Metabolic Regulation of Hormone Secretion in Beta-Cells and Alpha-Cells of Female Mice: Fundamental Differences.

It is unclear whether the secretion of glucagon is regulated by an alpha-cell-intrinsic mechanism and whether signal recognition by the mitochondrial metabolism plays a role in it. To measure changes of the cytosolic ATP/ADP ratio, single alpha-cells and beta-cells from NMRI mice were adenovirally transduced with the fluorescent indicator PercevalHR. The cytosolic Ca2+ concentration ([Ca2+]i) was measured by use of Fura2 and the mitochondrial membrane potential by use of TMRE. Perifused islets were used to measure the secretion of glucagon and insulin. At 5 mM glucose, the PercevalHR ratio in beta-cells was significantly lower than in alpha-cells. Lowering glucose to 1 mM decreased the ratio to 69% within 10 minutes in beta-cells, but only to 94% in alpha-cells. In this situation, 30 mM glucose, 10 mM alpha-ketoisocaproic acid, and 10 mM glutamine plus 10 mM BCH (a nonmetabolizable leucine analogue) markedly increased the PercevalHR ratio in beta-cells. In alpha-cells, only glucose was slightly effective. However, none of the nutrients increased the mitochondrial membrane potential in alpha-cells, whereas all did so in beta-cells. The kinetics of the PercevalHR increase were reflected by the kinetics of [Ca2+]i. increase in the beta-cells and insulin secretion. Glucagon secretion was markedly increased by washing out the nutrients with 1 mM glucose, but not by reducing glucose from 5 mM to 1 mM. This pattern was still recognizable when the insulin secretion was strongly inhibited by clonidine. It is concluded that mitochondrial energy metabolism is a signal generator in pancreatic beta-cells, but not in alpha-cells.

An Immersible Microgripper for Pancreatic Islet and Organoid Research.

To improve the predictive value of in vitro experimentation, the use of 3D cell culture models, or organoids, is becoming increasingly popular. However, the current equipment of life science laboratories has been developed to deal with cell monolayers or cell suspensions. To handle 3D cell aggregates and organoids in a well-controlled manner, without causing structural damage or disturbing the function of interest, new instrumentation is needed. In particular, the precise and stable positioning in a cell bath with flow rates sufficient to characterize the kinetic responses to physiological or pharmacological stimuli can be a demanding task. Here, we present data that demonstrate that microgrippers are well suited to this task. The current version is able to work in aqueous solutions and was shown to position isolated pancreatic islets and 3D aggregates of insulin-secreting MIN6-cells. A stable hold required a gripping force of less than 30 µN and did not affect the cellular integrity. It was maintained even with high flow rates of the bath perfusion, and it was precise enough to permit the simultaneous microfluorimetric measurements and membrane potential measurements of the single cells within the islet through the use of patch-clamp electrodes.

Glucagonotropic and Glucagonostatic Effects of KATP Channel Closure and Potassium Depolarization.

The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell-intrinsic signal recognition. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect.

Fresh and cultured mouse islets differ in their response to nutrient stimulation.

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs-Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.

A 3D microfluidic perfusion system made from glass for multiparametric analysis of stimulus-secretioncoupling in pancreatic islets.

Microfluidic perfusion systems (MPS) are well suited to perform multiparametric measurements with small amounts of tissue to function as an Organ on Chip device (OOC). Such microphysiolgical characterization is particularly valuable in research on the stimulus-secretion-coupling of pancreatic islets. Pancreatic islets are fully functional competent mini-organs, which serve as fuel sensors and transduce metabolic activity into rates of hormone secretion. To enable the simultaneous measurement of fluorescence and oxygen consumption we designed a microfluidic perfusion system from borosilicate glass by 3D femtosecond laser ablation. Retention of islets was accomplished by a plain well design. The characteristics of flow and shear force in the microchannels and wells were simulated and compared with the measured exchange of the perfusion media. Distribution of latex beads, MIN6 cell pseudo islets and isolated mouse islets in the MPS was characterized in dependence of flow rate and well depth. Overall, the observations suggested that a sufficient retention of the islets at low shear stress, together with sufficient exchange of test medium, was achieved at a well depth of 300 µm and perfusion rates between 40 and 240 µl/min. This enabled multiparametric measurement of oxygen consumption, NAD(P)H autofluorescence, cytosolic Ca2+ concentration, and insulin secretion by isolated mouse islets. After appropriate correction for different lag times, kinetics of these processes could be compared. Such measurements permit a more precise insight into metabolic changes underlying the regulation of insulin secretion. Thus, rapid prototyping using laser ablation enables flexible adaption of borosilicate MPS designs to different demands of biomedical research.

Acute metabolic amplification of insulin secretion in mouse islets: Role of cytosolic acetyl-CoA.

OBJECTIVE: Stimulation of the ß-cell metabolism by glucose and other fuels triggers insulin release by enhancing the mitochondrial ATP production and acutely amplifies the secretory response by increase in mitochondrial export of metabolites. We aimed to narrow down the uniform final reaction steps mediating fuel-induced acute amplification of insulin secretion. MATERIAL/METHODS: Insulin secretion and metabolic parameters were measured in isolated mouse islets exposed to the sulfonylurea glipizide in high concentration (closing all ATP-sensitive K(+) channels) during the entire experiment. Fuel-induced effects were examined after treating the islets for one hour with medium devoid of fuels. This experimental design prevented acute amplification, but only when glucose was the sole fuel. RESULTS: Strong amplification of insulin secretion by α-ketoisocaproate or glucose combined with α-ketoisovalerate (supplying mitochondrial oxaloacetate) was abolished within 14min after transition to medium devoid of fuels. After transition from medium containing glucose plus α-ketoisovalerate to medium containing solely glucose or α-ketoisovalerate, amplification (strong or weak, respectively) occurred until the end of the experiment. Glucose (alone or combined with α-ketoisovalerate) increased the total acetyl-CoA content as intensely as α-ketoisocaproate. Low concentrations of α-ketoisovalerate or α-ketoisocaproate were sufficient for saturation of acetyl-CoA increase, but caused no or only weak amplification, respectively. No acetyl-CoA increases occurred in the absence of glipizide. CONCLUSIONS: Glucose and other fuels regulate acute amplification of insulin secretion by controlling the supply of acetyl-CoA to the ß-cell cytosol. Cytosolic acetyl-CoA does not amplify by serving as substrate for syntheses of metabolic intermediates, but amplifies by acting as substrate for cytosolic protein acetylation.