. Glucose and leucine decreased palmitate oxidation and increased esterification. Strikingly, BCH had no effect on palmitate oxidation or esterification. Thus BCH activates the KATP channel-independent pathway of glucose signaling without raising citrate levels, without decreasing fatty acid oxidation, and without mimicking the effects of glucose and leucine on esterification. The results indicate that increased flux through the TCA cycle is sufficient to activate the KATP channel-independent pathway.

Effects of Caffeine and Acetylcholine on Glucose-Stimulated Insulin Release from Islet Transplants in Mice

In mouse islet grafts under the kidney capsule, the potentiating responsiveness to acetylcholine was markedly attenuated after a few weeks. The question arose as to whether transplanted islets show an decreased responsiveness to potentiators in general. The effect of caffeine on glucose-induced insulin secretion was, therefore, examined. Intrastrain transplantation was performed in NMRI and BALB/c mice, and islet grafts were removed and perifused in vitro after 3 and 12 wk. In grafts from both NMRI and BALB/c mice, 16.7 mmol/L glucose induced a biphasic insulin release. When 1 or 5 mmol/L caffeine was included in the perifusion medium, there was a marked potentiation of the glucose-induced insulin release that was at least as responsiveness as fresh untransplanted islets. In the absence of caffeine, 3-wk-old BALB/c grafts reacted less strongly to acetylcholine than did untransplanted islets. The addition of 1 mmol/L caffeine did not enhance the potentiating effect of acetylcholine, whether in untransplanted or transplanted islets. Rather, the interaction between caffeine and acetylcholine appeared negative. We concluded that the glucose-induced insulin secretion exhibits a diminished potentiatory responsiveness to acetylcholine but not to caffeine. The displacement and denervation of transplanted islets is likely to affect either the cholinergic receptors or their mediated influence on intracellular calcium. Copyright © 1997 Elsevier Science Inc.

L-leucine or its keto acid potentiate but do not initiate insulin release in chicken

In the isolated perfused chicken pancreas, 20 and 40 mM L-leucine or 10–40 mM alpha-ketoisocaproic acid (alpha-KIC) did not initiate insulin release. In the presence of 14 mM glucose (a noninsulinotropic concentration), 20 mM L-leucine and 10 mM alpha-KIC evoked a slight biphasic insulin release. The response to 20 mM L-leucine was further increased when 14 mM glucose was combined with 10 mM L-glutamine (10 mM glutamine alone did not induce insulin release and did not potentiate the response to 10 mM L-leucine). At 1 mM, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BrcAMP) alone caused a slight and progressive increase in insulin secretion but did not sensitize the pancreas to either 20 mM L-leucine or 10 mM alpha-KIC, whereas it facilitated a marked insulin release in response to 14 mM glucose. On the other hand, 10–40 mM K+ or 20 mM L-arginine induced a rapid monophasic insulin output. In conclusion, L-leucine or alpha-KIC, which do not initiate insulin release alone and are not potentiated by 8-BrcAMP, may not be regarded as primary insulinotropic agents in the chicken. This result, together with the previously documented resistance of the chicken pancreas to glucose alone or to D-glyceraldehyde, strongly suggests that the mechanisms initiating insulin secretion are different in chickens and mammals, whereas potentiating mechanisms (low glucose concentration, arginine, acetylcholine, and cAMP) and membrane depolarization events (K+ and arginine) are present in both chickens and mammals.

Development of the biphasic response to glucose in fetal and neonatal rat pancreas

A study on the development of biphasic insulin release and sensitivity to inhibitors has been performed using perifused rat pancreas at 19.5 days of gestation (3 days before birth) and at 3 days after birth. In the fetal pancreas, 16.7 mM glucose caused a marked stimulation of insulin release that did not, however, manifest a biphasic response and was not inhibited by verapamil, a Ca2+ channel blocker. This suggested that the immature response was due to either a lack of voltage-dependent Ca2+ channels or their failure to open in response to glucose. Depolarizing concentrations of KCl stimulated insulin release, which was inhibited by verapamil, demonstrating that functional Ca2+ channels were present. In the presence of 16.7 mM glucose, quinine, which blocks glucose-sensitive k+ channels, potentiated the response of the fetal pancreas that now became sensitive to verapamil, demonstrating that functional K+ channels were also present in the fetal pancreatic beta-cell. The immaturity of the response is not due specifically to a defect in glucose metabolism; rather the metabolism of nutrient secretagogues fails to couple with the K+ channel in the fetal islet and thus fails to depolarize the beta-cell membrane. Three days after birth the pattern of response to high glucose is biphasic. Insulin release in fetal pancreas was inhibited by epinephrine and somatostatin.