Furthermore, according to another exemplary embodiment, the above-described ACBPS-D 202 can be applied towards pancreatic tissues that are to be used for transplantation. Generally, such a tissue is placed in a “protected site”, such as under the renal capsule. A problem that transplants surgeons commonly face is that of not knowing the viability of the cells, or how effectively they can produce insulin. However, biphasic current may be applied to the proposed tissue and the insulin produced thereby can be measured. Then, the cells may be stimulated to have a higher membrane potential which “up-regulates” insulin production and makes for a more active and better transplant. As one of ordinary skill would recognize, similar electrical current therapy may also be applied to virtually any endocrine tissue of the body.

Returning to the Figures, FIG. 3 provides a flow diagram of an exemplary method for applying electrical current therapy. While the method of FIG. 3 is directed to glucose sensing, it can be appreciated that, in view of FIG. 2A and FIG. 2B, glucose sensing and voltage sensing may occur concurrently or independently.

At step 335 of method 330, glucose in the blood of a patient can be sensed by a glucose sensor of the ACBPS-D. In an embodiment, the sensed glucose can be detected and quantified.

At step 340 of method 330, the quantified glucose data can be evaluated by a comparator to determine if the concentration of glucose in the blood of the patient is above a threshold at which insulin secretion would be required. If it is determined that the blood sugar level is too high at step 340, the ACBPS-D can deliver an electric current therapy (e.g., biphasic therapy) to the pancreas via electrodes at step 345 of method 330. If it is determined that the blood sugar level is below the threshold, the method 330 loops back to step 335 and glucose continues to be sensed.

In an embodiment, the comparator may evaluate, at step 340 of method 330, a current value of the quantified glucose data to a threshold to determine if an increase in insulin release, by electrical stimulation, is needed. Accordingly, the threshold may be a pre-determined blood glucose concentration threshold corresponding to a healthy blood glucose level in a patient. The healthy blood glucose level may be determined for each patient, individually, based on, among other things, age and life expectancy, conditions a person may have, cardiovascular disease or diabetes complications, hypoglycemia awareness, and duration of diabetes, if present. In an example, if the comparator determines, at step 340 of method 330, that the current value of the quantified glucose data is greater than the pre-determined blood glucose concentration threshold, then an electric current therapy may be generated and applied. For instance, the applied electric current therapy may be a pulsed therapy having a magnitude of 25 volts. In another instance, the applied electric current therapy may be a pulsed therapy having a variable magnitude between 10 volts and 25 volts, the variable magnitude being time dependent and based on a current deviation of the current value of the quantified glucose data and the pre-determined blood glucose concentration threshold.

The above described comparisons, performed by the comparator at step 340 of method 330, may be based on an analog signal corresponding to the current value of the quantified glucose data and an analog signal corresponding to the pre-determined blood glucose concentration threshold. To this end, it can be appreciated that processing circuitry of the ACBPS-D may be configured to evaluate a raw analog signal. Moreover, the processing circuitry of the ACBPS-D may be configured to convert each analog signal to standard units of glucose measurement according to a calibration curve, the converted signals then being compared to determine a deviation of the present glucose level.

As indicated, the electric current therapy applied to the pancreas at step 345 of method 330 may be based on a deviation of the current value of the quantified glucose data and the pre-determined blood glucose concentration threshold that is a target value for the patient. In example, if the deviation, represented by an error value, is approximately one standard deviation from the target value, an applied electric current therapy may have a magnitude of approximately one. Further, if the deviation is approximately two standard deviations from the target value, the applied electric current therapy may have a magnitude of approximately two. Of course, the above examples can be appreciated as merely exemplary of a system for graded responses to the comparison of the current blood glucose value and the target value. For instance, percentages, scaling, or any other suitable system for evaluating blood glucose levels and generating an appropriate electrical response may be considered without straying from the spirit of the present disclosure.

According to an embodiment, following application of electrical stimulation via the electrodes at step 345 of method 330, the method 330 returns to step 335 and glucose continues to be sensed until the comparator determines that the blood sugar level is equal to or minimally-below the pre-determined blood glucose concentration threshold.

For the sake of simplicity, the above discussion is presented as a sequence of steps that are performed serially. However, one of ordinary skill in the art would recognize that the steps of FIG. 3 may be performed in parallel such that glucose sensing at step 335 and electrical current therapy at step 345 may occur simultaneously. Other orders of performing these steps are also possible without departing from the scope of the present disclosure.

Non-Limiting Experimental Results

The effects of electrical stimulation on insulin release from rat insulinoma (INS-1) cells were evaluated. In brief, the anodal/cathodal biphasic stimulation (ACBPS) electrical waveform resulted in a voltage- and stimulation duration-dependent increase in insulin release. ACBPS elicited insulin release both in the presence and absence of glucose. Basal and BPS-induced insulin secretion can be inhibited by mitochondrial poisons and calcium channel blockers, indicating insulin release was dependent on ATP and the influx of calcium. ACBPS parameters that released insulin caused no detectable plasma membrane damage or cytotoxicity although temporary morphological changes could be observed immediately after ACBPS. ACBPS did not alter the plasma membrane transmembrane potential, but did cause pronounced uptake of MitoTracker Red into the mitochondrial membrane, indicating an increased mitochondrial membrane potential. The ATP:ADP ratio following ACBPS did not change but guanosine triphosphate (GTP) levels increased and increased GTP levels have previously been associated with insulin release in INS-1 cells. These results provide evidence that, in the case of INS-1 cells, ACBPS promotes insulin release without causing cytotoxicity.

As background, some evidence suggests that the ACBPS waveform increases morbidity and mortality in a chronically-paced patient. In practice, electrical stimuli can be delivered as either monophasic or biphasic waveforms, and with anodal as well as cathodal polarities. In cardiac tissues and whole heart acute studies, ACBPS increases the speed of conduction and contractility. In addition, ACBPS may also promote healing of infarcts as judged by improved chronic hemodynamics.

In isolated cell studies, biphasic stimulation may be more advantageous than monophasic stimulation since it generates lower levels of electrolysis products at the electrodes and thus can be applied for longer time periods and at higher voltages. Stimulation with biphasic waveforms in which the initial phase is anodal (ACBPS) stimulate only at the end of the initial anodal pulse, coincident with the start of the cathodal phase. In cellular studies, ACBPS stimulation has been shown to elicit effects that differ from those induced by monophasic pulses.

Pancreatic beta cells are electrically-active and have similar ion channels to those found on cardiac myocytes but lack contractile elements. Pancreatic beta cells are thus an alternative cell model to investigate the effects of electrical stimulation on plasma membrane-dependent activities. These cells have been used extensively to study the glucose-stimulated insulin secretion (GSIS) pathway, which is regulated, at least in part, by plasma membrane voltage-dependent ion channels. The canonical GSIS pathway involves the uptake of glucose and consequential production of ATP. An increase in intracellular ATP results in the closure of ATP-sensitive potassium channels in the plasma membrane, leading to membrane depolarization and activation of voltage-sensitive calcium channels. The influx of calcium into the cell promotes the fusion of insulin-containing vesicles with the plasma membrane and, thereby, the subsequent release of insulin. A role for ion channels in insulin secretion from beta cells, therefore, is well established. However, ATP-independent mechanisms of GSIS have also been identified and mitochondrial GTP has been characterized as a critical signal for insulin secretion.

It is thought that electrical currents, including ACBPS, have the potential to alter the electronic flux of the plasma membrane of pancreatic beta cells and modulate insulin release either by promoting or inhibiting the actions of plasma membrane ion channels. The results described below explore the manner in which ACBPS can induce insulin secretion from rat insulinoma cells.

Materials and Methods

Cells

A rat insulinoma cell line INS-1 was obtained and were grown in 6-well polystyrene culture plates as attached monolayers in RPMI1640 culture medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 100 IU/mL penicillin and 100 μg/mL streptomycin (i.e., complete medium). Cells were maintained and experiments were conducted in a humidified atmosphere of 5% CO₂in air at 37° C. Select studies were carried out in glucose-free RPMI1640 culture medium supplemented with reagents as described above. In these experiments, the regular medium was exchanged for glucose-free medium 16 hours prior to the electrical stimulation experiment.

Reagents

Antimycin A, oligomycin A, verapamil, nifedipine, valinomycin and gramicidin A were obtained from Santa Cruz Biotechnology and dissolved in dimethyl sulfoxide (DMSO). Mitotracker Red CMXRos and DiBac₄(3) were obtained from ThermoFisher Scientific, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Research Products International.

Electrical Stimulation

ACBPS was applied to cells using a C-Pace EP cell culture stimulator in combination with a C-dish electrode array for a 6-well plate. Control cells were mock stimulated using an identical 6-well C-dish electrode array that was not connected to the stimulator. Unless otherwise stated, the biphasic stimulation parameters were anodal followed by cathodal at 10 Hz (frequency), 5 milliseconds (ms) (total pulse duration), and 25 V (pulse magnitude).

For instance, as it relates to the insulin release study of FIG. 4A, ACBPS may be applied at variable voltages of between 10 V and 25 V (pulse magnitude). And, as it relates to the release study of FIG. 4B, ACBPS may be applied at 25 V (pulse magnitude) and in 5 ms pulse durations over a period of between 15 minutes and 30 minutes, a control being maintained for a period of 30 minutes without electrical stimulation in the form of ACBPS to evaluate a baseline of insulin release.

Insulin Release

Insulin release from INS-1 cells into the culture medium was determined by ELISA (Rat Insulin ELISA Kit) per the manufacturer's instructions. For these studies, cells were grown to approximately 95% confluency in 6-well plates containing 3 mL complete medium/well. 16 hours before stimulation, the medium was replaced with 3 mL fresh complete medium. Immediately prior to stimulation, the medium was aspirated, the cells were washed twice with 2 mL phosphate buffered saline (PBS), and then 3 mL fresh complete medium was added to each well. Cells were subjected to ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration (i.e. anodal duration/cathodal duration), 10-25 V magnitude) or mock stimulation for the indicated times. At the end of the stimulation periods, a 0.5 mL sample of medium was removed from each well and stored frozen at −70° C. for later analysis. In some studies, cells were grown for 16 hours in glucose-free medium prior to stimulation. To examine a role for electrolysis products in promoting insulin release, an additional series of experiments were conducted that involved the application of ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) or no stimulation (mock) for 30 min to 6-well plates containing only complete medium (3 mL per well) (i.e., no cells). At the end of the stimulation period, the “pre-stimulated” medium was transferred to cells that had been washed twice with PBS (as above). The cells were allowed to incubate with the pre-stimulated medium for 30 min, after which a 0.5 mL sample of medium was removed from each well and stored frozen at −70° C. for latter analysis. The insulin concentration of the culture medium was measured by colorimetric ELISA using 2 μL of the culture medium as described by the manufacturer. Culture medium samples and insulin standards were allowed to bind for 16 hours at 4° C. The concentration of insulin in each medium sample was estimated by interpolation on a standard curve (0.156-10 ng/mL) obtained using purified recombinant rat insulin provided in the ELISA kit.

Insulin release from INS-1 cells was also confirmed by immunostaining for intracellular insulin. In these studies, INS-1 cells were grown to 95% confluency on 18 mm glass coverslips. The coverslips were washed twice with 2 mL PBS, then placed in the well of a 6-well plate containing 3 mL per well complete medium and subjected to mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. Immediately following the stimulation period, the medium was aspirated, the coverslips rinsed twice with 2 mL of PBS, and then incubated in 2 mL freshly-prepared 3.7% (v/v) formaldehyde in PBS for 15 min at 25° C. (to fix the cells). The cells were permeabilized by incubation of the coverslips in 2 mL 0.1% (v/v) Triton X100 in PBS for 10 min at 25° C., and subsequently incubated in 2 mL blocking buffer (RPMI1640 containing 10% fetal bovine serum diluted 1:1 with 10 mM Tris-HCl, pH 7.8, 150 mM NaCl and 0.2% Tween-20 (TBST)). Rabbit anti-insulin antibody (Abcam, #ab63820), diluted 1:1000 in 2 mL blocking buffer, was added to each coverslip and allowed to incubate for 16 hours at 4° C. The coverslips were rinsed extensively in TBST prior to incubation with Alexa Fluor-488-conjugated goat anti-rabbit IgG secondary antibody diluted 1:1000 in 2 mL blocking buffer containing 1 μg/mL DAPI (MilliporeSigma, Burlington, MA) for 30 min at 25° C. In some experiments, phalloidin (1 μg/mL, phalloidin CruzFluor 555 conjugate) was added to blocking buffer containing secondary antibodies and DAPI. Following the addition of the secondary antibody, the cells were rinsed extensively with TBST, dipped in deionized-distilled H₂O, inverted, and then mounted on a glass slide. Cells were visualized at 600× magnification using a Nikon C-1 confocal microscope.

Cytotoxicity

Plasma membrane integrity and cytotoxicity were assessed in INS-1 cells using Trypan blue staining. Cells were grown to 95% confluency in 6-well plates and then underwent mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. Immediately following the stimulation period, the medium was aspirated and 1 mL trypsin solution (0.25% trypsin; 0.1% EDTA) was added to each well. After 30 s, 2 mL complete medium was added to each well, the cells were dislodged, transferred to a 15 mL Falcon tube and collected by centrifugation (800 rpm for 4 min). The cell pellet was suspended in 1 mL Hank's balanced salt solution (containing Ca⁺⁺ and Mg⁺⁺, Invitrogen), gently vortexed, and a 50 μL aliquot was transferred to a 1.7 mL microfuge tube containing 50 μL 0.4% Trypan blue solution. The cells were then gently mixed and 10 μL of the cell suspension was applied to a hemocytometer. One hundred cells were counted and the percentage of Trypan blue-positive cells was determined. In addition, Trypan blue staining was conducted 4 hours after the end of the stimulation period. In these experiments, cells underwent mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. At the end of the stimulation period the medium was replaced with 3 mL per well of fresh complete medium, and the cells were placed in a cell incubator for 4 hours. The cells were collected, stained with Trypan blue and analyzed as described above.

Cytotoxicity was also examined using the intracellular reduction of MTT. INS-1 cells were grown to 95% confluency in 6-well plates, and then underwent mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 mc) pulse duration, 25 V magnitude) for 30 min. Immediately following the stimulation period, the medium in each well was replaced by 3 mL fresh complete medium containing MTT (1 mg/mL) for 15 min. The MTT-containing medium was then aspirated and the cells were solubilized by the addition of DMSO (1 mL per well). After agitation of the 6-well plates on an orbital shaker for 10 min, 100 μL aliquots from each well were transferred to a 96-well plate and the absorbance was determined in triplicate at a wavelength of 550 nm using a Thermo Max microplate reader.

Transmembrane Potential Assay

The transmembrane potential (TMP) was estimated using DiBAC₄(3) staining as previously described. For these studies, INS-1 cells were grown to 95% confluency in 6-well plates. The medium was aspirated, replaced with 3 mL fresh complete medium per well, and subjected to mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. Immediately following the stimulation period, the medium was aspirated from each well and replaced with 2 mL per well staining buffer (20 mM HEPES, 120 mM NaCl, 2 mM KCl, 2 mM CaCl₂), 1 mM MgCl₂, and 5 mM glucose [pH 7.4]) containing 5 μM DiBAC₄(3) with DMSO (mock and ACBPS), valinomycin (100 nM), gramicidin (100 nM) or KCl (140 mM). The cells were allowed to incubate in staining buffer at 37° C. for 30 min. Thereafter, the fluorescence intensity of DiBAC₄(3) stained INS-1 cells (Ex 490 nm/Em 516 nm) was measured using a BioTek Synergy H1 plate reader programmed to acquire 49 (7×7) individual measurements per well.

MitoTracker Red Staining

The effect of ACBPS stimulation on the inner mitochondrial membrane potential in cells was examined using MitoTracker Red staining. For these studies, cells were grown to 95% confluency on 18 mm glass coverslips, then placed in a 6-well dish containing 3 mL complete medium and subjected to mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. At the indicated times after the stimulation period, the medium was aspirated, and 100 nM MitoTracker Red CMXRos (diluted in 3 mL complete medium) was added to each coverslip and incubated for 15 min at 37° C. The coverslips were then rinsed twice with 2 mL PBS, and subsequently fixed by incubation with 2 mL freshly-prepared 3.7% (v/v) formaldehyde in PBS for 15 min at 25° C. The coverslips were washed twice in 2 ml PBS and the cells were then permeabilized by incubation with 2 mL 0.1% (v/v) Triton X100 in PBS for 10 min at 25° C. The coverslips were then rinsed twice with 2 mL PBS, and subsequently incubated with 2 mL fresh PBS containing 1 μg/mL DAPI for 5 min at 25° C. The coverslips were washed twice in 2 mL PBS, rinsed once in deionized-distilled H₂O, and mounted on a glass slide. For studies combining MitoTracker Red staining and insulin immunostaining, cells were fixed, permeabilized, blocked and immunostained for insulin (as described above) after incubation with MitoTracker Red. Cells were visualized at 600× magnification using a Nikon C-1 confocal microscope.

Cellular Respiration

Oxygen consumption was measured in INS-1 cells after ACBPS. Cells, grown to 95% confluency in 6-well plates, underwent mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. Immediately following the stimulation period, the medium was aspirated and replaced with 3 mL fresh complete medium. At the indicated times after stimulation, the cells were gently dislodged, transferred to a 15 mL Falcon tube, and pelleted at 800 rpm for 4 min. The cell pellet was suspended in 12 mL fresh complete medium at 37° C., cell number was determined using a hemocytometer, and 3 mL aliquots were assayed for oxygen consumption using a YSI Biological Oxygen Monitor. Cellular oxygen uptake was measured over a 15 min period and the rate of oxygen consumption was calculated from 5 min to 15 min. Calculations of dissolved oxygen were corrected for temperature (37° C.) and pressure (29.9 mmHg).

Intracellular Nucleotides

Nucleotide concentrations in INS-1 cells were determined by LC-MS analysis. Cells, grown to 95% confluency in 6-well plates (in duplicate), underwent mock stimulation or ACBPS (10 Hz, (2.5 ms)/(2.5 ms) pulse duration, 25 V magnitude) for 30 min. Immediately following the stimulation period, the medium was aspirated, the cells washed twice with PBS, and subsequently lysed with 500 μL ice-cold 70% methanol in water containing 20 μL internal standard and vortexed for 10 sec. The resulting extract was centrifuged at 8000×g for 5 min at 4° C. The resulting supernatant was transferred to a new centrifuge tube and stored on ice. The remaining pellet was suspended in 500 μL ice-cold methanol by vortexing for 10 sec. The suspension was then centrifuged at 8000×g for 5 min at 4° C. The supernatant was removed and combined with the 70% methanol supernatant which was then centrifuged at 18,000×g for 15 min. The resulting supernatant was transferred to a new tube and dried in a vacuum centrifuge at 55° C. Each dried sample was reconstituted in 100 μL 50% methanol in water and centrifuged at 18,000×g for 10 min at 4° C. Samples were separated high performance liquid chromatography (HPLC) using a 100×2 mm 5 μm Luna NH₂column operated in HILIC mode. Mass spectrometric analysis was performed on a triple quadrupole mass spectrometer in positive ionization mode. The drying gas was 300° C. at a flow rate of 12 mL per min. The nebulizer pressure was 30 psi. The capillary voltage was 4000 V. Results were quantified using calibration curves to obtain the on-column concentration, followed by normalization of the results to protein concentration. Protein concentrations were determined using cell homogenates obtained from duplicate plates of cells treated with Mock stimulation or ACBPS.

Results

As shown in FIG. 4A in FIG. 4B, ACBPS of INS-1 cells resulted in an increase in insulin release into the culture medium that was pulse amplitude (FIG. 4A) and stimulation period-dependent (FIG. 4B).

With reference to FIG. 5A and FIG. 5B, basal insulin release was inhibited by the mitochondrial poisons antimycin A and oligomycin A (FIG. 5A) and by the calcium channel blocker verapamil (FIG. 5B). ACBPS-induced insulin release was also inhibited by the mitochondrial poisons antimycin A or oligomycin A, as shown in FIG. 5A, and by both calcium channel blockers, verapamil and nifedipine, as shown in FIG. 5B.

With reference to FIG. 6A and FIG. 6B, basal insulin release from INS-1 cells was reduced when cells were grown and mock stimulated in glucose-free medium (e.g., compare mock of FIG. 6A with mock of FIG. 6B). Under similar glucose-free conditions, ACBPS continued to stimulate insulin release, as shown in FIG. 6A. No increase in insulin release was observed when INS-1 cells were exposed to culture medium previously subjected to ACBPS, as shown in FIG. 6B, indicating that the insulin release observed during ACBPS does not result from electrolysis products generated from the tissue culture medium.

With reference to FIG. 6C and FIG. 6D, and in view of FIG. 5A and FIG. 5B, data indicating the requirement of mitochondrial activity and calcium channels for insulin release clearly demonstrated that ACBPS was not causing insulin release as a result of toxicity. We confirmed that ACBPS did not increase Trypan blue uptake by INS-1 cells measured immediately or 4 hours after the stimulation period, as shown in FIG. 6C. Similarly, ACBPS did not affect MTT-formazan production measured immediately following the stimulation period, as shown in FIG. 6D. While ACBPS did not cause plasma membrane damage (see Trypan blue assay) or cell loss (see MTT assay), a temporary change in cell morphology was observed immediately after ACBPS. Specifically, INS-1 cells subjected to ACBPS appeared to temporarily lose contact with their neighboring cells but did not detach from the coverslip. This change in cell-to-cell contact induced by ACBPS was confirmed by immunocytochemical studies which showed the phenomenon occurring immediately after the stimulation period (To) and recovering 24 hours (T₂₄) after stimulation (FIG. 4). In these studies, INS-1 cells were also examined for insulin content by immunostaining and these data, shown in FIG. 7A and FIG. 7B, confirmed the ELISA results in showing a reduced level of intracellular insulin staining immediately following ACBPS and recovery of insulin levels 24 hours, thereafter, relative to cells that underwent mock stimulation.

To determine if the effects of ACBPS were mediated by changes in the plasma membrane potential, DiBAC₄(3) dye uptake and fluorescence was measured in INS-1 cells subjected to ACBPS. This anionic dye accumulates in the plasma membrane of depolarized cells but is excluded from the plasma membrane of hyperpolarized cells. As shown in FIG. 8A, cells exposed to ACBPS did not show any increase in DiBAC₄(3) fluorescence relative to mock stimulation cells. To validate the utility of DiBAC₄(3) as a tool to estimate changes in plasma membrane potential, control experiments were also conducted in INS-1 cells treated with valinomycin (control for plasma membrane hyperpolarization), or with gramicidin or KCl (positive controls for plasma membrane depolarization). As expected, with continued reference to FIG. 8A, valinomycin induced a decrease in DiBAC₄(3) fluorescence while gramicidin and KCl elicited increased DiBAC₄(3) fluorescence.

Because mitochondria play a central role in regulating insulin release from pancreatic beta cells, ACBPS-induced changes in the mitochondrial inner membrane potential of INS-1 cells were examined using MitoTracker Red staining. In these studies, shown in FIG. 8B and FIG. 9A, ACBPS induced a dramatic increase in the intensity of MitoTracker Red staining immediately following the stimulation period. The increase in MitoTracker Red staining of INS-1 cells was long lived, as shown in FIG. 9A, with increased staining being observed 4 hours after the stimulation period. MitoTracker Red staining returned to mock stimulation levels by 24 hours. In these same studies the levels of intracellular insulin decreased immediately following ACBPS, were still depressed at 4 hours, but returned to normal after 24 hours post stimulation.

To further examine the effect of ACBPS on mitochondrial function in INS-1 cells, cellular respiration was measured, as shown in FIG. 9B. In these experiments, cells subjected to ACBPS demonstrated a decrease in cellular respiration and, similar to MitoTracker Red staining, the decrease in oxygen consumption was detectable immediately after ACBPS, returning to mock stimulation levels by 24 hours after ACBPS.

Given the importance of cellular nucleotides in controlling insulin secretion from beta cells, the impact of ACBPS on intracellular levels of critical nucleotides (including ATP and GTP) were analyzed in FIG. 9C. ACBPS did not alter ATP but did significantly increase cellular GTP levels. Stimulation also increased the intracellular levels of CTP and NAD (P)⁺. ACBPS did not change (α=0.05) intracellular levels of adenosine, AMP, cytidine, CMP, CDP, guanosine, GMP, GDP, uridine, UMP, UDP, UTP, FAD, NADH or NAD⁺.

Analysis

In these studies, ACBPS was shown to increase the rate of insulin release from INS-1 cells in a manner that was dependent on the pulse magnitude and time period of stimulation. ACBPS-induced insulin release occurred in the absence of glucose, excluding the possibility that the secretion of insulin involved the increased cellular uptake of glucose. The amounts of insulin released by INS-1 cells during ACBPS were similar to those shown to be induced by glucose, the physiological stimulus for insulin secretion. Both basal and ACBPS-induced insulin release were inhibited by antimycin A, oligomycin A, nifedipine and verapamil, indicating roles for mitochondria and calcium channels in the release process. These results argue against the possibility that ACBPS was eliciting insulin release by non-specific damage to the plasma membrane of the cell. The Trypan blue and MTT assay results also demonstrate that ACBPS did not compromise the plasma membrane or induce adverse cytotoxicity to INS-1 cells.

As described, insulin release from beta cells is dependent upon plasma membrane depolarization and the opening of voltage-dependent calcium channels. To further examine the mechanism by which ACBPS promoted insulin release, the capacity of ACBPS to alter the INS-1 transmembrane potential was examined using the slow-responsive dye DiBAC