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Amiloride derivatives enhance insulin release in pancreatic islets from diabetic mice
BMC Endocrine Disordersvolume 5, Article number: 9 (2005)
Amiloride derivatives, commonly used for their diuretic and antihypertensive properties, can also cause a sustained but reversible decrease of intracellular pH (pHi). Using dimethyl amiloride (DMA) on normal rodent pancreatic islets, we previously demonstrated the critical influence of islet pHi on insulin secretion. Nutrient-stimulated insulin secretion (NSIS) requires a specific pHi-range, and is dramatically enhanced by forced intracellular acidification with DMA. Furthermore, DMA can enable certain non-secretagogues to stimulate insulin secretion, and induce time-dependent potentiation (TDP) of insulin release in mouse islets where this function is normally absent. The present study was performed to determine whether pHi-manipulation could correct the secretory defect in islets isolated from mice with type 2 diabetes.
Using two mouse models of type 2 diabetes, we compared a) pHi-regulation, and b) NSIS with and without treatment with amiloride derivatives, in islets isolated from diabetic mice and wild type mice.
A majority of the islets from the diabetic mice showed a slightly elevated basal pHi and/or poor recovery from acid/base load. DMA treatment produced a significant increase of NSIS in islets from the diabetic models. DMA also enabled glucose to induce TDP in the islets from diabetic mice, albeit to a lesser degree than in normal islets.
Islets from diabetic mice show some mis-regulation of intracellular pH, and their secretory capacity is consistently enhanced by DMA/amiloride. Thus, amiloride derivatives show promise as potential therapeutic agents for type 2 diabetes.
As is widely known, nutrient-stimulated insulin secretory response in the pancreatic β cell consists of three distinct components. These include: a) an initial peak (first phase) triggered by Ca2+, b) augmentation of the Ca2+-triggered response (second phase), and c) a memory that persists after removal of the nutrient, causing enhancement of subsequent secretory responses (time-dependent potentiation) [1–4]. The first phase of the insulin response is initiated by ATP derived from glucose metabolism, which leads to membrane depolarization through closure of ATP-dependent K+ channels (KATP channels) and consequent entry of extracellular Ca2+ through voltage-gated Ca2+ channels. This influx of Ca2+ triggers the release of a small pool of secretory granules, thus producing the initial peak of the insulin response . This peak is followed by a sustained second phase of insulin release that lasts through the duration of exposure to the nutrient, mediated through augmentation of the Ca2+-triggered first-phase response . Time-dependent potentiation (TDP), a positive memory induced during this acute response, magnifies subsequent secretory responses to all secretagogues [2, 4–6].
Islet intracellular pH (pHi) exerts a strong influence on all aspects of the insulin secretory response, as well as other related functions such as Ca2+ influx and K+ retention [7–17]. These functions are enhanced by a decrease of pHi, and inhibited by an increase of pHi. Our recent work on isolated mouse islets has demonstrated that NSIS requires a specific pHi-range, and is greatly enhanced by decreasing islet pHi using DMA . The basal pHi in mouse islets ranges from 6.9 to 7.2. Treatment with DMA, an inhibitor of the Na+/H+ exchanger, brings islet pHi to the lower range of 6.4–6.8. This decrease of pHi in turn causes a dramatic enhancement in insulin secretion induced by metabolic secretagogues, and enables certain non-secretagogues to stimulate insulin release . Another remarkable effect of DMA is to enable glucose to induce TDP, a function normally absent in mouse islets . Hence, DMA has a consistently favorable effect on all aspects of the insulin response in isolated mouse islets. Previous studies have shown similarly positive effects of amiloride derivatives on insulin secretion in other species as well [8, 12–14, 16].
Thus, it is possible that: a) a mis-regulation of intracellular pH may contribute to the defect in insulin secretion found in type 2 diabetes; and b) Forcing the islet pHi to a lower range may correct/improve this secretory defect. To test these hypotheses, we monitored insulin secretion and islet pHi in two mouse models of type 2 diabetes. Regulation of islet pHi and the influence of amiloride derivatives on insulin secretion and TDP in isolated islets from diabetic mice were compared to similar parameters in islets from wild type mice.
Results from both models of type 2 diabetes demonstrate some abnormalities in pHi-regulation, and consistently favorable effects of DMA/amiloride on both direct insulin secretion and TDP in isolated islets. These results suggest the potential value of amiloride derivatives in treatment of type 2 diabetes
Mouse models of type 2 diabetes i.e. strains NON/LtJ (Diabetic NON mice; stock number 002423) and KK/Upj-A<y>/J (Diabetic KK mice; stock number 002468) were purchased from Jackson Laboratories, Bar Harbor, ME. C57BL6 mice (Harlan Sprague Dawley, Indianapolis, IN) were used as wild type controls. All animals were males, and the control mice were 7–9 weeks of age. The diabetic KK and NON mice were used at 9–20 weeks and 5–9 months, respectively, in order to allow time for NIDDM to develop. The body weight range was 20–30 g for the control mice, and 40–80 g for the diabetic mice. The animals were fed standard laboratory chow, and cared for according to the guidelines of the Vanderbilt Institutional Animal Care and Use Committee.
Pancreatic islets were isolated in Hanks Balanced Salt solution, and HEPES-buffered Krebs Ringer Bicarbonate solution (KRBH) was used for the static incubations in secretion experiments. The components of KRBH are as follows: 128.8 mM NaCl; 4.8 mM KCl; 1.2 mM KH2PO4; 1.2 mM MgSO4; 2.5 mM CaCl2; 5 mM NaHCO3 -; 10 mM HEPES; and 0.1% bovine serum albumin. The medium pH was maintained at 7.4. Basal KRBH used for pre-incubation and non-stimulated controls contained 2.8 mM glucose, while the stimulating media contained either 16.7 mM glucose, or other secretagogues as indicated in the presence of 2.8 mM glucose. The non-glucose secretagogues included 20 mM alpha-ketoisocaproate (αKIC), or 20 mM 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH). αKIC, a mitochondrially-metabolized secretagogue, is a metabolic product of the amino acid leucine. BCH is a non-metabolizable analog of leucine, and stimulates insulin secretion presumably through allosteric activation of mitochondrial dehydrogenases. For forced decrease of islet pHi, 40 μM DMA was added to the medium to produce intracellular acidification. In preparation for imaging experiments, islets were cultured in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 0.1 g/L streptomycin and 11 mM glucose.
Isolation of islets
A modified version of the collagenase digestion method described by Lacy and Kostianovsky  was used. Mice were terminally anesthetized with intra-peritoneal injection of Ketamine/Xylazine (80/20 mg/Kg). Pancreas was removed, placed in ice-cold Hanks solution and minced with scissors. Collagenase (3 mg/ml) was added and the mixture shaken in a 37°C water bath until the tissue was adequately digested. The mixture was then centrifuged, supernatant removed and the pellet re-suspended in Hanks solution. Centrifugation and re-suspension were repeated several times to remove exocrine tissue. The final pellet was re-suspended either in basal KRBH medium for secretion experiments or RPMI medium for islet culture. Islets were hand picked under a stereomicroscope.
Culture of islets
The method described by Arkhammar et al. 1998  was used with minor modifications [7, 20, 21]. 35 mm culture dishes with glass-bottomed wells (Mat-Tek corporation) were used. The dishes were pre-prepared by coating the wells with human extracellular matrix (BD Biosciences). Freshly isolated islets were placed carefully in each well, covered with RPMI medium containing 11 mM glucose, and cultured at 37°C in 95% O2 and 5% CO2. Under these conditions, the cells in the islet spread out within 14 days, greatly reducing the islet thickness and making it particularly suitable for imaging with confocal microscopy. Islets cultured under these conditions exhibit normal responses of Ca2+, NAD(P)H and insulin release to glucose stimulation, as described in previous studies [19–21] and confirmed in our preliminary experiments.
Intracellular pH measurements
As described in our previous studies , islet pHi was monitored by confocal microscopy, using carboxy-seminaphthorhodofluor-5 (SNARF5) , a pH-sensitive fluorescent indicator. Prior to imaging, cultured islets were maintained in RPMI medium containing 5 mM glucose for at least 48 hours. On the day of the experiment, RPMI medium was removed, and islets were washed and placed in basal KRBH medium. SNARF5-AM (5 μM final concentration) was added and incubated for one hour at 37°C. Loaded islets were placed on a warmed stage in a humidified and temperature-controlled chamber at 37°C, and monitored with a F-Fluar 40 × 1.3 NA oil immersion lens of a LSM510 confocal laser-scanning microscope (Zeiss). Islets were excited at 514 nm with an argon laser, and the emission fluorescence was collected in the band-widths 568–589 nm and 621–643 nm (peak emission at 580 and 630 nm) using the Meta detector (Zeiss). Time-series images (2.56 μs/pixel) were collected for 5–20 minutes as was suitable for each experiment. A stable baseline was obtained before the actual recording for each experimental condition started. LSM software was used to calculate the ratio between the two emission fluorescence values from selected well-loaded regions in each islet. This ratio was proportional to the islet pHi. The results were analyzed using LSM software, Graphpad Prism, and Microsoft Excel. One representative recording for each experimental condition is shown in the results section, and the value n denotes the number of recordings done with different islets for each condition. A standard curve was prepared by fixing the islet pHi at known values (ranging from 5.5 to 9, with 5–10 islets for each pH), using a KRBH medium containing 100 mM K+ and 20 mM nigericin to equilibrate the pH inside and outside cells.
All incubations were done in a 37°C water bath. Freshly isolated islets were pre-incubated for one hour in basal KRBH containing 2.8 mM glucose. Islets were then divided into groups and stimulated with different compounds as indicated in the results section, for one hour. Control group was maintained in basal glucose. Each group consisted of 4–5 tubes containing 4 islets per tube. At the end of the stimulation period, samples were collected for insulin measurement by radio-immuno-assay (performed by the DRTC Core facility at Vanderbilt University). Islet insulin content was measured after freezing islets overnight in 1% Triton-X. Insulin secretion was expressed as fractional release, i.e. the percentage of total insulin content released over the period of stimulation. In the experiments monitoring TDP, after pre-incubation islets were exposed to high (16.7 mM) glucose with or without intracellular acidification for 40 minutes, while the control group was exposed to basal glucose. Subsequently all groups were rested in basal glucose for 20 minutes, and stimulated with high glucose for 40 minutes prior to collection of samples for insulin assay. The value n denotes the number of times each experiment was repeated using islets from different mice.
In vivo treatment
Effect of oral treatment with amiloride derivatives on insulin secretion from isolated islets was monitored in diabetic NON mice. Mice were divided into three groups, i.e. DMA-treated; amiloride treated; and untreated control. DMA (estimated dose: 1 mg/Kg/day) or amiloride (estimated dose: 5 mg/Kg/day) was administered in drinking water to the test groups for one week. At the end of the week mice were euthanized and insulin secretion was measured from isolated islets as described earlier.
Values are expressed as mean ± SEM. Groups were compared using paired Student's t test. In secretion studies, n denotes the number of times each experiment was repeated with islets from different mice. In imaging experiments for pHi, n denotes the number of islets imaged for each condition.
Results and Discussion
The influence of islet pHi on insulin secretion has been reported in a number of studies [7–17]. Our recent work in isolated mouse islets has demonstrated the critical dependence of NSIS on an appropriate pHi, and the ability of pHi-lowering agents to dramatically enhance all components of the insulin response . Type 2 diabetes is characterized by a progressive decline in insulin secretion in the β cell in addition to peripheral insulin resistance. Since islet pHi plays a critical role in NSIS in normal islets, it is likely that a mis-regulation of pHi may contribute to this secretory defect, which could be corrected through manipulation of islet pHi. Thus, the present study was aimed at exploring the beneficial effects of pHi-lowering drugs on insulin release from isolated islets in type 2 diabetes.
We used two mouse models of type 2 diabetes obtained from Jackson laboratories: the KK/Upj-A<y>/J mice (Diabetic KK mice) and the NON/LtJ mice (Diabetic NON mice). Diabetic KK mice are a congenic strain developed as a model for obesity and type 2 diabetes. They develop obesity, hyperglycemia (300–500 mg/dl), insulin resistance and hyperinsulinemia (10 ng/ml) by 8 weeks of age . Diabetic NON mice are an inbred strain, originally developed as a control strain for the well-known IDDM model of NOD/LtJ. NON mice are genetically related closely to NOD mice but have a diabetes resistant MHC haplotype H2 nb 1(Kb, Anb1, Ek, Db), and are homozygous for the retinal degeneration allele Pde6b rd 1. NON mice have genes predisposing to type 2 diabetes, evidenced by early impaired glucose tolerance and development of maturity onset obesity and hypoinsulinemia. At 20 weeks of age NON males exceed 40 g in body weight, with blood glucose and insulin levels reported around 200 mg/dl and 1 ng/ml, respectively .
Basal intracellular pH in wild type (WT) mouse islets ranges from 6.9 to 7.2. The strong buffering mechanisms in the β cells ensure quick recovery from the pHi-changes induced by treatment with a weak acid or weak base (acid/base load) [7, 8], and Fig. 1A]. Approximately 70% of the islets isolated from diabetic mice showed abnormalities in their pHi-regulation. In diabetic KK mice, the basal islet pH was higher than that in WT mice (Fig. 1B). Islets isolated from both diabetic strains showed slower recovery from acid/base load, sometimes followed by overcompensation (Fig. 1:B&C). Thus, type 2 diabetes is associated with some mis-regulation of islet pHi. Regardless of whether these abnormalities play a role in insulin secretion, treatment with amiloride derivatives is likely to improve NSIS in diabetes.
DMA normally produces a dramatic increase in nutrient stimulated insulin secretion from isolated islets [7, 8]. In this study we monitored the effect of DMA on the insulin response to glucose and αKIC in islets isolated from diabetic mice. DMA consistently enhanced insulin secretion in response to both secretagogues (glucose and αKIC), in islets from both diabetic models as well as from WT mice (Fig. 2).
Since in-vitro treatment of isolated islets with DMA enhances NSIS, we next tested the corresponding effects of oral administration of DMA or amiloride to diabetic mice. Diabetic KK mice, being hyperinsulinemic, exhibited above-normal NSIS in the in-vitro experiments. Therefore, the in-vivo administration of amiloride was done only in the diabetic NON mice, which exhibited a significant impairment in NSIS. Diabetic NON mice were divided into three groups, i.e. DMA-treated; amiloride treated; and untreated control. DMA (1 mg/Kg/day) or amiloride (5 mg/Kg/day) was administered in drinking water to the test groups for one week prior to isolation of islets for insulin measurement. Mice were housed two to a cage and their behavior was monitored. All animals were observed to drink frequently, and drug dose was calculated using estimated water intake. Islets isolated from mice pre-treated with DMA or amiloride consistently exhibited a significantly stronger insulin response to all three secretagogues tested (16.7 mM glucose, 20 mM αKIC or BCH) (Fig. 3). Thus, oral treatment with low doses of DMA or amiloride enhances insulin release in islets from diabetic NON mice, with no conspicuous adverse effects on fluid or blood pressure regulation.
One problem we encountered with the diabetic NON mice was that the basal insulin release from isolated islets was high. Consequently the defect in NSIS was not obvious due to the elevated basal insulin release. However, the increase in insulin release produced by high glucose in NON mice (2 fold or less compared to basal glucose) is much smaller than that in the other two strains (10–12 fold compared to basal glucose), and non-glucose secretagogues such as αKIC and BCH do not stimulate above-basal insulin release in NON islets. Furthermore, the increased basal insulin release found in isolated islets may not directly translate into elevated plasma insulin, because the islet number per pancreas is diminished in NON mice, and the islets show abnormalities in size and shape as well. Thus, NON mice have a marked secretory defect, which is corrected to a significant degree by amiloride derivatives.
DMA treatment also unmasks TDP, a function normally absent in mouse islets . TDP is defined as an enhancement of the insulin secretory response in the β cell, induced by a previous exposure to glucose or certain other secretagogues. TDP can be measured by comparing the glucose-induced insulin response in islets previously exposed high glucose or basal glucose. As shown in figure 4, treatment with DMA enables glucose to induce TDP in islets from both diabetic strains, albeit to a lesser degree than in WT islets.
These results show that islets isolated from diabetic mice exhibit some abnormalities in their pHi-regulation, and that several aspects of their insulin response are significantly improved by amiloride derivatives. Thus, treatment with low doses of amiloride derivatives shows promise in the therapy of type 2 diabetes. The next step is to determine whether these drugs can normalize blood glucose and insulin levels. These in vivo studies will be conducted in a more suitable model of type 2 diabetes with a more pronounced secretory defect, with parallel in-vitro work on human islets. Currently we are looking into suitable rodent models of NIDDM, and one mouse model that shows promise is the strain GKlox/w + Rip-Cre developed by another group at Vanderbilt . The current study shows the potential value of amiloride in improving insulin release in diabetes, and recently-developed better amiloride analogs such as pyrazinoylguanidine [26, 27] may be good candidates for correcting the secretory defect in human diabetes.
This study demonstrates the presence of abnormalities in islet pHi-regulation in type 2 diabetes, and the ability of DMA and amiloride to significantly improve NSIS in isolated islets from diabetic mice. Thus, treatment with low doses of amiloride derivatives has potential therapeutic value for enhancing NSIS in human diabetes, and merits further investigation through in vivo studies.
nutrient-stimulated insulin secretion
- pHi :
HEPES-buffered Krebs Ringer Bicarbonate solution
carboxy-seminaphthorhodofluor-5 acetoxy methyl ester
standard error of mean
- Diabetic NON mice:
Major histocompatibility complex.
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The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/5/9/prepub
This work was supported by the NIH grants DK67821 (to SCG) and DK53434 (to DWP). Some assays and data analysis were performed in part through the use of the DRTC Hormone Core Resource and VUMC Cell Imaging Core Resource (supported by NIH grants CA68485, DK20593 and DK58404).
The author(s) declare that they have no competing interests.
SCG designed the study and carried out the insulin secretion experiments, in-vivo treatments and intracellular pH-measurements. WSH prepared the cultured islets used in pHi measurements. DWP participated in the design and coordination of the study.