Endoplasmic Reticulum Stress (ERS) and Diabetes Mellitus

Outline What is ERS ? Why is ERS ? When does ERS happen ? Where does ERS happen ? How does ERS happen ?

What is ERS ?

Physiological functions of Endoplasmic Reticulum The lumen of the ER provides a specialized environment for posttranslational modification and folding of secreted, transmembrane, and resident proteins of the various compartments of the endomembrane system.

Disequilibrium between ER load and folding capacity Nature of ERS Disequilibrium between ER load and folding capacity

Cell adaptation Unfolded protein response (UPR) The first one consists of translational attenuation to reduce the load of new protein synthesis and to prevent further accumulation of unfolded proteins.. The second involves upregulation of the genes encoding ER chaperone proteins to increase protein folding activity and to prevent protein aggregation The third is degradation of proteins misfolded in the ER and this is called ER-associated degradation (ERAD). The fourth is apoptosis which occurs when functions of the ER are extensively impaired.

Why is ERS ?

Something wrong with gene (blueprint) means mutation. Something wrong with protein (manufacture) means ERS

When does ERS happen ?

Lead to ERS To be more specific Anything cause Inhibition of protein glycosylation, Reduction of formation of disulfide bonds, Calcium depletion from the ER lumen, Impairment of protein transport from the ER to the Golgi, Expression of malfolded proteins, etc To be more specific Amino acid starvation, Virus infection; Hypoxia and Hypoglycaemia etc. Experimental toxins (tunicamycin, thapsin etc). Lead to ERS

Where does ERS happen ?

Cystic fibrosis (connective tissue); Hemophilia (blood); Adiposis hepatica (liver); Coronary artery disease (heart); Alzheimer's disease (brain). Diabetes:  cell liver Perphery adipose muscle Almost all tissues especially for professional secretory cells.

How does ERS happen ?

WFS1 X Wolfram sydrome PERK X Wolcott-Rallison syndrome Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis, 2002,7:335-45.

Hand 1 PERK/BIP-eIF2---translation attenuation ---ATF4 Pancrea ER kinase. or Pancreatic enriched kinase，PEK PERK is a type I transmembrane serine/threonine kinase localized in the ER. Like IRE1α, activation of PERK is triggered by the dissociation of Bip from its ER lumenal domain and this leads to oligomerization and trans-autophosphorylation. The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. The exocrine and endocrine pancreas developed normally in Perk-/- mice. These findings suggest a special role for translational control in protecting secretory cells from ER stress.

Wolcott-Rallison syndrome 是一种婴儿糖尿病，伴有胰腺发育不全，骨营养不良，由PERK基因突变引起。小鼠中PERK基因敲除也有类似的表现。出生时具有几乎正常的胰岛形态，但是接下来的几周里，会有进行性的破坏。分离出来的胰岛在高糖环境下，胰岛素分泌能力较野生型更强。

PERK(-/-)的细胞更易受到内质网应激的伤害。 eIF2a51氨基酸的突变小鼠出生后8小时死于低血糖。 eIF2a的磷酸化可以由PERK等激酶调节。而通常情况下由于相当数量的BIP与PERK在内质网膜上结合，导致PERK活性较低。当ERS时，翻译受到抑制，但是这种抑制也是特异性的，因为同时也有蛋白受到诱导表达，如ATF4。但是能引起ATF4表达增高的还有其他的因素，如上游的其他能磷酸化eIF2a的激酶，如GCN等。eIF2a的51位丝氨酸磷酸化位点的突变会导致胰腺的发育障碍。这证明除了PERK之外，其他的eIF2a激酶可能会影响胰腺的发育。

S1P Hand 3-----ATF6 p50ATF6 S2P Moriand colleagues发现在ERS条件下，两种类似的转录因子ATF-6a和ATF-4b，可以通过ER膜蛋白水解激活。在unstressed 细胞中，ATF6在内质网膜上处于一种失活的状态，ERS时，蛋白与ER结合的氮端被水解，之后转移到细胞核激活ERS的基因表达。水解ATF6的蛋白水解酶与修剪SREBP-1和-2的蛋白酶同源，均为site-1,site-2 protease.

Hand 3----ERAD WFS1,是位于内质网的一种蛋白，与SEL-1和Hrd3p具有相似的一段序列，该基因的突变，可能与线虫酵母等生物处理错误折叠的蛋白有关，可以造成Wolfram syndrome,一种特殊类型的糖尿病。

Hand 4-----IRE1/BIP--XBP-1 --TRA2---JNK ---Caspase12 IRE1蛋白属于type1 ER resident protein，具有与PERK相似的结构。IRE1也是激酶，而且也受到BIP的负向调节，ERS时可以自我激活，对底物mRNA的特异位点进行特异剪切，去除内含子，之后通过连接酶连接。这些通过剪切的mRNA可以编码为转录因子继而启动ERS的相关基因表达。在哺乳动物和酵母中这些基因不同。在前者IRE1激活后X-box binding protein1 mRNA受到剪接，并导致XBP-1蛋白的表达。XBP-1是一种在具有分泌功能细胞中高表达的转录因子。与B细胞转换为浆细胞过程中内质网的分泌功能调节有关。 目前IRE1a-/-和XBP-1-/-都是致死性的，所以其功能还有待条件敲除的进一步验证。

哺乳动物中，IRE1蛋白有两种亚型，其中a广泛表达，而b只选择性的表达于小肠上皮。 caspase-12主要分布于内质网膜上，在ERS时被剪切激活。属于caspase-1家族上游，有文献报道，m-calpain 可以剪切caspase-12。而caspase-7也可能具有这个作用。caspase-12-/-的小鼠和细胞在暴露于内质网应激诱导剂时死亡显著减少。 CHOP-/-的细胞对ERS具有抵抗，而过表达CHOP则会导致细胞死亡。

Akita mouse 自发性糖尿病，由于INS2的96位半胱氨酸突变为了酪氨酸，使胰岛素原2的两个二硫键之一形成障碍，滞留于内质网。但是敲除INS2的小鼠没有代谢异常。 Akita小鼠出生时有形态正常的胰岛，但随着生长会有高血糖，低胰岛素和进展性的beta细胞mass减少。电镜下内质网形态肿胀，发现bip和chop都表达增加。当Aktia小鼠被导入CHOP-/-背景后，糖尿病发病延迟，而且减少胰岛细胞死亡。但是并不能完全阻止发病。 目前被公认的ERS 动物模型。

IRE1alpha phosphorylation is coupled to insulin biosynthesis in response to transient exposure to high glucose; inactivation of IRE1alpha signaling by siRNA or inhibition of IRE1alpha phosphorylation hinders insulin biosynthesis. IRE1 activation by high glucose does not accompany XBP-1 splicing and BiP dissociation but upregulates its target genes such as WFS1. Thus, IRE1 signaling activated by transient exposure to high glucose uses a unique subset of downstream components and has a beneficial effect on pancreatic beta cells. In contrast, chronic exposure of beta cells to high glucose causes ER stress and hyperactivation of IRE1, leading to the suppression of insulin gene expression. IRE1 signaling is therefore a potential target for therapeutic regulation of insulin biosynthesis. Lipson KL, Fonseca SG, Ishigaki S, et al. --Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1. Cell Metab, 2006,4:245-54

Obesity causes endoplasmic reticulum (ER) stress Obesity causes endoplasmic reticulum (ER) stress. This stress in turn leads to suppression of insulin receptor signaling through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). Mice deficient in X-box-binding protein-1 (XBP-1), a transcription factor that modulates the ER stress response, develop insulin resistance. These findings demonstrate that ER stress is a central feature of peripheral insulin resistance and type 2 diabetes at the molecular, cellular, and organismal levels. Pharmacologic manipulation of this pathway may offer novel opportunities for treating these common diseases Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science, 2004,306:457-61.

Chop-promoter luciferase assay in the presence of cotransfected Foxo1 support a putative target, Chop. Martinez SC, Cras-Meneur C, Bernal-Mizrachi E, et al. Glucose regulates Foxo1 through insulin receptor signaling in the pancreatic islet beta-cell. Diabetes, 2006,55:1581-91.

最近对ERS参与胰岛素抵抗又有了新的证据11，在3T3细胞中，内质网应激可以导致Glut4转录水平的降低。这可能是因为CHOP10, an inhibitor of the activity and expression of C/EBP , 可以引起C/EBP的活性和表达减少，而后者则被认为可以增加Glut4的表达。