Effect Of Various Concentration Of Glucose On Primary Culture β Cell Intracellular Ca～(2+) Concentration And Insulin Secretion

BackgroundNow further studies are required to unravel the mechanisms primingβ-cells functional impairment. As the development ofβ-cells research, we found that the stimulus-secretion coupling that drives insulin release involve in Ca²⁺ influx, intracellular Ca²⁺ concentration ([Ca²⁺]_i) rise, in which Ca²⁺ signals play a key role as a messenger. Whether [Ca²⁺]_i change play an important role in the begin of development ofβ-cells functional impairment result from high glucose damage is unknown now.Ca²⁺ concentration rise, evoking by glucose, first occur beneath the plasma membrane. L type Ca²⁺ channels is associated with hot spots of exocytosis in areas. This results in high [Ca²⁺]_i near the plasma membrane Ca²⁺ channels and adjacent Ca²⁺-sensitive targets in the exocytotic machinery. Thus Ca²⁺ concentration in different domains ofβ-cells has different effect on insulin secretion. There are several advantages of developing spatial-restricted Ca²⁺ signals rather than global increases in Ca²⁺ to control insulin release.But in the past research, because of technique restriction, sub-plasma-membrane Ca²⁺ ( [Ca²⁺] SPM ) could not be localization and measurement, only be expressed as bulk cytoplasmic Ca²⁺ concentration. There is no report about the relationship between sub-plasma-membrane Ca²⁺ gradients and insulin secretion now. The present aims to investigate the relationship among glucose, [Ca²⁺] SPM, [Ca²⁺]_i and insulin secretion using primarily cultured rat pancreaticβ-cells incubated with various concentration glucose for different time respectively.AIMDevelop the technique to measure [Ca²⁺]_SPM ofβ-cells. Find way to simultaneous record [Ca²⁺] SPM and [Ca²⁺]_i. Using cultured primary rat pancreaticβ-cells incubated with various concentration glucose for different time, we investigate the relationship among glucose, [Ca²⁺] SPM, [Ca²⁺]_i and insulin secretion.MethodsMale SD rat islets were isolated using digestion by ductal injection of collagenaseâ…¤and treated with trypsin to obtain isolated cells. Afterβ-cells were loaded with fluorescent probe FFP-18-AM , fluorescent images were collected using laser scanning confocal microscope (LSCM) with an excitation of 340nm to monitor changes of [Ca²⁺]_SPM ofβ-cells. Whenβ-cells were loaded with fluorescence probe FFP-18-AM and Fluo-3-AM at the same time, fluorescent measurements of Ca²⁺ concentration from sub-plasma-membrane domain and bulk cytoplasmic domain ofβ-cells were performed. Cultured primary rat pancreaticβ-cells incubated with RPMI 1640 including 5.6, 8.3, 11.1, 16.7 and 33.3mmol/L glucose for 0, 30,60, 120 and 240 min,respectively(n=8). Then the changes of [Ca²⁺]_SPM and [Ca²⁺]_i ofβ-cells loaded Fluo-3 and FFP-18 were monitored and recorded using LSCM . The secretion amount of insulin was measured by using a RIA-kit.Results1. The number of isolated functional islets whose diameter between 100 and 300μm was 337.80±21.36 per rat. 70% cells was verifiedβ-cells by anti-insulin antibody immunofluorescence.2. Afterβ-cells were loaded with FFP-18-AM, Fluorescent images shows that Ca²⁺ located near-plasmalemma presented nonuniformity blue fluorescence. [Ca²⁺]_SPM ofβ-cells was transient increase from (938.04±74.38) to (1259.93±123.03) within 10s whenβ-cells were stimulated by 16.7mmol/L glucose(P﹤0.01).3. Fluo-3-loadedβ-cells could be observed that Ca²⁺ located bulk cytoplasmic presented green fluorescence. And FFP-18-loadedβ-cells could be observed that Ca²⁺ located sub-plasma-membrane presented blue fluorescence. Azure was presented sub-plasma-membrane domain because of two fluorescent images overlap, which is the first time to simultaneous recordβ-cells [Ca²⁺]_i from sub-plasma-membrane domain and bulk cytoplasmic domain.4. After cultured primary rat pancreaticβ-cells was incubated with RPMI 1640 including 5.6mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_SPM ofβ-cells were (609.84±72.65), (607.20±69.31), (608.33±82.75), (634.76±46.34) and (646.59±81.53), which have no difference(P>0.05);β-cells was incubated with RPMI 1640 including 8.3mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_SPM ofβ-cells were(608.84±32.63), (657.50±37.41), (753.33±17.44), (780.87±46.07) and (798.45±17.73), which have significant difference every two time point(P<0.01), basal [Ca²⁺]_SPM increased with the incubation time of glucose of RPMI 1640(P<0.01);β-cells was incubated with RPMI 1640 including 11.1mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_SPM ofβ-cells was (609.21±42.35), (758.03±28.16), (831.81±39.60), (946.51±26.68) and (1001.54±51.04),which has significant difference every two time point(P<0.01), basal [Ca²⁺]_SPM increased with the incubation time of glucose of RPMI 1640(P<0.01);β-cells was incubated with RPMI 1640 including 16.7mmol/L glucose for 0, 30, 60, 120 and 240 min, respectively, the basal [Ca²⁺]_SPM ofβ-cells were(610.64±62.68), (844.981±35.13), (1148.52±77.49), (1235.32±33.82) and (1375.00±39.76),which have significant difference every two time point(P<0.01), basal [Ca²⁺]_SPM increased with the incubation time of glucose of RPMI 1640(P<0.01);β-cells was incubated with RPMI 1640 including 33.3mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_SPM ofβ-cells were (609.64±52.65), (1211.41±28.33), (1267.49±25.15), (1303.57±23.20) and (1573.25±39.21),which have significant difference every two time point(P<0.01), basal [Ca²⁺]_SPM increased with the incubation time of glucose of RPMI 1640(P<0.01);the basal [Ca²⁺]_SPM increased with the incubation glucose concentration step up among groups(P<0.01).5. After cultured primary rat pancreaticβ-cells was incubated with RPMI 1640 including 5.6mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_i ofβ-cells were (398.92±42.96), (383.79±32.17), (399.56±15.69), (383.50±36.51) and (393.50±34.21) ,which have no difference(P>0.05);β-cells was incubated with RPMI 1640 including 8.3mmol/L glucose for 0, 30,60,120 and 240 min respectively, the basal [Ca²⁺]_i ofβ-cells were (398.92±42.96), (451.29±9.80), (509.99±7.04), (534.22±16.40) and (646.00±13.88), which have significant difference every two time point(P<0.01);β-cells was incubated with RPMI 1640 including 11.1mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_i ofβ-cells were (398.92±42.96), (607.86±10.47), (616.54±24.82), (724.98±25.94) and (751.63±37.91), which have significant difference every two time poin(tP<0.01), except that the basal [Ca²⁺]_i between at 30min and at 60min was no difference(P>0.05);β-cells was incubated with RPMI 1640 including 16.7mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_i ofβ-cells were (398.92±42.96), (696.97±14.75), (708.26±28.65), (830.92±23.70) and (870.492±22.51),which have significant difference every two time point(P<0.01), except that the basal [Ca²⁺]_i between at 30min and at 60min was no difference(P>0.05);β-cells was incubated with RPMI 1640 including 33.3mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the basal [Ca²⁺]_i ofβ-cells were (398.92±42.96), (955.41±33.96), (1069.94±38.26), (1123.40±33.14) and (1307.96±55.38), which have significant difference every two time poin(tP<0.01), basal [Ca²⁺]_i increased with the incubation time of glucose of RPMI 1640(P<0.01); the basal [Ca²⁺]_i increased with the incubation glucose concentration step up among groups(P<0.01).6. After cultured primary rat pancreaticβ-cells was incubated with RPMI 1640 including 5.6mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the insulin secretion ofβ-cells were (25.24±3.23), (24.73±1.99), (25.32±2.49), (26.09±1.60) and (27.02±1.55)μU/mL, which have no difference(P>0.05);β-cells was incubated with RPMI 1640 including 8.3mmol/L glucose for 0, 30, 60, 120 and 240 min respectively, the insulin secretion ofβ-cells were (25.24±3.23), (42.26±4.67), (89.25±2.95), (120.29±4.46) and (125.92±3.20)μU/mL, which have significant difference every two time point(P<0.01), except that the insulin secretion between at 120min and at 240min was no difference(P>0.05);β-cells was incubated with RPMI 1640 including 11.1mmol/L glucose for 0, 30, 60, 120 and 240 min, respectively, the insulin secretion ofβ-cells were (25.24±3.23), (80.71±4.63), (125.83±2.56), (146.99±3.17) and (149.85±6.11)μU/mL, which have significant difference every two time point(P<0.01)except that the insulin secretion between at 120min and at 240min was no difference(P>0.05);β-cells was incubated with RPMI 1640 including 16.7mmol/L glucose for 0, 30, 60, 120 and 240 min, respectively, the insulin secretion ofβ-cells were (25.24±3.23), (98.52±2.52), (156.11±5.95), (163.14±3.08) and (166.17±4.85)μU/mL, which have significant difference every two time point(P<0.01)except that the insulin secretion between at 120min and at 240min was no difference(P>0.05);β-cells was incubated with RPMI 1640 including 33.3mmol/L glucose for 0, 30, 60, 120 and 240 min, respectively, the insulin secretion ofβ-cells were (25.24±3.23), (105.92±4.66), (169.49±2.88), (181.27±4.62) and (184.94±7.51)μU/mL, which have significant difference every two time point(P<0.01)except that the insulin secretion between at 120min and at 240min was no difference(P>0.05); With the incubation time of glucose of RPMI 1640 went by insulin secretion rised(P<0.01);the insulin secretion increased with the incubation glucose concentration step up in all groups(P<0.01).Conclusion1. Using fluorescent probe FFP-18 could observe the changes of [Ca²⁺]_SPM ofβ-cells. It is the first time to simultaneous recordβ-cells Ca²⁺ concentration from bulk cytoplasmic domain and sub-plasma-membrane domain by using two different fluorescent probe FFP-18/AM and Fluo-3/AM.2. [Ca²⁺]_SPM, [Ca²⁺]_i and insulin release all have glucose dose-response relationship. So maybe high glucose effect onβ-cells insulin release function by changing of Basic [Ca²⁺]_SPM and [Ca²⁺]_i.