指導教授：周涵怡臺灣大學：口腔生物科學研究所戴珮涵Tai, Pei-HanPei-HanTai2014-11-252018-07-092014-11-252018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/260690Salt-inducible Kinase 2 (SIK2) 是屬於AMPK家族的一員，先前的研究已指出SIK2在維持身體的代謝恆定中扮演很重要的角色。另外，在胰島β cell中，經由血糖的刺激，活化cAMP-PKA後可以調控SIK2而促使與胰島細胞存活相關的基因，IRS2的表現，進而增加胰島β cell細胞的存活率。而先前我們實驗室的研究更發現，SIK2不只表現在細胞質裡，在Rinm5F胰臟癌細胞為模式的研究中，被PKA 磷酸化的SIK2蛋白直接位於胰島素囊泡中。然而SIK2在胰島素囊泡中的功能則尚未釐清。在本實驗裡，我們認為SIK2藉由其磷酸酶活性的調控參與在胰島素囊泡的運送過程。當加抑制劑使SIK2磷酸酶的活性被抑制時，會有較多的胰島素囊泡被運送並且釋放。另外一方面，在生理狀況下的胰島細胞，SIK2是藉由被PKA的磷酸化在Ser-587的位置，而使得其SIK2本身的磷酸酶活性下降。因此，當胰島細胞經由SIK2抑制劑 (compound C)，及PKA 活化的促進劑 (glucose)刺激後，會有較高的SIK2-pS587表現伴隨著胰島素分泌的增加。綜合來說，SIK2本身的功能是藉由限制胰島素囊泡的運送而抑制胰島素分泌。 此外，為了瞭解SIK2 對於胰島素分泌的調控，是否具有生理上的價值。我們製造胰島β cell專一性的SIK2基因轉殖鼠。而當這些基因轉殖鼠在正常進食的情況下，年紀到達11周的時候，顯示出了較高的禁食血糖，及較少的胰島素分泌。 當我們直接給與代謝壓力時，20周大的老鼠，餵食12周的高脂食物，則顯示出葡萄糖清除率明顯的降低。這些現象可能都是由於高度表現的SIK2蛋白抑制了胰島素的分泌。我們也發現了胰島我們也發現了胰島 α cell的數量有增加的趨勢，而這則是糖尿病早期的症狀之一。除此之外，在胰島 β cell專一性的SIK2基因轉殖鼠中，我們做了胰島素相關基因的測試，發現與胰島素製造有關的Ins 1基因有顯著的下降。 根據我們的研究結果，當我們在胰島 β cell高度表現SIK2時，會影響正常的血糖恆定。這個研究結果，可能可以提供糖尿病治療一個新的標的，進而改善胰島素分泌的問題。Salt-inducible Kinase 2 (SIK2), is a member of AMPK family, has been reported to play an important role in metabolism and it also regulatesβcell survival by driving IRS-2(insulin receptor substrate 2) gene expression upon glucose stimulation. The SIK2 expression was previously found in not only cytosol but also insulin vesicles; however, the specific function of SIK2 in pancreaticβcells remains unclear. Here we hypothesized that inhibition of SIK2 kinase activity may lead to the insulin vesicle translocation from reserve pool to readily releasable pool. Vesicle dynamic were imaged in single pancreatic RINm5F cells by total internal reflection fluorescence microscope. Inactivation of SIK2 kinase by treated relatively inhibitor, compound C, resulted in unconstrained reserved pool insulin vesicle mobilization and concomitant increase in insulin release. On the other hand, SIK2 kinase activity is physically inhibited while phosphorylation at Ser-587 by cAMP-PKA in pancreaticβcells. An hour treatment of 0.3 uM compound C or 16.7 mM glucose stimulation made isolated islets increase insulin secretion and higher SIK2-pS587 expression. Therefore, we concluded that SIK2 kinase activity is critical for forbidding insulin secretion through restricting their vesicle movement. Furthermore, for examine the physiological relevance of SIK2 in pancreatic βcells, the β cell-specific sik2 transgenic mice were generated by using bacterial artificial chromosomes (BACs) system. Glucose-stimulated insulin secretion test from 11-week-old transgenic mice fed with regular diet showed higher fasting blood glucose and lower insulin secretion compared to control. Moreover, under metabolic stress, 20-week-old transgenic mice had been fed with high fat diet for 12 week showed significant decreased in glucose clearance rate. This phenotype might result from suppression of insulin vesicle transportation by over-expressed SIK2 protein. The slightly increasedα cells, which was the phenomenon in early stage of diabetes was also observed. The gene expression profile was examined by real-time PCR, and over-expression of sik2 inβcell resulted in down-regulated Ins1 mRNA level. We speculated that this may result from an inhibitory effect on the CREB activity by over-expression of sik2. Above phenotypes indicated that theβcell specific sik2 transgenic mice might have the tendency for development abnormal glucose homeostasis. Taken together, our results uncovered a novel function of SIK2 in the regulation of insulin secretion inβ-cells via insulin transcription and insulin vesicle transportation. These findings suggested that SIK2 is an attractive target for developing new strategies for diabetes.口試委員會審定書 1 ACKNOWLEDGMENTS 2 中文摘要 3 ABSTRACT 5 CONTENTS 7 Chapter 1 Introductions 1.1 Diabetes Mellitus 10 1.2 Regulation of insulin secretion 11 1.2.1.Insulin synthesis. 11 1.2.2 Insulin is secreted in a biphasic pattern 12 1.2.3 Mechanisms involved in first phase (triggering pathway) 13 1.2.4 Mechanisms involved in second phase (amplifying pathway) 13 1.3 PKA function in pancreatic β cell 14 1.4 Salt-inducible kinase (SIK) 15 1.4.1 The expression of SIK2 protein in pancreatic β cells. 17 Chapter 2 Specific Aim 19 Chapter 3 Material and Method 3.1 Generation of transgenic mice 20 3.2 DNA extraction from mouse tail to genotyping 21 3.3 Immunohistochemistry 21 3.4 Western blot analysis 22 3.5 Immunofluorescence staining 23 3.6 Isolation of pancreatic islets 25 3.7 Reverse transcriptase PCR and real-time PCR 26 3.8 Cell culture and treatment 26 3.9 High fat diet 27 3.10Glucose Stimulated Insulin Secretion 27 3.11Fasting blood glucose detection 28 3.12 TIRF Microscopy and living image 28 3.13 Statistical analysis 29 Chapter 4 Results 4.1 SIK2 was expressed in murine pancreatic islets 30 4.2 SIK2 regulated the processes of insulin secretion through its kinase activity in vitro 31 4.3 SIK2 altered the dynamics of insulin vesicle transportation 33 4.4 SIK2 pS587 co-localized with insulin containing vesicles in pancreatic islet cell upon glucose stimulation 35 4.5 Inhibition of SIK2 kinase activity promotes insulin secretion 36 4.6 Highly expression of SIK2-pS587 related to increased insulin secretion. 37 4.7 Generation of β cell specific sik2 transgenic mice 38 4.8 Over-expression of sik2 gene in pancreatic β cell showed the tendency of higher fasting blood glucose in female mice. 39 4.9 The decrease of glucose clearance rate in Ins-sik2 BAC transgenic on HFD treatment 40 4.10 Over-expression of sik2 gene inβcells down-regulated Ins1 mRNA level 41 Chapter 5 Discussion 5.1 The mechanism of SIK2 regulate insulin transcription 44 5.2 SIK2 may involve in the balance of insulin vesicle dynamic inβcells 45 5.3 SIK2 plays a functional role to response several stresses in cells 46 5.4 PP2A may play a potential role of dephosphorylation of SIK2 protein 47 5.5 Role of SIK2 in insulin secretion……………………………………………… 48 5.6 Future perspectives SIK2 regulation in clinical applications 49 Chapter6 Conclusion 50 Chapter7 Reference 51 Chapter8 Figures, Appendix and Table 564044914 bytesapplication/pdf論文使用權限：同意有償授權(權利金給回饋學校)SIK2磷酸酶活性胰島素囊泡運送禁食血糖[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/260690/1/ntu-103-R00450005-1.pdf