Abstract
In this study, we investigated the use of a novel oxygen biosensor system to detect changes in oxygen consumption rates (OCRs) by islets in response to glucose. Islets from non-human primate and human pancreata were seeded into an oxygen biosensor system microplate and exposed to basal (2.8 or 5.6 mM) or high (16.7 or 33.3 mM) glucose over either a long-term or a short-term culture. Our data clearly demonstrated that non-human primate islets cultured in high glucose conditions exhibited significant increases in OCRs over a 168 h extended culture period (P<0.05), which indicates an accelerated rate of β-cell metabolism triggered by glucose over time. Significant increases in OCRs (P<0.01) were also attained in both non-human primate and human islets exposed to high glucose conditions in a 120 min short-term incubation period. OCRs exhibited by human islets exposed to different glucose concentrations correlated with insulin secretion (r2=0.7681, P<0.01). Moreover, the OCR stimulation index (i.e. OCR at high glucose/OCR at basal glucose) was significantly greater in human islets displaying high viabilities as opposed to islets exhibiting low viabilities (P<0.05). Together these data demonstrate that this novel oxygen biosensor system documents significant increases in islet oxygen consumption upon acute and chronic exposure to high glucose concentrations. Importantly, this methodology rapidly and robustly detects changes in OCRs by islets in response to high glucose stimulation that correlate well with the metabolic activities and functional viability of islets and clearly delineates significant differences in OCR stimulation index between high and low viability human islets, and therefore may prove to be an effective approach for quickly assessing the functional viability of islets prior to transplantation.
Introduction
Islet transplantation can result in insulin independence in patients diagnosed with type 1 diabetes (Shapiro et al. 2003). To achieve a successful islet transplant, islets are first isolated from an organ donor pancreas, followed by infusion of purified functional islets, in suffcient quantities, into the hepatic portal vein of an immunosuppressed transplant recipient (Shapiro et al. 2000).
Although islet isolation procedures generate adequate yields of relatively good quality islets with the ability to reverse type 1 diabetes, there is presently no pre-release potency assay available that unequivocally predicts whether a given islet product will result in a successful transplant. At present, the quality control testing that is routinely performed on the final product prior to an islet transplant includes islet count, viability, purity and islet morphology, none of which is predictive of islet function. Test results that come the closest to measuring the function of islets are only available after a clinical islet transplant is completed. These tests include the glucose-stimulated insulin secretion assay (Ashcroft et al. 1971, Andersson et al. 1976, Gray et al. 1984, de Haan et al. 2004) and the transplantation of human islets into diabetic and non-diabetic animal models (Lake et al. 1988, Gaber et al. 2004).
The importance of establishing a potency assay cannot be overemphasized. The availability of an assay to objectively determine the effectiveness of a product prior to an islet infusion minimizes the possibility of a patient undergoing unnecessary procedures and increases the likelihood of a successful transplant. Consequently, an alternative assay or assays are needed, which are simple, fast and capable of rapidly and robustly assessing the overall functional viability of an islet preparation before transplantation.
It is well known that glucose-stimulated insulin secretion of β-cells is of vital importance in maintaining glucose homeostasis. Signals that stimulate insulin release are derived from the intracellular metabolism of glucose, rather than from a ligand–receptor interaction (Malaisse et al. 1979). This process triggers an acceleration of β-cell metabolism (Newgard & McGarry 1995, Rasmussen et al. 1995, Matschinsky 1996, Prentki 1996), that ultimately leads to insulin exocytosis (Malaisse et al. 1979, 1983, Hutton & Malaisse 1980, Longo et al. 1991, Newgard & McGarry 1995, Prentki et al. 1997, Rorsman 1997). Increases in oxygen consumption upon glucose stimulation in single islets are reported (Jung et al. 1999, 2000), which provides direct evidence for an accelerated rate of β-cell metabolism that accompanies increases in insulin secretion (Ortsater et al. 2000). Together these observations suggest that detection of the islet oxygen consumption rate (OCR) in response to glucose may provide an in vitro means to rapidly and robustly assess the functional viability of an islet preparation prior to clinical transplantation.
Cellular oxygen consumption is conventionally detected by an oxygraph method, which consists of a Clark-type oxygen electrode fitted to an airtight chamber to monitor cellular respiration (Backman & Wadso 1991). Despite widespread use, this approach has certain disadvantages such as drift of calibration, invasiveness, problems with sterilization, and cost. The BD Biosciences Oxygen Biosensor System (BD OBS) (BD Biosciences, Bedford, MA, USA) is an alternative innovative system that is coupled to a new cell culture technology that enables repeated and non-invasive monitoring of OCRs by cells. In the present study, we investigated the use of the BD OBS to detect changes in OCRs by islets in response to different glucose concentrations over short- and long-term culture periods. The results of these experiments are encouraging and suggest that this methodology may prove to be a fast an effective way to quickly assess the functional viability of islets prior to clinical transplantation.
Research Design and Methods
Non-human primate and human islet isolation
Non-human primate pancreata were obtained from the Washington National Primate Research Center under an approved protocol in accordance with the policies of the Institutional Animal Care and Use Committee at the University of Washington.
Non-human primate islets were isolated from Macaca nemestrina by intra-ductal infusion of 1.5 mg/ml collagenase P (Boehringer Mannheim GmbH, Mannheim, Germany) containing 10 U/ml DNase I (Sigma-Aldrich, Saint Louis, MO, USA), followed by a modified two-step digestion procedure (Une et al. 1997, Kenmochi et al. 2000). Islets were purified using a continuous density gradient of Biocoll Separating Solution (Biochrom KG, Berlin, Germany).
Human pancreata were obtained from LifeCenter Northwest and Tissue Transformation Technology. Organs were procured from brain-dead organ donors after obtaining informed consent from the family members of the donors. Human islets were isolated using Liberase-HI enzyme (Roche Applied Science, Indianapolis, IN, USA) by techniques previously described (Ricordi et al. 1988, Shapiro et al. 2000). Islets from non-human primate and human pancreata were cultured in CMRL 1066 (Mediatech Inc., Herndon, VA, USA) containing basal (5.6 mM) glucose concentration, supplemented with 0.625% human albumin (Baxter, Glendale, CA, USA) and 1% antibiotic–antimycotic solution (i.e. 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B) (Mediatech) overnight at 37 °C in a humidified incubator at 5% CO2.
Islet quality control assessments
After culture, islets were harvested and evaluated for total islet equivalents (IEs), purity and viability. Islets were enumerated by staining with dithizone (DTZ) (Sigma-Aldrich) according to a previously described procedure (Ricordi 1991). A graduated ocular was used to count and categorize each islet according to size. Total IEs were calculated by normalizing the data such that the average size of each islet equaled 150 μm. Islet purity was estimated as the ratio of DTZ-stained islets vs acinar tissue. Islet viability was assessed fluorometrically by examining the proportion of islets stained with fluorescein diacetate (Molecular Probes, Inc., Eugene, OR, USA) and propidium iodide (Molecular Probes).
Detection of oxygen consumption
The BD OBS (BD Biosciences) is a microplate-based culture and homogeneous assay platform, which allows kinetic monitoring of dissolved oxygen concentrations at the well bottom of an OBS microplate. This is possible due to the presence of an oxygen-sensitive fluorescent dye, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) chloride, embedded in a gas-permeable silicone polymer matrix affixed to each well bottom of a standard Falcon microplate (Fig. 1). The oxygen-sensitive fluorophore in the BD OBS is quenched by molecular oxygen in a predictable fashion. When monitored on a standard fluorescence microplate reader, the intensity and lifetime of the emitted fluorescence varied inversely with oxygen concentration, in a manner consistent with quenching mechanisms described by the Stern–Volmer theory. Thus, oxygen is depleted and fluorescence intensity increases when cells grow or survive, whereas fluorescence intensity decreases when islet cells die. Under static culture conditions, applying the laws of Fickian diffusion to the system allows equilibrium oxygen concentration to be converted to an equilibrium OCR.
Non-human primate or human islets were seeded into the wells of a BD OBS microplate and cultured in CMRL 1066 culture medium supplemented with different concentrations of glucose. The OBS microplate was read on the fluorescence plate reader using a bottom-reading configuration by an Flx 800 Microplate Fluorescence Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 485 nm excitation and 620 nm emission. For a 168 h extended culture period, an OBS microplate seeded with islets was maintained at 37 °C in a humidified incubator with 5% CO2 and fluorescence intensity was read at 24, 48, 72, 96, 120, 144 and 168 h. For a 120 min short-term incubation, an OBS microplate seeded with islets was incubated at room temperature and fluorescence intensity was kinetically read at 30, 60, 90 and 120 min.
Fluorescence data collection and normalization and calculation of oxygen concentration and OCR
Fluorescence data were collected at selected time points. Since fluorescence intensity measurements can vary with the concentration of fluorophore in each well and machine drift, fluorescence data were normalized using a two-step normalization protocol (Guarino et al. 2004). Briefly, each well’s fluorescence value was first normalized by the value in the same well’s pre-blank reading and then against the value of no-cell negative controls for the corresponding time point.
The normalized relative fluorescence (NRF) value was used to calculate equilibrium oxygen concentration at the well bottom, [O2]=(DR/NRF−1)/Ksv. Dynamic range (DR) is a characteristic property of the sensor and is constant for a given set of experimental conditions (temperature, wavelengths, etc.). DR is determined using 100 mM sodium sulfite (Sigma-Aldrich) as a positive control. The maximum possible fluorescence intensity occurs at zero oxygen. The sodium sulfite depletes oxygen at the well bottom to zero, DR is expressed as the ratio between the maximum fluorescence intensity and the fluorescence intensity at ambient conditions for a given well. Ksv is a function of the fluorescence plate reader and is determined from DR, which is expressed as Ksv= (DR − 1)/[O2]a. Governed by Henry’s law, assuming that the medium and water have the same oxygen diffusion properties and the atmosphere is standard air, the oxygen concentration at the surface, i.e. [O2]a is the ambient oxygen concentration in the medium at equilibrium, which is 257 μM at 25 °C under normal atmospheric condition and 195 μM at 37° in 5% CO2 environment.
The Fickian diffusion theory has been used to model the oxygen gradient and pericellular oxygen concentration of static monolayer cell culture (Metzen et al. 1995, Mamchaoui & Saumon 2000). Under static culture conditions, the BD OBS uses a variation of a model derived from Fick’s Law to relate the OCR at the well bottom, the oxygen concentration gradient in the well, and the rate of oxygen diffusion (Guarino et al. 2004) (Fig. 2). At equilibrium, the rate of oxygen diffusion will equal the rate of oxygen consumption, which is expressed as OCR=DSlΔp/h, in which D is the diffusion coefficient of oxygen in the medium (assume the same as in water, 3.3 × 10−5 cm2/s); S is the surface area of the medium exposed to the atmosphere, which is 0.31 cm2 in a typical well of a 96-well microplate; h is the diffusion path length between the atmosphere and the cells, which is 0.65 cm (typically 200 μl of culture medium); l is a unit conversion factor, which is 6.0 × 107 when OCR is expressed as femtomoles of oxygen consumed per minute; and Δp is the difference in oxygen concentration between the air/medium interface and the medium/cell-matrix interface, which is expressed as [O2]a − [O2].
Measurements of insulin secretion
Using the BD OBS microplate, it is possible to concurrently measure insulin secretion and OCR in response to glucose stimulation. Immediately after reading fluorescence intensities of the wells of the OBS microplate seeded with islets, aliquots of supernatants from each well were collected and assayed for insulin levels by a 1–2–3 Human Insulin ELISA kit according to the manufacturer’s instructions (ALPCO, Windham, NH, USA).
Statistical analysis
All results are expressed as means ± s.e.m. Correlation analysis was performed by the Pearson product-moment correlation. Statistical significance was determined using the Student’s t-test (Statistica 5.0 for Windows). Differences were considered significant where P<0.05.
Results
Effect of islet seeding density on OCR and correlation between OCR and seeding density in non-human primate islets
To determine the effect that islet seeding density has on islet oxygen consumption, non-human primate islets were serially titrated into the wells of an OBS microplate. Islets were seeded at 10–200 IE per well in a total volume of 200 μl CMRL 1066 culture medium containing a basal (5.6 mM) glucose concentration. As shown in Fig. 3A, OCR exhibited by non-human primate islets cultured with the basal glucose concentration increased as islet seeding density increased from 10 to 200 IE at the 24 h culture. At islet seeding densities between 10 and 125 IE, OCR slightly decreased over a 168 h extended culture period. However, at islet seeding density of 150 and 200 IE, OCRs significantly declined at the 168 h culture compared with the 24 h culture (P<0.05, P<0.01 respectively).
Correlation analysis revealed that islet seeding density closely correlated with OCR at both 24 h culture (r2= 0.9572, P<0.01) and 168 h culture (r2=0.9447, P<0.01) (Fig. 3B). However, OCR per IE as calculated from the slope of the line decreased from 801.06 fmol/min per IE at 24 h of culture to 452.67 fmol/min per IE at 168 h of culture, suggesting that there was either a decrease in islet cell mass or a decline in islet viability after an extended culture period.
Significant increases in OCRs in non-human primate islets exposed to high glucose concentrations
To investigate the impact of exposing islets to various glucose concentrations on oxygen consumption over an extended culture period, 100 IE of non-human primate islets were seeded into the wells of an OBS microplate in 200 μl CMRL 1066 culture medium containing basal (5.6 mM) or high (16.7 or 33.3 mM) glucose concentration. As a control, non-human primate pancreatic exocrine cells were also tested. After seeding the wells of an OBS microplate with islets or pancreatic exocrine cells, the microplate was maintained at 37 °C in a humidified incubator with 5% CO2 and the fluorescence intensity was monitored at 24, 48, 72, 96, 120, 144 and 168 h. Compared with islets cultured with the basal glucose concentration, no human primate islets cultured with the high glucose concentrations exhibited significantly higher OCRs over the 168 h extended culture period (P<0.05) (Fig. 4A). Furthermore, OCRs exhibited by non-human primate islets cultured with the high glucose concentrations decreased significantly (P<0.05), while OCRs from islets cultured with the basal glucose concentration remained essentially steady. Pancreatic exocrine cells cultured under various glucose concentrations also consumed oxygen; however, there was no significant difference noted in OCRs in response to changes in glucose concentrations (Fig. 4B).
To determine whether changes in OCRs by islets in response to high glucose stimulation were true responses, the OCRs of the islets were assessed under three consecutive incubation conditions in which islets were first exposed to a low (2.8 mM) level of glucose followed by a high (16.7 mM) concentration of glucose, and then returned to the low glucose condition. Our results demonstrated that stimulated OCRs of the islets during challenge with the high glucose concentration significantly increased when compared with that at the low glucose concentration (P<0.01). Moreover, although OCRs did not exactly return to the basal OCR levels obtained at the initial low glucose concentration prior to stimulation with high glucose, OCRs did significantly decrease after removal of high glucose stimulation (P<0.01) (Fig. 5).
Significant increases in OCR in human islets in response to high glucose stimulation
To ascertain the impact of exposing human islets to various glucose concentrations on oxygen consumption over a short-term incubation, 200 IE of human islets in 200 μl CMRL 1066 culture medium containing basal (5.6 mM) glucose concentration or high (16.7 or 33.3 mM) glucose concentration were seeded into the wells of an OBS microplate. As a control, human pancreatic exocrine cells were also seeded into separate wells of the microplate. The OBS microplate seeded with human islets and pancreatic exocrine cells was incubated at room temperature and kinetically monitored at 30, 60, 90, and 120 min. OCRs exhibited by human islets exposed to basal glucose concentration gradually increased between 30 and 120 min (P<0.05), while OCRs of islets exposed to high glucose concentrations more markedly increased over the same time period (P<0.01). Moreover, human islets exposed to the high glucose concentrations exhibited significantly higher OCRs when compared with islets exposed to the basal glucose concentration (P<0.01) at each time point (Fig. 6A). Human pancreatic exocrine cells also consumed oxygen over the short-term incubation; OCRs did not increase when pancreatic exocrine cells were stimulated with the high glucose concentrations (Fig. 6B).
Correlation between increased OCRs and increased insulin secretions in response to various glucose concentrations in human islets
To investigate insulin secretion concurrently with detection of oxygen consumption in human islets in response to various glucose concentrations, after the final fluorescence reading was obtained at 120 min, aliquots of supernatant were collected for insulin assays. Human islets exposed to high glucose concentrations exhibited significantly greater increases in both OCRs (P<0.05) (Fig. 7A) and insulin secretion levels (P<0.01) (Fig. 7B) compared with islets exposed to the basal glucose condition. Moreover, OCRs exhibited by human islet in response to different glucose stimulation significantly correlated with insulin secretion (r2=0.7681, P<0.01) (Fig. 7C).
Significant difference in OCR stimulation index between viable and low viability human islets
To demonstrate the utility of this methodology for determining the functional viability of human islets, human islets were divided into two groups (i.e. viable group (viability>80%) and low viability group (viability<65%)) according to an islet viability assessment. OCRs of human islets from two groups were evaluated and an OCR stimulation index of human islets was calculated by dividing the OCR of human islets in response to high glucose stimulation at 120 min by the OCR at the basal glucose condition (i.e. OCR at high glucose/OCR at basal glucose). Our data showed that the OCR stimulation index of human islets with high viability was significantly greater than that with low viability (P<0.05) (Fig. 8).
Discussion
Information on oxygen dynamics is of special importance for evaluating metabolic activity and functional viability of pancreatic islets because β-cell membrane potential and secretory activity is stimulated by metabolism rather than by cell surface receptors (Malaisse et al. 1979, Jung et al. 1999). There is a tight link between metabolism and secretion in pancreatic islets. A common approach for studying the link between metabolism and secretion has been to measure oxygen uptake of islets as a function of conditions that affect insulin secretion (Hutton & Malaisse 1980, Sener & Malaisse 1984, Dionne et al. 1989, 1993). A number of investigators have studied the relationship among oxygen consumption, insulin secretion and other biochemical changes in pancreatic islets by developing methods to detect oxygen consumption in islets (Dionne et al. 1991, Longo et al. 1991, Papas et al. 2001, Sweet et al. 2002). However, those methods are mainly based on the principle of detecting changes in oxygen concentration between the inflow and outflow within a sealed perifusion column, which requires a specific apparatus with well-defined geometry. Those methods have some drawbacks similar to conventional oxygraph methods, such as the need for an absolute seal and avoidance of trapped bubbles, drift of calibration, and cost. In contrast, the BD OBS offers a microplate-based culture and assay platform that enables real-time, kinetic monitoring of dissolved oxygen with great ease, convenience and rapid throughput.
In the present study, we demonstrate for the first time that changes in OCRs by non-human primate and human islets exposed to different glucose concentrations are detectable using a novel BD oxygen biosensor system. We first investigated the effect of islet seeding density on OCR and correlation between OCR and seeding density in non-human primate islets. Our data show that OCRs exhibited by non-human primate islets cultured with a basal level of glucose varied directly with islet seeding densities. At higher islet seeding densities, the OCRs significantly declined after an extended culture period (i.e. 168 h). These data indicate that at high islet seeding densities it becomes more difficult to maintain islets in culture over time. This is possibly due to limitations of surface area and oxygen accessibility to islets within the wells of the OBS microplate, causing a gradual decline in cellular metabolic activities (Matsumoto et al. 2003).
The pancreatic islet β-cell plays a vital role in the regulation of energy metabolism (Panten et al. 1986, Malaisse 1996, 1997). Inter- and intracellular signaling by the β-cells in response to glucose and other metabolites and hormones, as well as the interaction of these cells with their microenvironment, are all thought to be critical for proper functioning of the β-cells. Glucose-stimulated insulin secretion is a fundamental property of the pancreatic islet β-cell (Newgard et al. 2002). Therefore, measurements of insulin release from islets after stimulation with different concentrations of glucose is commonly used to assess vital islet functions in a static incubation system (Ashcroft et al. 1971, Andersson et al. 1976, Gray et al. 1984) or in a continuous perifusion system. According to the fuel hypothesis, extracellular elevations in glucose concentrations lead to intracellular increases in glucose consumption rates and OCRs, causing an increase in the rate of ATP synthesis, which results in an increase in the intracellular ATP/ADP ratio. Increases in intracellular ATP/ADP ratio directly closes the K+ ATP channels, resulting in membrane depolarization, influx of Ca2+ and thereby an increase in [Ca2+ ]i triggering insulin exocytosis (Detimary et al. 1998, Ainscow & Rutter 2002, Kennedy et al. 2002, MacDonald & Wheeler 2003). It is well established that oxygen consumption increases in islets in response to glucose stimulation (Hutton & Malaisse 1980, Sener & Malaisse 1984). However, real-time and kinetic detection of oxygen consumption in cultured islets upon both acute and chronic exposure to different glucose concentrations has been technically difficult until now. The BD OBS possesses substantial advantages described previously to detect both acute and chronic changes in oxygen consumption in cultured islets in response to glucose stimulation.
Our data clearly demonstrated that non-human primate islets cultured in the high glucose concentrations exhibited significant increases in OCRs over an extended culture period, which indicates an accelerated rate of β-cell metabolism triggered by glucose over time. Importantly, although pancreatic exocrine cells consumed oxygen, no significant increases in OCRs in response to high glucose stimulation were observed. This excludes the possibility that pancreatic exocrine cells contributed to changes in OCRs with high glucose stimulation. Furthermore, our data also showed that OCRs exhibited by non-human primate islets cultured in the high glucose concentrations decreased significantly after an extended culture period, suggesting impaired metabolic activities and functional viability of the islet cells resulting from the exposure to the high glucose concentrations (Marshak et al. 1999, Federici et al. 2001, Piro et al. 2002).
We initially utilized this OBS to investigate the impact of glucose on oxygen consumption in non-human primate islets cultured in various glucose concentrations over a long-term culture period. It was observed that the significant increases in the OCRs in non-human primate islets in response to high glucose stimulation could be also detected even in a 120 min short-term incubation period. Stimulated OCRs of the islets significantly increased during challenge with the high glucose. Moreover, OCRs did significantly decrease after removal of high glucose stimulation. These data indicate that increased OCRs of islets in response to high glucose stimulation detected by BD OBS are true responses, which provides a substantial potential to further utilize this methodology to assess the functional viability of human islets prior to transplantation. Human islets exhibit significant increases in both OCRs and insulin secretions in response to high glucose stimulation with a good correlation between the two parameters, suggesting that increased OCRs in response to high glucose stimulation can be a marker of the functional viability of islets, which reflects the changes of metabolic activity and functional viability of islets. Most importantly, our results also show that the OCR stimulation index is significantly greater in human islets displaying high viabilities as opposed to islets exhibiting low viabilities. These results demonstrate that BD OBS has the sensitivity to distinguish differences in responses of OCRs to high glucose stimulation between viable and low viability human islets.
In conclusion, our data demonstrate that this novel oxygen biosensor system documents significant increases in islet oxygen consumption upon acute and chronic exposure to high glucose concentrations. Importantly, this methodology rapidly and robustly detects changes in OCRs by islets in response to high glucose stimulation that correlate well with the metabolic activities and functional viability of islets and clearly delineates significant differences in OCR stimulation index between high and low viability human islets, and therefore may prove to be an effective approach for quickly assessing the functional viability of islets prior to transplantation.
A gas-permeable and hydrophobic matrix is permanently attached to the well bottom of a standard BD Falcon microplate. An oxygen-sensitive fluorescent compound (tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) chloride) is embedded in the matrix. Islets were seeded into the well of an OBS microplate as shown by an inverted fluorescence microscope. Original magnification × 40.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
Schematic representation of a well of an OBS. The oxygen-sensitive fluorophores embedded in the well bottom of an OBS microplate can be bottom-read when an excitation filter at 485 nm is used in conjunction with an emission filter at 590–630 nm. Oxygen quenches the fluorophores in a concentration-dependent manner consistent with the quenching mechanism described by the Stern–Volmer theory. Fluorescence is inversely proportional to the concentration of oxygen in the vicinity of the fluorophores. The concentration of oxygen in the vicinity of the dye is in equilibrium with that in the liquid medium. Under static culture conditions, Fick’s Law allows equilibrium oxygen concentration to be converted to an equilibrium OCR.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
Effect of islet seeding density (10–200 IE/well) on OCR (A) and correlation between OCR and islet seeding density (B) in non-human primate islets cultured with basal (5.6 mM) glucose concentration over a 168 h extended culture period. Data are expressed as means ± s.e.m. of six individual detections in each group of islet seeding densities. #P<0.05, ##P<0.01, compared with the same group at the 24 h culture. For correlation analysis between OCR and islet seeding density, OCRs were plotted against islet seeding densities at the 24 h culture and the 168 h culture.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
Impact of exposing islets to various glucose (‘G’) concentrations on oxygen consumption in non-human primate islets (A) and pancreatic exocrine cells (B) over a 168 h extended culture period. Data are expressed as means ± s.e.m. of five individual detections in each group of glucose concentrations. *P<0.05, **P<0.01, compared with the basal (5.6 mM) glucose concentration at different time points; #P<0.05, ##P<0.01, compared with the same group at the 24 h culture.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
Response of oxygen consumption in non-human primate islets to glucose (‘G’) stimulation under three consecutive incubation conditions, i.e. low (2.8 mM) glucose, high (16.7 mM) glucose, and low (2.8 mM) glucose again. Data are expressed as means ± s.e.m. of six individual detections. **P<0.01, compared with low (2.8 mM) glucose concentration; ##P<0.01, compared with high (16.7 mM) glucose concentration.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
Impact of exposing islets to various glucose (‘G’) concentrations on oxygen consumption in human islets (A) and pancreatic exocrine cells (B) over a short-term incubation. Data are expressed as means ± s.e.m. of five individual detections in each group of glucose concentrations. **P<0.01, compared with the basal (5.6 mM) glucose concentration at different time points; ## P<0.01, compared with the same group at 30 min.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
OCR (A), insulin secretion (B), and correlation between OCR and insulin secretion (C) in human islets. Data are expressed as means s.e.m. of three individual detections in duplicates in each group. *P<0.05, **P<0.01, compared with the basal (5.6 mM) glucose (‘G’) concentration. For correlation analysis between OCR and insulin secretion, the OCRs were plotted against insulin secretion levels.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
Stimulation index of OCR between viable and low viability human islets. Data are expressed as means ± s.e.m. of five individual preparations in each group. *P<0.05, compared with the low viability group.
Citation: Journal of Endocrinology 185, 3; 10.1677/joe.1.06092
The authors would like to thank Yoshiko Tamura and Jondell Clever-Hendrix for their technical assistance.
Funding
The authors gratefully acknowledge financial support in part by a grant from the National Center for Research Resources, National Institutes of Health (RO1) U42#RR16604–01. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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