摘要
一、研究背景
近年来,流行病学调查显示,世界范围内的糖尿病发病率由2%上升到5%。特别是在一些发展中国家,例如中国,经济的迅速发展伴随着生活模式和生活环境的变化,使糖尿病的发病率每年增长3%。1型糖尿病(type1diabetes,T1D)又名胰岛素依赖型糖尿病(IDDM)或青少年糖尿病,是儿童与青少年常见的内分泌疾病。1型糖尿病是易感个体在环境因素的诱导下,由Thl细胞介导,以免疫性胰岛炎和胰岛p细胞受损为主要特征的自身免疫性疾病。2型糖尿病主要发生于成人,是由于体内高糖高脂毒性对β细胞的长期损害所致,表现为胰岛素抵抗和肥胖。外源性胰岛素治疗不能像自身分泌的内源性胰岛素那样精确地调节血糖水平,所以,糖尿病患者常伴有多种并发症：糖尿病肾病、酮症酸中毒、非酮症性高渗性昏迷、糖尿病性心脏病、糖尿病性脑血管病变、糖尿病性肢端坏疽、糖尿病性神经病变等。并且,糖尿病病情迁延,难以根除,需长期用药,导致家庭和社会负担沉重。我国用于治疗糖尿病的医疗支出约占医疗卫生总费用4%-5%。
1型和2型糖尿病的发病机制至今尚未明确,但大量的研究证实氧化应激(Oxidative stress)和内质网应激(ER stress)在β细胞损坏导致糖尿病过程中发挥重要作用。氧化应激是导致p细胞损伤、凋亡的主要病理基础。在正常状态下机体可产生少量活性氧自由基(reactive oxygen species,ROS),同时存在自由基清除体系,使自由基的产生和清除保持平衡。少量活性氧自由基参与代谢,如细胞内的信号转导,调控细胞的生长、分裂、分化、迁移、凋亡及衰老等生理活动,但过多活性氧自由基可导致组织、细胞发生病理改变,造成的机体伤害。胰腺p细胞抗氧化防御能力低下,细胞内自由基清除酶(Cu/Zn超氧化物歧化酶、Mn超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶)和ROS清除蛋白(硫氧化还原蛋白)水平较低,使较低的氧自由基浓度即可使胰腺p细胞受损。
内质网应激是导致p细胞损伤、凋亡的另一个重要机制。p细胞作为内分泌细胞,拥有高度发达的内质网系统,同时也使p细胞对内质网应激高度敏感。在氧化应激等情况下,内质网内未折叠蛋白增多,发生未折叠蛋白反应(Unfold protein response, UPR),该反应首先激活膜上的3个信号蛋白：PERK (PKR-like ER kinase,双链RNA依赖的蛋白激酶样ER激酶),IRE-1(inositol requiring enzyme1,跨膜蛋白激酶1)和ATF6(Activating transcription factor6,活化转录因子6),通过其下游信号通路抑制蛋白质的合成,上调分子伴侣和折叠蛋白的表达,加速未折叠蛋白的降解等多种方式缓解内质网应激状态。当UPR超过细胞的承受能力,使细胞不能重新回到稳态时,则会启动细胞凋亡程序。通过CHOP通路、IRE1-TRAF2-ASK1-caspasel2通路和Ca2+通路,使细胞发生凋亡。
氧化应激和内质网应激所致p细胞损伤是引发及推动1型和2型糖尿病的重要环节,两者分别／共同作用,使p细胞失去分泌胰岛素的功能。本课题组的前期及其它课题组研究结果显示,在氧化应激和内质网应激状态下,SUMO高表达,多种抵抗应激的蛋白被SUMO化,显示蛋白SUMO化是一种重要的转录后抵抗应激的调节机制。
SUMO化修饰过程与泛素化类似,包含E1活化酶,E2结合酶以及E3连接酶三个酶的级联反应。E1活化酶是一种异源二聚体,在哺乳动物中为SAE1/SAE2,E2结合酶为UBC9,UBC9是SUMO化修饰中唯一的E2结合酶,UBC9的表达变化直接影响到SUMO化功能,因此,UBC9是研究β细胞SUMO化功能的良好靶点。UBC9基因缺乏会导致小鼠胚胎期死亡。E3酶主要包括三类:PIAS家族、核孔蛋白RanBP2/Nup358和Pc2。通过三种酶的级联反应SUMO分子被转移至底物蛋白。
本研究以UBC9为靶点,以诱导型条件性UBC9基因敲除小鼠,在成年小鼠p细胞内特异性地敲除UBC9基因作为模型,研究SUMO化修饰对p细胞功能的影响,进行了以下工作：
二、诱导型UBC9基因敲除小鼠模型的建立
本研究用ubc9fl/fl小鼠和他莫昔芬诱导型β细胞特异性RipCreER转基因小鼠进行杂交,建立RipCreER+ubc9fl/fl小鼠。对8周龄RipCreER+ubc9fl/fl小鼠腹腔注射他莫昔芬(50mg/kg/天),每天一次,连续5天,以注射玉米油溶剂(含10%乙醇)的同窝同性别小鼠作为对照,诱导成年小鼠的p细胞U BC9基因敲除。注射完成5天后处死小鼠,由胆总管向胰腺注入胶原酶P,消化挑拣胰岛。提取胰岛的DNA和蛋白,进行PCR和Western blot分析胰岛的基因组型别及UBC9的表达,验证基因敲除效果。结果显示：对照组小鼠胰岛UBC9基因完整,表达UBC9蛋白高,而他莫昔芬组诱导组胰岛中染色体UBC9基因部分缺失,未检出UBC9蛋白表达,表明他莫昔芬诱导后,RipCreER+ubc9fl/fl.小鼠UBC9表达缺失；并且雌性小鼠的诱导完全缺失的比率为50%,雄性小鼠的诱导完全缺失的比率为75%。
三、UBC9缺失导致小鼠早期发生糖耐量异常,晚期罹患1型糖尿病
1、UBC9基因敲除小鼠早期发生糖耐量异常
(1)UBC9基因敲除小鼠糖耐量降低：他莫昔芬或玉米油注射完毕后第9天(D9)行葡萄糖耐量实验,实验前小鼠空腹16小时,腹腔注射2g/kg葡萄糖溶液后于0min、30min、60min、90min、120min和180min采集小鼠尾部血样检测各时间点的血糖值。结果显示：①他莫昔芬组小鼠空腹血糖值较对照油组明显降低；②对照组小鼠血糖30min升到峰值,60min迅速下降,90min基本恢复到正常,而他莫昔芬组小鼠血糖30min峰值明显升高,60-120min血糖缓慢下降,180min时尚未恢复正常。
(2)UBC9基因敲除小鼠胰岛素耐受正常：胰岛素的分泌减少与胰岛素抵抗均可造成糖耐量下降,为了进一步明确他莫昔芬组引起糖耐量下降的原因,我们比较了两组小鼠的胰岛素耐受情况。腹腔注射0.75U/kg胰岛素,于注射后0min、15min、30min、45min和60min检测血糖。结果显示：两组小鼠0min的随机血糖值无差异,30min时血糖均降到最低点,60min时血糖基本恢复正常,各时间点组间差异不显著。
以上两个实验表明：胰岛β细胞特异性UBC9基因缺失导致p细胞分泌胰岛素的功能明显下降,造成糖耐量异常。
2、UBC9基因敲除小鼠晚期血糖自发升高,血清胰岛素水平下降,出现自发糖尿病
(1)UBC9基因敲除小鼠平均血糖值升高：监测他莫昔芬组和对照组小鼠的随机血糖值,一周2次。D7到D42天两组小鼠平均血糖持续在正常水平,他莫昔芬组血糖略高于对照组,但组间差异不显著；D42天之后,他莫昔芬组血糖值持续上升,D70天达478.6±12.4mg/dl,较对照组166.3±6.0mg/dl明显升高。
(2)UBC9基因敲除小鼠血清胰岛素水平下降：采集小鼠心脏血液,ELISA检测血清胰岛水平,与对照组相比,他莫昔芬组小鼠血清胰岛素水平在D45天之后明显下降。
(3)UBC9基因敲除小鼠糖尿病晚期发病率100%：他莫昔芬组小鼠在D43天开始出现血糖异常,D55天有66.7%小鼠血糖达到糖尿病标准,D65天所有小鼠均出现血糖异常和糖尿病相关症状,而对照组小鼠无1例发病。
以上结果显示：胰岛p细胞特异性UBC9缺失可导致胰岛素分泌水平下降,造成血糖升高,长时间血糖升高在晚期造成小鼠罹患糖尿病。
四、UBC9缺失导致胰岛形态和功能异常,p细胞发生凋亡
1、胰腺组织内胰岛团变小,数目减少：小鼠胰腺石蜡切片HE染色示：对照组胰腺组织胰岛团大而圆,胞浆胞核着色均匀；他莫昔芬组胰岛团随着UBC9敲除时间的后移逐渐变小,并出现凋亡小体。胰岛素免疫组化染色和免疫荧光染色示：对照组胰岛素阳性染色的β细胞团大、圆,占整个切片胰腺组织面积的2%左右；他莫昔芬组胰岛素阳性染色细胞团逐渐减小,D45天仅占切片胰腺组织面积的0.9%；D75天减小到0.2%以下。结果显示：胰岛p细胞特异性UBC9缺失导致胰岛β细胞形态异常,数量减少。
2、胰岛细胞功能异常：胶原酶P消化获得胰岛,体外培养过夜,进行葡萄糖刺激的胰岛素分泌(GSIS)实验,检测胰岛在低糖(3.3mM)和高糖(16.7mM)刺激下胰岛素的释放量。他莫昔芬组胰岛分泌能力明显下降。
3、胰岛内p细胞发生凋亡：TUNEL和cleaved caspase3染色检测显示他莫昔芬组D45天左右胰岛内2.3%的β细胞发生凋亡,对照组内仅见0.2%的凋亡细胞,组间差异显著。结果提示：胰岛p细胞特异性UBC9缺失使p细胞更容易发生凋亡。
五、UBC9缺失小鼠对STZ诱导糖尿病的耐受力降低
他莫昔芬和对照组小鼠腹腔内连续5天注射低剂量STZ(40mg/kg),两组小鼠随机血糖值均缓慢上升,他莫昔芬组血糖明显高于对照组,上升到近600mg/dl,而对照组血糖仅上升到近400mg/dl。结果提示：胰岛p细胞特异性UBC9缺失的p细胞对STZ耐受力明显降低。
六、UBC9缺失导致β细胞凋亡的机制
1、SUMO-NADPH氧化酶-ROS途径对p细胞凋亡的影响
(1)UBC9基因缺失早期p细胞内ROS升高：UBC9基因敲除后D10天,胶原酶消化分离胰岛,培养过夜,细胞因子(IL-1β、TNF-a和IFN-y)混合刺激6小时,25μM羧基-H2DCFDA中孵育30min,他莫昔芬组胰岛内ROS荧光强度明显高于对照组。结果说明：胰岛p细胞特异性UBC9缺陷在早期可导致p细胞内ROS表达增强。
(2)UBC9基因缺失后期β细胞内ROS降低：UBC9基因敲除后D30-D45天,胰岛培养过夜,细胞因子刺激6小时,H2DCFDA中孵育30min,他莫昔芬组胰岛内ROS荧光强度明显低于对照组。结果说明：胰岛p细胞特异性UBC9缺陷导致β细胞大量损伤、凋亡,在诱导后期胰岛内ROS表达减弱。
2、Ubc9基因缺陷导致β细胞内质网应激
我们检测了p细胞内ER stress相关蛋白Grp78、Grp58、eIf2a、ATF6、IREla和CHOP等蛋白的表达,结果显示：ER stress可能介导了p细胞凋亡。
七、结论
1、UBC9基因缺失早期小鼠发生糖耐量异常,后期血糖升高,血清胰岛素水平下降,基因敲除小鼠全部自发糖尿病；
2、UBC9基因缺陷导致小鼠胰岛内p细胞凋亡,引发胰岛形态和功能异常；
3、UBC9基因缺陷小鼠对STZ诱导糖尿病的耐受力降低；
4、UBC9基因缺陷小鼠p细胞内ROS和ER stress的异常表达是导致p细胞损伤、凋亡的重要原因。
综上所述,SUMO化修饰对胰岛内p细胞的功能和活性起保护作用,该机制可能是通过SUMO1抑制NADPH氧化酶产生的ROS发挥的,同时,ER stress参与其中。去除SUMO化修饰,将导致p细胞凋亡,发生糖尿病。因此,SUMO化修饰的唯一E2结合酶UBC9可能成为临床治疗糖尿病的干预靶点。
1Research background
Recent epidemiologic studies revealed that the incidence for diabetes in most regions of worldwide has been increasing by2%to5%. Particularly, in some developing countries such as in China, the rapid economic development along with changes in life style and presumably, the living environment have rendered China with an annual increase of3%for diabetes prevalence. For type1diabetes (T1D, once also known as juvenile diabetes or insulin-dependent diabetes mellitus), the disease is manisfested by the autoimmune responses resulted from the breakdown of peripheral tolerance. Similar as other autoimmune diseases, a characteristic feature for T1D is the selective targeting of a specific type of cells, the insulin secreting (3cells of the islets of Langerhans in the pancreas, by a certain population of autoreactive immune-cells. In contrast, type2diabetes usually occurs in adults and is chacaterized by insulin resistance associated with obesity, which eventually leads complete loss of P mass due to glucotoxicity and lipotoxicity. Given that T1D is typically developed in children and juveniles, its impact on quality of life is far more significant than that of type2diabetes. Although exogenous insulin therapy partly compensates the function of β cells, it cannot regulate blood glucose as accurately as the action of endogenous insulin. As a result, long-term improperly control of blood glucose homeostasis predisposes diabetic patients to the development of diverse complications such as diabetic retinopathy, nephropathy, neuropathy, foot ulcers, and cardiovascular diseases. Moreover, duo to its long treatment cycle, the patients are necessary for lifelong medication, which constitutes a significant burden for both families and society. Indeed, it has been estimatd that the annual medical expenses for diabetic patients in China now is around4to5%of the total health costs.
Although the underlying mechanisms leading to both T1D and T2D have yet to be fully addressed, extensive studies, however, have consistently demonstrated that both oxidative stress and endoplasmic reticulum (ER) stress play a critical role in β cell destruction during the course of diabetest. Neverlethess, the exact regulatory mechanisms underlying β cell destruction are largely remained elusive.
In type1diabetes, autoimmune responses against beta cell self antigens lead to the production of copious amounts of inflammatory cytokines, which then induce oxidative stress to mediate beta cell destruction. Indeed, animals with beta cell specific expression of antioxidant enzymes are resistant to developing T1D, and beta cells deficient in Nox2, the main intracellular ROS producer, are protected from cytokine-or alloxan-induced apoptosis. As such, oxidative stress resulting from autoimmune responses is believed to be the leading cause for beta mass loss in T1D pathogenesis. Unlike T1D, beta mass loss in T2D is slower manifested by a period of beta cell dysfunction. Sustained exposure of isolated islets to high glucose induces increases intracellular ROS, which renders beta cells undergoing apoptosis. It has now become evident that oxidative stress caused by chronic exposure to elevated glucose or fatty acid contributes to beta cell death in type2diabetes. Collectively, beta cell death caused by oxidative stress is likely the major mechanism for beta mass loss for both type1and type2diabetes.
Under physiological condition, low levels of reactive oxygen species (ROS) are necessary for the maintenance of normal metabolism such as intracellular signal transduction, regulation of cell growth, division, differentiation, migration, apoptosis and senescence. In general, the suppression systems are responsible for the clearance of free radicals, so the production and clearance of free radicals maintain a balance. However, under certain pathological states, excessive production of superoxide (02-), hydrogen peroxide(H2O2), hydroxyl radical(OH) and reactive nitrogen species (RNS) such as NO, N02, ONOO-will break the balance between oxidation and clearance, leading to tissue injury. Particularly, pancreatic beta cells possess poor antioxidant capacity along with low level of intracellular free radical scavenging enzymes such as Cu/Zn superoxide dismutase, Mn superoxide dismutase, and catalase and glutathione peroxidase, and therefore, they are particularly vulnerable to oxidative stress.
ER stress is also another important mechanism for beta-cell damage and apoptosis. As endocrine cells, beta cells have a highly developed endoplasmic reticulum system and beta cells are highly sensitive to endoplasmic reticulum stress. In circumstances such as oxidative stress, unfold protein increases in the endoplasmic reticulum, unfold protein response (UPR) occurs which then activates3signaling proteins, the PKR-like ER kinase (PERK), the inositol requiring enzyme1(IRE-1)and the activating transcription factor6(ATF6), and by which to reduce protein synthesis, increase production of molecular mate and expression of fold protein, accelerate the degradation of unfold proteins and ease the endoplasmic reticulum stress. Apoptosis of cells by CHOP pathway, IRE1-TRAF2-ASK1-caspase12pathway and Ca2+pathway would initiate once the UPR is not strong enough to rebuild ER homeostasis.
The pancreatic β cells are equipped with highly developed endoplasmic reticulum (ER) to fulfill the requirement for secreting large amount of insulin. This physiological feature renders β cells particularly vulnerable to ER stress. Exhaustion of β cells is essential for the onset of diabetes, which requires the residual β cells for compensated insulin secretion. While this compensated action is beneficial for control of blood glucose homeostasis, it also increases ER burden associated with the induction of unfolded protein response (UPR) and ER stress, which further exacerbates β cell death. Although the implication of ER stress in β cell death has been extensively emphasized, the underlying mechanisms, however, are yet to be fully elucidated. As such, understanding the role of ER stress in the loss of β mass and dissecting the mechanisms underlying ER stress would be important for developing therapeutic approaches aimed at prevention and intervention of diabetes.
Post-translational attachment of a small ubiquitin-like modifier (SUMO) to the lysine (K) residue(s) of a target protein (defined as sumoylation) is an evolutionarily conserved regulatory mechanism. Four SUMO proteins have been identified in humans. To be functionally active, SUMO needs to be hydrolyzed by a SUMO-specific protease (SENP) to expose its C-terminal diglycine motif, a prerequisite for its covalent conjugation to the substrate proteins. The sumoylation process involves a SUMO-activating enzyme (E1, Uba2/Aos1), a single SUMO-conjugating enzyme (E2, Ubc9), and a SUMO-E3ligase (such as the PIAS family or RanBP2). Sumoylation is a reversible process and, in some cases, dynamic cycles of sumoylation/desumoylation of target proteins are required for cellular processes.
Over the past few years, sumoylation is not only found to be an important regulator of the normal function of many vital cellular proteins, but also found to be a major player in the pathogenesis of human diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, cancer and cardiovascular diseases. Particularly, studies including ours have consistently demonstrated that sumoylation regulates the capacity of cells against oxidative stress. Our recent studies further revealed that sumoylation regulates cell viability through repressing intracellular ROS generation. While these results are important and exciting, the mechanisms by which sumoylation regulates oxidase activity/ROS generation, and the related impact on
beta cells, a ROS vulnerable target, are yet to be elucidated.
2Establishment of induciable UBC9gene knockout mouse model
Ubc9is the only SUMO conjugating enzyme essential for sumoylation, and alterations in Ubc9expression directly reflect the capacity of sumoylation function. Therefore, Ubc9is an ideal target for dynamic modulation of β-cell sumoylation function. As loss of Ubc9leads to embryonic lethality, we used the strategy to generate a conditional Ubc9knockout model. The knock-in allele contains a neo-FRT and two loxP sites flanking Ubc9exons2-4, which can be identified by probe5'and probe3'. Seven chimeric mice were obtained in the B6background. The neo-FRT site in the chimeric mice was then deleted by crossing with Flp transgenic mice, and we have now obtained the Ubc9flox mice with germline transmissions.
The Rip-CreER mice were obtained from Dr. Douglas Melton (Harvard University), in which recombinant Cre can be induced specifically in beta cells by tamoxifen. We then crossed Ubc9-flox with Rip-CreER to generate Cre-Ubc9fl/fl mice, and the mice were then induced for beta cell specific Ubc9deficiency at8wk old with tamoxifen (i.p.,50mg/kg body weight) for5consecutive days. Littermates injected with equal volumes of com oil (control vehicle) were used as controls.
To isolate pancreatic islets, the mice were sacrificed5days after the last injection. In brief, the pancreas was inflated via the bile duct with a digestion buffer containing collagenase P. The distended pancreas was removed and digested at37℃for20to30-min with shaking. Islets were then hand-picked under a dissecting microscope. PCR and Western blot analysis of islet lysates after day2of last induction revealed a significant reduction for Ubc9as compared with that of corn oil induced mice, confirming that Ubc9deficiency was only limited to beta cells.
3Ubc9deficiency leads to abnormal glucose tolerance and diabetes onset
3.1Early occurrence of impaired glucose tolerance in Ubc9knockout mice
3.1.1Impaired glucose tolerance in Ubc9knockout mice
Glucose tolerance test started day9(D9) after Tamoxifen or corn oil injection and mice were fasted for16hours before the experiment. Blood glucose levels were recorded after Omin,30min,60min,90min,120min and180min after intraperitoneal injection with glucose solution (2g/kg).
It was found that fasting blood glucose levels of Tamoxifen mice were significantly lower than that of the control oil group;②blood glucose level reaches the highest peak at30min in control group, but rapidly declined within60min and returned to normal level around90min. In contrat, blood glucose level significantly increased at30min in tamoxifen group, and mot importantly, the blood glucose level failed to decline as that of control mice, after180min of challenge, the blood glucose level still failed to return to normal level.
3.1.2Ubc9deficiency does not result in insulin resistance
Insulin resistance of two groups of mice was then compared to further clarify whether the failure of glucose tolerance in tamoxifen group was due to less insulin secretion or insulin resistance in vivo. After intraperitoneal injection of0.75U/kg insulin Omin,15min,30min,45min and60min, blood glucose levels were recorded. It was noted that there was no difference in random blood glucose levels in both groups of mice, blood sugar dropped to the lowest point at30min and returned to normal level at60min, and no significant differences were detected at each time point examined. Together, these results suggest that loss of Ubc9impairs insulin secretion in beta cells.
3.2Loss of Ubc9renders mice to the development of spontaneous diabetes
3.2.1Loss of Ubc9resulted in the increase of blood glucose levels
Random blood glucose values of tamoxifen and control group of mice were monitored every other day for80days. We failed to observe a significant difference for the average blood glucose levels between two groups of mice in the first30days of Ubc9deficiency. However, a progressive increase for blood glucose leve in Tamoxifen group was noted as compared with that of control group, and the mice started to show onset of diabetes around day43, all mice developed spontaneous diabetes on day65(478.6±12.4mg/dl in Tamoxifen group vs.166.3±6.0mg/dl in control group).
3.2.2Serum insulin levels declined in UBC9gene knockout mice
To further demonstrate that the increase of blood glucose levels in Ubc9deficiency mice was due to the decrease of insulin secretion, we assayed serum insulin levels using the heart blood specimens by ELISA. In consistent with our expectation, much lower levels of insulin were noted in mice in Tamoxifen group as compared with that of mice in control group after day45of Tamoxifen injection.
3.2.3Incidence of diabetes in Ubc9deficiency mice
We have monitored diabetes incidence using12Ubc9deficency mice and12control mice. One Ubc9deficient mice developed diabetes after day43of Tamoxifen induction;7mice showed diabetes onset between days50to55after Tamoxifen induction, and all mice developed spontaneous diabetes on day65of Ubc9deficiency. In sharp contrast, noen of the control mice developed diabetes.
4Ubc9plays an essential role for beta cell viability
4.1Ubc9deficiency affects the morphology, number and size of the islet
Next, histological analysis was conducted by HE staining of the pancreatic sections. In line with the above results, mice with Ubc9deficiency diplayed shrinked islets with loss of beta mass as compared with that of control mice. Insulin staining demonstrated a significantly lower number of insulin producing beta cells in Ubc9deficient sections as control sections. TUNEI assay further revealed a massive beta cell apoptosis after loss of Ubc9. To further confirm these results, we did caspase3staining and demonstrated that these caspase3positive cells were also positive for insulin, indicating that these caspase3positive cells were insulin producing beta cells.
4.2Impared beta cell function in Ubc9deficient mice
Pancreatic islets were hand picked under a dissection microscope as described earlier after collagenase P digestion. The isolated islets were cultured overnight in vitro, and then subjected to glucose-stimulated insulin secretion (GSIS) analysis by stimulation with low glucose (3.3mM) and high glucose (16.7mM). In consistent with the above results, the capacity of pancreatic islets for secretion of inulin after glucose chanllenge was significantly reduced in Ubc9deficient mice as compared with that of control mice.
5Loss of Ubc9renders mice with higher sensitivity to stretozotocin (STZ)-induced toxicity
To assess whether Ubc9impacts STZ-induced cytoxicity, low-dose of STZ (40mg/kg) were administered via tail vein for5consecutive days for mice in both tamoxifen group and control group. An increase for blood glucose was noted for mice in both groups. However, the increase of blood glucose was much faster and the glucose levels were much higher in mice of Tamoxifen group (600mg/dl) as compared with that of control mice, suggesting that loss of Ubc9enhances the sensitivity of beta cells to STZ toxicity. It show:the STZ tolerance of beta cells with UBC9defect is significantly reduced.
6Ubc9regulation of beta cell viability involves ROS accumulation and ER stress
6.1Loss of Ubc9results in ROS accumulation in beta cells
6.1.1Early UBC9gene deletion leads to ROS increase
To dissect the mechanisms by which Ubc9regulates beta cell viability, we first examined intracellular ROS accumulation. For this purpose, pancreatic islets were siloated from mice on day10after Tamoxifen induction. The isolated islets were first cultured overnight, then subjected to cytokine stimulation (IL-1β, TNF-α and IFN-gamma) for6hours. Intracellular ROS accumulation was assessed by incubation with25mM carboxy-H2DCFDA for30min. Interestingly, ROS fluorescence intensity was significantly higher in islets from tamoxifen induced mice than that of control mice, indicating that Ubc9deficiency leads ROS accumulation in beta cells.
6.1.2ROS reduction in later stage of Ubc9deficiency mice
To further demonstrate that ROS accumulation beta cell specific, islets isolated from mice after days30and45of Ubc9deficeincy were subjected to ROS accumulation assay, in which the amount of functional beta cells in Ubc9deficient mice should much less as compared with that of control mice due to apoptosis. Indeed, ROS fluorescence intensity in islets from tamoxifen induced mice was significantly lower than that of control mice, demonstrating that ROS accumulation is beta cell specific.
6.2Ubc9deficency induces ER stress in beta cells
Given the role of ER stress played in beta cell apoptosis, we next examined the ER stress related signaling pathways. For this purpose, islets were isolated from mice after day10of Tamoxifen induction, and cell lysates were prepared and subjected to Western blot analysis of Grp78、Grp58、elf2a、ATF6、IRE1a and CHOP expressions. It was noted tht loss of Ubc9significantly enhanced IRE1a and Elf2a expression along with higher levels of phosphorylated IRE1a and Elf2a. In consistent with these results, much higher levels of Grp58and Grp78were noted in Ubc9deficient islets. Together, these data support that loss of Ubc9induces ER stress, which then synergizes with ROS-induced oxidative stress to promote beta cells undergoing apoptosis.
7Conclusions
(1) Loss of Ubc9renders pancreatic beta cells undergoing progressive apoptosis along with the development of spontaneous diabetes;
(2) Ubc9deficiency results in islet morphological change and functional abnormality due to beta cell death;
(3) Mice deficient in Ubc9display higher sensitivity to STZ-induced cytoxicity;
(4) Ubc9regulation of beta cell viability is associated with modulation of ROS accumulation and ER stress in beta cells.
引文
1. Guo B, Yang SH, Witty J, Sharrocks AD. Signalling pathways and the regulation of SUMO modification. Biochem Soc Trans.2007 Dec;35(Pt 6):1414-8.
2. Tempe D, Piechaczyk M, Bossis G. SUMO under stress. Biochem Soc Trans. 2008 Oct;36(Pt 5):874-8.
3. Verger A, Perdomo J, Crossley M. Modification with SUMO. A role in transcriptional regulation. EMBO Rep.2003 Feb;4(2):137-42.
4. Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004;73:355-82.
5. Praefcke GJ, Hofmann K, Dohmen RJ. SUMO playing tag with ubiquitin. Trends Biochem Sci.2012 Jan;37(1):23-31.
6. Danciu TE, Chupreta S, Cruz O, Fox JE, Whitman M, Iniguez-Lluhi JA. Small ubiquitin-like modifier (SUMO) modification mediates function of the inhibitory domains of developmental regulators FOXC1 and FOXC2. J Biol Chem.2012 May 25;287(22):18318-29.
7. Guo D, Li M, Zhang Y, Yang P, Eckenrode S, Hopkins D, et al. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet.2004 Aug;36(8):837-41.
8. Li M, Guo D, Isales CM, Eizirik DL, Atkinson M, She JX, et al. SUMO wrestling with type 1 diabetes. J Mol Med (Berl).2005 Jul;83(7):504-13.
9. Maison C, Romeo K, Bailly D, Dubarry M, Quivy JP, Almouzni G. The SUMO protease SENP7 is a critical component to ensure HP1 enrichment at pericentric heterochromatin. Nat Struct Mol Biol.2012 Apr;19(4):458-60.
10. Krumova P, Weishaupt JH. Sumoylation fights "aggregopathies". Cell Cycle. 2012 Feb 15;11(4):641-2.
11. Wei W, Yang P, Pang J, Zhang S, Wang Y, Wang MH, et al. A stress-dependent SUMO4 sumoylation of its substrate proteins. Biochem Biophys Res Commun. 2008 Oct 24;375(3):454-9.
12. Wang CY, Yang P, Li M, Gong F. Characterization of a negative feedback network between SUMO4 expression and NFkappaB transcriptional activity. Biochem Biophys Res Commun.2009 Apr 17;381(4):477-81.
13. Wang X, Hai CX. ROS acts as a double-edged sword in the pathogenesis of type 2 diabetes mellitus:is Nrf2 a potential target for the treatment? Mini Rev Med Chem.2011 Oct;11(12):1082-92.
14. Hur J, Sullivan KA, Schuyler AD, Hong Y, Pande M, States DJ, et al. Literature-based discovery of diabetes-and ROS-related targets. BMC Med Genomics.2010;3:49.
15. Bitar MS, Al-Mulla F. ROS constitute a convergence nexus in the development of IGF 1 resistance and impaired wound healing in a rat model of type 2 diabetes. Dis Model Mech.2012 May;5(3):375-88.
16. Nerup J, Mandrup-Poulsen T, Molvig J, Helqvist S, Wogensen L, Egeberg J. Mechanisms of pancreatic beta-cell destruction in type I diabetes. Diabetes Care. 1988 Nov-Dec;11 Suppl 1:16-23.
17. Park EJ, Choi KS, Kwon TK. beta-Lapachone-induced reactive oxygen species (ROS) generation mediates autophagic cell death in glioma U87 MG cells. Chem Biol Interact.2011 Jan 15;189(1-2):37-44.
18. Subasinghe W, Syed I, Kowluru A. Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic beta-cells:evidence for regulation by Racl. Am J Physiol Regul Integr Comp Physiol.2011 Jan;300(1):R12-20.
19. Lim YA, Grimm A, Giese M, Mensah-Nyagan AG, Villafranca JE, Ittner LM, et al. Inhibition of the mitochondrial enzyme ABAD restores the amyloid-beta-mediated deregulation of estradiol. PLoS One.2011;6(12):e28887.
20. Koek GH, Liedorp PR, Bast A. The role of oxidative stress in non-alcoholic steatohepatitis. Clin Chim Acta.2011 Jul 15;412(15-16):1297-305.
21. Suarez-Pinzon WL, Rabinovitch A. Approaches to type 1 diabetes prevention by intervention in cytokine immunoregulatory circuits. Int J Exp Diabetes Res. 2001;2(1):3-17.
22. Serreze DV, Chapman HD, Post CM, Johnson EA, Suarez-Pinzon WL, Rabinovitch A. Thl to Th2 cytokine shifts in nonobese diabetic mice:sometimes an outcome, rather than the cause, of diabetes resistance elicited by immunostimulation. J Immunol.2001 Jan 15;166(2):1352-9.
23. Kawasaki E, Abiru N, Eguchi K. Prevention of type 1 diabetes:from the view point of beta cell damage. Diabetes Res Clin Pract.2004 Dec;66 Suppl 1:S27-32.
24. Kawasaki E, Eguchi K. Is Type 1 diabetes in the Japanese population the same as among Caucasians? Ann N Y Acad Sci.2004 Dec;1037:96-103.
25. Kubisch HM, Wang J, Luche R, Carlson E, Bray TM, Epstein CJ, et al. Transgenic copper/zinc superoxide dismutase modulates susceptibility to type I diabetes. ProcNatl Acad Sci U S A.1994 Oct 11;91(21):9956-9.
26. Chen H, Yu M, Li M, Zhao R, Zhu Q, Zhou W, et al. Polymorphic variations in manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPX1), and catalase (CAT) contribute to elevated plasma triglyceride levels in Chinese patients with type 2 diabetes or diabetic cardiovascular disease. Mol Cell Biochem.2012 Apr;363(1-2):85-91.
27. Ivanovic-Matic S, Mihailovic M, Dinic S, Martinovic V, Bogojevic D, Grigorov I, et al. The absence of cardiomyopathy is accompanied by increased activities of CAT, MnSOD and GST in long-term diabetes in rats. J Physiol Sci.2010 Jul;60(4):259-66.
28. Xiang FL, Lu X, Strutt B, Hill DJ, Feng Q. NOX2 deficiency protects against streptozotocin-induced beta-cell destruction and development of diabetes in mice. Diabetes.2010 Oct;59(10):2603-11.
29. Seeler JS, Dejean A. Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol.2003 Sep;4(9):690-9.
30. Muller S, Ledl A, Schmidt D. SUMO:a regulator of gene expression and genome integrity. Oncogene.2004 Mar 15;23(11):1998-2008.
31. Zunino R, Schauss A, Rippstein P, Andrade-Navarro M, McBride HM. The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. J Cell Sci.2007 Apr 1;120(Pt 7):1178-88.
32. Martin S, Nishimune A, Mellor JR, Henley JM. SUMOylation regulates kainate-receptor-mediated synaptic transmission. Nature.2007 May 17;447(7142):321-5.
33. Rajan S, Plant LD, Rabin ML, Butler MH, Goldstein SA. Sumoylation silences the plasma membrane leak K+ channel K2P1. Cell.2005 Apr 8;121(1):37-47.
34. Benson MD, Li QJ, Kieckhafer K, Dudek D, Whorton MR, Sunahara RK, et al. SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5. Proc Natl Acad Sci U S A.2007 Feb 6;104(6):1805-10.
35. Dai C, Brissova M, Hang Y, Thompson C, Poffenberger G, Shostak A, et al. Islet-enriched gene expression and glucose-induced insulin secretion in human and mouse islets. Diabetologia.2012 Mar;55(3):707-18.
36. Lin X, Liang M, Liang YY, Brunicardi FC, Feng XH. SUMO-1/Ubc9 promotes nuclear accumulation and metabolic stability of tumor suppressor Smad4. J Biol Chem.2003 Aug 15;278(33):31043-8.
37. Rajan S, Torres J, Thompson MS, Philipson LH. SUMO downregulates GLP-1-stimulated cAMP generation and insulin secretion. Am J Physiol Endocrinol Metab.2012 Mar 15;302(6):E714-23.
38. Liu LB, Omata W, Kojima I, Shibata H. The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in3T3-L1 adipocytes. Diabetes.2007 Aug;56(8):1977-85.
39. Ohlsson H, Karlsson K, Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J.1993 Nov;12(11):4251-9.
40. Mosley AL, Ozcan S. The pancreatic duodenal homeobox-1 protein (Pdx-1) interacts with histone deacetylases Hdac-1 and Hdac-2 on low levels of glucose. J Biol Chem.2004 Dec 24;279(52):54241-7.
41. Mosley AL, Corbett JA, Ozcan S. Glucose regulation of insulin gene expression requires the recruitment of p300 by the beta-cell-specific transcription factor Pdx-1. Mol Endocrinol.2004 Sep;18(9):2279-90.
42. Stanojevic V, Habener JF, Thomas MK. Pancreas duodenum homeobox-1 transcriptional activation requires interactions with p300. Endocrinology.2004 Jun;145(6):2918-28.
43. Lu X, Yi J. SUMO-1 enhancing the p53-induced HepG2 cell apoptosis. J Huazhong Univ Sci Technolog Med Sci.2005;25(3):289-91.
44. Sudharsan R, Azuma Y. The SUMO ligase PIAS1 regulates UV-induced apoptosis by recruiting Daxx to SUMOylated foci. J Cell Sci.2012 Dec 1;125(Pt 23):5819-29.
45. Shao R, Rung E, Weijdegard B, Billig H. Induction of apoptosis increases SUMO-1 protein expression and conjugation in mouse periovulatory granulosa cells in vitro. Mol Reprod Dev.2006 Jan;73(1):50-60.
46. Lee YS, Jang MS, Lee JS, Choi EJ, Kim E. SUMO-1 represses apoptosis signal-regulating kinase 1 activation through physical interaction and not through covalent modification, EMBO Rep.2005 Oct;6(10):949-55.
47. O'Sullivan-Murphy B, Urano F. ER stress as a trigger for beta-cell dysfunction and autoimmunity in type 1 diabetes. Diabetes.2012 Apr;61(4):780-1.
48. Gurzov EN, Ortis F, Cunha DA, Gosset G, Li M, Cardozo AK, et al. Signaling by IL-lbeta+IFN-gamma and ER stress converge on DP5/Hrk activation:a novel mechanism for pancreatic beta-cell apoptosis. Cell Death Differ.2009 Nov;16(11):1539-50.
49. Chen H, Qi L. SUMO modification regulates the transcriptional activity of XBP1. Biochem J.2010 Jul 1;429(1):95-102.
50. Uemura A, Taniguchi M, Matsuo Y, Oku M, Wakabayashi S, Yoshida H. UBC9 Regulates the Stability of XBP1, a Key Transcription Factor Controlling the ER Stress Response. Cell Struct Funct.2013 Apr 18;38(1):67-79.
1. Guo B, Yang SH, Witty J, Sharrocks AD. Signalling pathways and the regulation of SUMO modification. Biochem Soc Trans.2007 Dec;35(Pt 6):1414-8.
2. Tempe D, Piechaczyk M, Bossis G. SUMO under stress. Biochem Soc Trans. 2008 Oct;36(Pt 5):874-8.
3. Verger A, Perdomo J, Crossley M. Modification with SUMO. A role in transcriptional regulation. EMBO Rep.2003 Feb;4(2):137-42.
4. Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004;73:355-82.
5. Praefcke GJ, Hofmann K, Dohmen RJ. SUMO playing tag with ubiquitin. Trends Biochem Sci.2012 Jan;37(1):23-31.
6. Danciu TE, Chupreta S, Cruz O, Fox JE, Whitman M, Iniguez-Lluhi JA. Small ubiquitin-like modifier (SUMO) modification mediates function of the inhibitory domains of developmental regulators FOXC1 and FOXC2. J Biol Chem.2012 May 25;287(22):18318-29.
7. Guo D, Li M, Zhang Y, Yang P, Eckenrode S, Hopkins D, et al. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet.2004 Aug;36(8):837-41.
8. Li M, Guo D, Isales CM, Eizirik DL, Atkinson M, She JX, et al. SUMO wrestling with type 1 diabetes. J Mol Med (Berl).2005 Jul;83(7):504-13.
9. Maison C, Romeo K, Bailly D, Dubarry M, Quivy JP, Almouzni G. The SUMO protease SENP7 is a critical component to ensure HPl enrichment at pericentric heterochromatin. Nat Struct Mol Biol.2012 Apr; 19(4):458-60.
10. Krumova P, Weishaupt JH. Sumoylation fights "aggregopathies". Cell Cycle. 2012 Feb 15;11(4):641-2.
11. Wei W, Yang P, Pang J, Zhang S, Wang Y, Wang MH, et al. A stress-dependent SUMO4 sumoylation of its substrate proteins. Biochem Biophys Res Commun. 2008 Oct 24;375(3):454-9.
12. Wang CY, Yang P, Li M, Gong F. Characterization of a negative feedback network between SUMO4 expression and NFkappaB transcriptional activity. Biochem Biophys Res Commun.2009 Apr 17;381(4):477-81.
13. Wang X, Hai CX. ROS acts as a double-edged sword in the pathogenesis of type 2 diabetes mellitus:is Nrf2 a potential target for the treatment? Mini Rev Med Chem.2011 Oct;11(12):1082-92.
14. Hur J, Sullivan KA, Schuyler AD, Hong Y, Pande M, States DJ, et al. Literature-based discovery of diabetes-and ROS-related targets. BMC Med Genomics.2010;3:49.
15. Bitar MS, Al-Mulla F. ROS constitute a convergence nexus in the development of IGF1 resistance and impaired wound healing in a rat model of type 2 diabetes. Dis Model Mech.2012 May;5(3):375-88.
16. Nerup J, Mandrup-Poulsen T, Molvig J, Helqvist S, Wogensen L, Egeberg J. Mechanisms of pancreatic beta-cell destruction in type I diabetes. Diabetes Care. 1988Nov-Dec;11 Suppl 1:16-23.
17. Park EJ, Choi KS, Kwon TK. beta-Lapachone-induced reactive oxygen species (ROS) generation mediates autophagic cell death in glioma U87 MG cells. Chem Biol Interact.2011 Jan 15;189(1-2):37-44.
18. Subasinghe W, Syed I, Kowluru A. Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic beta-cells:evidence for regulation by Racl. Am J Physiol Regul Integr Comp Physiol.2011 Jan;300(1):R12-20.
19. Lim YA, Grimm A, Giese M, Mensah-Nyagan A.G, Villafranca JE, Ittner LM, et al. Inhibition of the mitochondrial enzyme ABAD restores the amyloid-beta-mediated deregulation of estradiol. PLoS One.2011;6(12):e28887.
20. Koek GH, Liedorp PR, Bast A. The role of oxidative stress in non-alcoholic steatohepatitis. Clin Chim Acta.2011 Jul 15;412(15-16):1297-305.
21. Suarez-Pinzon WL, Rabinovitch A. Approaches to type 1 diabetes prevention by intervention in cytokine immunoregulatory circuits. Int J Exp Diabetes Res. 2001;2(1):3-17.
22. Serreze DV, Chapman HD, Post CM, Johnson EA, Suarez-Pinzon WL, Rabinovitch A. Thl to Th2 cytokine shifts in nonobese diabetic mice:sometimes an outcome, rather than the cause, of diabetes resistance elicited by immunostimulation. J Immunol.2001 Jan 15; 166(2):1352-9.
23. Kawasaki E, Abiru N, Eguchi K. Prevention of type 1 diabetes:from the view point of beta cell damage. Diabetes Res Clin Pract.2004 Dec;66 Suppl 1:S27-32.
24. Kawasaki E, Eguchi K. Is Type 1 diabetes in the Japanese population the same as among Caucasians? Ann N Y Acad Sci.2004 Dec;1037:96-103.
25. Kubisch HM, Wang J, Luche R, Carlson E, Bray TM, Epstein CJ, et al. Transgenic copper/zinc superoxide dismutase modulates susceptibility to type I diabetes. Proc Natl Acad Sci U S A.1994 Oct 11;91(21):9956-9.
26. Chen H, Yu M, Li M, Zhao R, Zhu Q, Zhou W, et al. Polymorphic variations in manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPX1), and catalase (CAT) contribute to elevated plasma triglyceride levels in Chinese patients with type 2 diabetes or diabetic cardiovascular disease. Mol Cell Biochem. 2012 Apr;363(1-2):85-91.
27. Ivanovic-Matic S, Mihailovic M, Dinic S, Martinovic V, Bogojevic D, Grigorov I, et al. The absence of cardiomyopathy is accompanied by increased activities of CAT, MnSOD and GST in long-term diabetes in rats. J Physiol Sci.2010 Jul;60(4):259-66.
28. Xiang FL, Lu X, Strutt B, Hill DJ, Feng Q. NOX2 deficiency protects against streptozotocin-induced beta-cell destruction and development of diabetes in mice. Diabetes.2010 Oct;59(10):2603-11.
29. Seeler JS, Dejean A. Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol.2003 Sep;4(9):690-9.
30. Muller S, Ledl A, Schmidt D. SUMO:a regulator of gene expression and genome integrity. Oncogene.2004 Mar 15;23(11):1998-2008.
31. Zunino R, Schauss A, Rippstein P, Andrade-Navarro M, McBride HM. The SUMO protease SENP5 is required to maintain mitochondrial morphology and function. J Cell Sci.2007 Apr 1;120(Pt 7):1178-88.
32. Martin S, Nishimune A, Mellor JR, Henley JM. SUMOylation regulates kainate-receptor-mediated synaptic transmission. Nature.2007 May 17;447(7142):321-5.
33. Rajan S, Plant LD, Rabin ML, Butler MH, Goldstein SA. Sumoylation silences the plasma membrane leak K+ channel K2P1. Cell.2005 Apr 8;121(1):37-47.
34. Benson MD, Li QJ, Kieckhafer K, Dudek D, Whorton MR, Sunahara RK, et al. SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5. Proc Natl Acad Sci U S A.2007 Feb 6;104(6):1805-10.
35. Dai C, Brissova M, Hang Y, Thompson C, Poffenberger G, Shostak A, et al. Islet-enriched gene expression and glucose-induced insulin secretion in human and mouse islets. Diabetologia.2012 Mar;55(3):707-18.
36. Lin X, Liang M, Liang YY, Brunicardi FC, Feng XH. SUMO-1/Ubc9 promotes nuclear accumulation and metabolic stability of tumor suppressor Smad4. J Biol Chem.2003 Aug 15;278(33):31043-8.
37. Rajan S, Torres J, Thompson MS, Philipson LH. SUMO downregulates GLP-1-stimulated cAMP generation and insulin secretion. Am J Physiol Endocrinol Metab.2012 Mar 15;302(6):E714-23.
38. Liu LB, Omata W, Kojima I, Shibata H. The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes. Diabetes.2007 Aug;56(8):1977-85.
39. Ohlsson H, Karlsson K, Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J.1993 Nov;12(11):4251-9.
40. Mosley AL, Ozcan S. The pancreatic duodenal homeobox-1 protein (Pdx-1) interacts with histone deacetylases Hdac-1 and Hdac-2 on low levels of glucose. J Biol Chem.2004 Dec 24;279(52):54241-7.
41. Mosley AL, Corbett JA, Ozcan S. Glucose regulation of insulin gene expression requires the recruitment of p300 by the beta-cell-specific transcription factor Pdx-1. Mol Endocrinol.2004 Sep;18(9):2279-90.
42. Stanojevic V, Habener JF, Thomas MK. Pancreas duodenum homeobox-1 transcriptional activation requires interactions with p300. Endocrinology.2004 Jun;145(6):2918-28.
43. Lu X, Yi J. SUMO-1 enhancing the p53-induced HepG2 cell apoptosis. J Huazhong Univ Sci Technolog Med Sci.2005;25(3):289-91.
44. Sudharsan R, Azuma Y. The SUMO ligase PIAS1 regulates UV-induced apoptosis by recruiting Daxx to SUMOylated foci. J Cell Sci.2012 Dec 1;125(Pt 23):5819-29.
45. Shao R, Rung E, Weijdegard B, Billig H. Induction of apoptosis increases SUMO-1 protein expression and conjugation in mouse periovulatory granulosa cells in vitro. Mol Reprod Dev.2006 Jan;73(1):50-60.
46. Lee YS, Jang MS, Lee JS, Choi EJ, Kim E. SUMO-1 represses apoptosis signal-regulating kinase 1 activation through physical interaction and not through covalent modification. EMBO Rep.2005 Oct;6(10):949-55.
47. O'Sullivan-Murphy B, Urano F. ER stress as a trigger for beta-cell dysfunction and autoimmunity in type 1 diabetes. Diabetes.2012 Apr;61(4):780-1.
48. Gurzov EN, Ortis F, Cunha DA, Gosset G, Li M, Cardozo AK, et al. Signaling by IL-lbeta+IFN-gamma and ER stress converge on DP5/Hrk activation:a novel mechanism for pancreatic beta-cell apoptosis. Cell Death Differ.2009 Nov;16(11):1539-50.
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