| Objectives: 1. To explore how uncarboxylated osteocalcin affects insulin secretion in rats. 2. To explore the relationships of uncarboxylated osteocalcin and action potentials, voltage-gated potassium channels,voltage-gated calcium channels, intracellular calcium concentration. 3. To observe if uncarboxylated osteocalcin increases insulin secretion and inhibits voltage-gated potassium channels by PLC-related signaling pathway. 4. To investigate if GPRC6 A is expressed in rat islets.Methods: 1. Rat islet isolation and culture The rat pancreas was digested by Collagenase P. The acquired islets were cultured in Hy Clone RPMI 1640 medium containing 11.1 mmol/l glucose, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum at 37 °C in 95% air with 5% CO2. 2. To observe the effects of different concentrations of uncarboxylated osteocalcin on insulin secretion in rats by insulin secretion experiments The islets were incubated in Krebs-Ringer bicarbonate-HEPES buffer containing 2.8 mmol/l glucose or 16.7 mmol/l glucose in the absence or presence of uncarboxylated osteocalcin(0.03 ng/ml, 0.3 ng/ml). After 30 min, the supernatant was collected and stored at-20 °C for measurement of insulin levels. The islet pellets in per tube were lysed with acid-ethanol solution and stored at-20 °C for test of insulin contents. 3. To observe the effects of uncarboxylated osteocalcin on insulin secretion under different concentrations of glucose by insulin secretion experiments The islets were incubated in Krebs-Ringer bicarbonate-HEPES buffer containing 2.8 mmol/l glucose, 11.1 mmol/l glucose or 16.7 mmol/l glucose in the absence or presence of uncarboxylated osteocalcin(0.3 ng/ml). After 30 min, the supernatant was collected and stored at-20 °C for measurement of insulin levels. The islet pellets in per tube were lysed with acid-ethanol solution and stored at-20 °C for test of insulin contents. 4. To explore the relationships of uncarboxylated osteocalcin and action potentials, voltage-gated potassium channels, voltage-gated calcium channels by patch-clamp experiments. 4.1 β cell isolation and culture The rat islets were digested by Dispase Ⅱ. The acquired β cells were cultured in Hy Clone RPMI 1640 medium containing 11.1 mmol/l glucose, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum at 37 °C in 95% air with 5% CO2. 4.2 Electrophysiology By use of whole-cell patch-clamp technique 5. To explore the relationship of uncarboxylated osteocalcin-regulated insulin secretion and voltage-gated potassium channels by the insulin secretion experiments. The islets were incubated in Krebs-Ringer bicarbonate-HEPES buffer containing 2.8 mmol/l glucose or 16.7 mmol/l glucose in the absence or presence of uncarboxylated osteocalcin(0.3 ng/ml), TEA(20 m M). After 30 min, the supernatant was collected and stored at-20 °C for measurement of insulin levels. The islet pellets in per tube were lysed with acid-ethanol solution and stored at-20 °C for test of insulin contents. 6. To explore the relationship of uncarboxylated osteocalcin and intracellular calcium concentration in the calcium imaging experiments. 6.1 β cell isolation and culture were same with the previous description. 6.2 Measurements of intracellular Ca2+ concentration([Ca2+]i) Ca2+-sensitive fluorescent dye, Fluo 4AM was applied. β cells were treated with 0.03 or 0.3 ng/ml uncarboxylated osteocalcin in the presence or absence of TEA(20 mmol/l) under 2.8 or 16.7 mmol/l glucose conditions. The change of intracellular Ca2+ concentration was represented by the change of fluorescence intensity. 7. To explore if uncarboxylated osteocalcin inhibits voltage-gated potassium channels by phospholipase-C related signaling pathway by patch-clamp experiments. β cell isolation, culture and experimental procedure of electrophysiology were same with the previous description. The drugs were dissolved in corresponding Kv extracellular solution.8. To explore the relationship of uncarboxylated osteocalcin-regulated insulin secretion and phospholipase-C by the insulin secretion experiments The islets were incubated in Krebs-Ringer bicarbonate-HEPES buffer containing 2.8 mmol/l glucose or 16.7 mmol/l glucose in the absence or presence of uncarboxylated osteocalcin(0.3 ng/ml), U-73122( 1μM). After 30 min, the supernatant was collected and stored at-20 °C for measurement of insulin levels. The islet pellets in per tube were lysed with acid-ethanol solution and stored at-20 °C for test of insulin contents. 9. To explore the relationship of uncarboxylated osteocalcin-regulated insulin secretion and phospholipase-C related signaling pathway by insulin secretion experiments Experimental procedure was same with the above description. U-73122(1μM)was replaced by Ro 31-8220(1 μM), FTS-A(50 μM) or PD 98059(20 μM). 10. To explore the relationship of uncarboxylated osteocalcin-regulated insulin secretion and adenylate cyclase by insulin secretion experiments Experimental procedure was same with the above description. The drug was replaced by SQ 22536(10 μM). 11. Real-Time PCR and general PCRResults: 1. The basal insulin secretion at 2.8 mmol/l glucose(2.8 G) was not affected by addition of uncarboxylated osteocalcin(0.03 ng/ml, 0.3 ng/ml)(P > 0.05 vs. 2.8 G). Compared to 16.7 mmol/l glucose(16.7 G), uncarboxylated osteocalcin(0.03 ng/ml, 0.3 ng/ml) further potentiated insulin secretion(P < 0.05 vs. 16.7 G). But uncarboxylated osteocalcin(0.03 ng/ml, 0.3 ng/ml) did not affect insulin content. 2. No significant increase in insulin secretion was observed in the presence of 0.3 ng/ml uncarboxylated osteocalcin under 2.8 G conditions(P > 0.05 vs. 2.8 G). In contrast, 0.3 ng/ml uncarboxylated osteocalcin potentiated insulin secretion by approximately 61% under 11.1 mmol/l glucose conditions(P < 0.05 vs. 11.1 G) and significantly potentiated insulin secretion by approximately 83% at 16.7 G(P < 0.01 vs. 16.7 G). But uncarboxylated osteocalcin(0.3 ng/ml) did not affect insulin content. 3. Uncarboxylated osteocalcin(0.03 ng/ml) lengthened action potential duration by nearly 52 % from 47.2 ± 4.8 ms of control(Ctrl) to 71.6 ± 8.2 ms of 0.03 ng/ml uncarboxylated osteocalcin(P < 0.05 vs. Ctrl). Uncarboxylated osteocalcin(0.3 ng/ml) lengthened action potential duration by nearly 61 % from 47.2 ± 4.8 ms of control(Ctrl) to 76.1 ± 4.8 ms of 0.3 ng/ml uncarboxylated osteocalcin(P < 0.05 vs. Ctrl). 4. The amplitude of action potential was not affected by addition of uncarboxylated osteocalcin(0.03 ng/ml, 0.3 ng/ml) compared to control(Ctrl). The amplitude of action potential was 77.2 ± 4.6 m V for 0.03 ng/ml uncarboxylated osteocalcin and 75.7 ± 4.3 m V for 0.3 ng/ml uncarboxylated osteocalcin, which did not differ from control(77.2 ± 3.0 m V for Ctrl, P > 0.05). 5. The current-voltage curves show that 0.03 ng/ml and 0.3 ng/ml uncarboxylated osteocalcin significantly suppressed KV channels compared to control(Ctrl). Uncarboxylated osteocalcin(0.03 ng/ml) produced approximately 31% inhibition of KV currents at 0 m V(15.3 ± 2.1 p A/p F for 0.03 ng/ml uncarboxylated osteocalcin, 22.3 ± 2.8 p A/p F for Ctrl, P < 0.05 vs. Ctrl). Uncarboxylated osteocalcin(0.3 ng/ml) produced approximately 39% inhibition of KV currents at 0 m V(13.7 ± 1.4 p A/p F for 0.3 ng/ml uncarboxylated osteocalcin, 22.3 ± 2.8 p A/p F for Ctrl, P < 0.05 vs. Ctrl). 6. The current-voltage curves show that 0.3 ng/ml uncarboxylated osteocalcin did not affect the characteristics of voltage-gated calcium channels compared to control(Ctrl). The current density was-4.2 ± 0.6 p A/p F for uncarboxylated osteocalcin at 0 m V, which did not differ from Ctrl(-4.0 ± 0.5 p A/p F for Ctrl, P > 0.05). 7. Under 16.7 G conditions, uncarboxylated osteocalcin(0.3 ng/ml) and tetraethylammonium chloride(TEA, 20 mmol/l) remarkably increased insulin secretion, respectively(P < 0.01 vs. 16.7 G). But in the presence of TEA, uncarboxylated osteocalcin had no such effect on insulin secretion at 16.7 G(P > 0.05 vs. 16.7 G + TEA). Uncarboxylated osteocalcin did not affect insulin content. 8. Compared to 2.8 mmol/l glucose, 16.7 mmol/l glucose increased [Ca2+]i(P < 0.05 vs. 2.8 G). Under 2.8 mmol/l glucose conditions, uncarboxylated osteocalcin(0.3 ng/ml) did not affect [Ca2+]i(P > 0.05 vs. 2.8 G). Under 16.7 mmol/l glucose conditions, 0.03 and 0.3 ng/ml uncarboxylated osteocalcin increased [Ca2+]i, respectively(P < 0.05 vs. 16.7 G). But in the presence of TEA at 16.7 mmol/l glucose, there was no significant change on [Ca2+]i by addition of 0.3 ng/ml uncarboxylated osteocalcin. 9. The current-voltage curves demonstrate that KV channels were significantly suppressed by 0.3 ng/ml uncarboxylated osteocalcin compared to control(Ctrl). U-73122 reversed the effect of uncarboxylated osteocalcin on KV channels at 80 m V(70.3 ± 5.7 p A/p F for Ocn, 97.9 ± 8.7 p A/p F for U-73 + Ocn, P < 0.05 vs. Ocn).10.The current-voltage curves demonstrate that KV channels were significantly suppressed by 0.3 ng/ml uncarboxylated osteocalcin compared to control(Ctrl). Ro 31-8220 alleviated the inhibition effect of uncarboxylated osteocalcin on KV channels at 80 m V(63.2 ± 7.0 p A/p F for Ocn, 95.7 ± 5.6 p A/p F for Ro + Ocn, P < 0.01 vs. Ocn). 11.The current-voltage curves show that compared to control, 0.3 ng/ml uncarboxylated osteocalcin significantly suppressed KV currents. FTS-A reversed the effect of uncarboxylated osteocalcin on KV channels at 80 m V(68.6 ± 4.4 p A/p F for Ocn, 96.0 ± 10.8 p A/p F for FTS-A + Ocn, P < 0.05 vs. Ocn). 12. PD 98059 reversed the effect of uncarboxylated osteocalcin on KV channels at 80 m V(75.1 ± 7.3 p A/p F for Ocn, 113.2 ± 11.3 p A/p F for PD + Ocn, P < 0.05 vs. Ocn). 13. Compared to 16.7 mmol/l glucose(16.7 G), uncarboxylated osteocalcin(0.3 ng/ml) further potentiated insulin secretion(P < 0.01 vs. 16.7 G). But the effect of uncarboxylated osteocalcin was suppressed by U-73122(1 μmol/l)(P < 0.01 vs. 16.7 G + Ocn). 14. Compared to 16.7 mmol/l glucose(16.7 G), uncarboxylated osteocalcin(0.3 ng/ml) further potentiated insulin secretion(P < 0.01 vs. 16.7 G). Ro 31-8220(1 μmol/l) inhibited the effect of uncarboxylated osteocalcin on insulin secretion(P < 0.01 vs. 16.7 G + Ocn). 15. Compared to 16.7 mmol/l glucose(16.7 G), uncarboxylated osteocalcin(0.3 ng/ml) further potentiated insulin secretion(P < 0.01 vs. 16.7 G). FTS-A(50 μmol/l) inhibited the effect of uncarboxylated osteocalcin on insulin secretion(P < 0.01 vs. 16.7 G + Ocn). 16. Compared to 16.7 mmol/l glucose(16.7 G), uncarboxylated osteocalcin(0.3 ng/ml) further potentiated insulin secretion(P < 0.01 vs. 16.7 G). PD 98059(20 μmol/l) inhibited the effect of uncarboxylated osteocalcin on insulin secretion(P < 0.01 vs. 16.7 G + Ocn). 17. Compared to 16.7 mmol/l glucose(16.7 G), uncarboxylated osteocalcin(0.3 ng/ml) further potentiated insulin secretion(P < 0.01 vs. 16.7 G). The effect of uncarboxylated osteocalcin was not affected by addition of SQ 22536(10 μmol/l)(P > 0.05 vs. 16.7 G + Ocn) 18. Real-Time PCR and general PCR indicate that there is no or rarely GPRC6 A gene expression in SD rats islets.Conclusions: 1. Uncarboxylated osteocalcin increases insulin secretion in SD rats, which relates to glucose concentration and may not be mediated by GPRC6 A.2. By inhibiting KV channels, uncarboxylated osteocalcin prolongs action potential duration, increases intracellular Ca2+ concentration and finally promotes insulin secretion. But uncarboxylated osteocalcin had no influence on the characteristics of voltage-gated calcium channels. 3. The PLC/PKC/Ras/MEK signaling pathway is involved in the effects of uncarboxylated osteocalcin on insulin secretion and KV channels. |
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