Endoplasmic Reticulum Stress Endoplasmic Reticulum Stress (ERS) and Diabetes Mellitus(ERS) and Diabetes Mellitus

OutlineOutline

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

What is ERS ?What is ERS ?

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.

Physiological functions of Endoplasmic Reticulum

Disequilibrium between

ER load and folding capacity

Nature of ERS

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 ERare extensively impaired.

Cell adaptation Unfolded protein response

(UPR)

Why is ERS ?Why is ERS ?

Something wrong with gene (blueprint) means mutation.

Something wrong with protein (manufacture) means ERS

When does ERS When does ERS happen ?happen ?

Anything causeAnything 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, etcTo be more specificTo be more specific Amino acid starvation, Virus infection; Hypoxia and HAmino acid starvation, Virus infection; Hypoxia and H

ypoglycaemia etc.ypoglycaemia etc. Experimental toxins (tunicamycin, thapsin etc).Experimental toxins (tunicamycin, thapsin etc).

Lead to ERSLead to ERS

Where does ERS Where does ERS happen ?happen ?

Cystic fibrosis (connective tissue);Cystic fibrosis (connective tissue); Hemophilia (blood);Hemophilia (blood); Adiposis hepatica (liver); Adiposis hepatica (liver); Coronary artery disease (heart);Coronary artery disease (heart); Alzheimer's disease (brain).Alzheimer's disease (brain).

Diabetes: Diabetes: cell cell liver liver Perphery adiposePerphery adipose muscle muscle

Almost all tissues especially for Almost all tissues especially for professional secretory cells.

How does ERS How does ERS happen ?happen ?

Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic beta-cells. Apoptosis, 2002,7:335-45.

WFS1WFS1XX

Wolfram sydromWolfram sydromee

PERKPERKXX

Wolcott-RallisoWolcott-Rallison syndromn syndromee

Hand 1 Hand 1 PERK/BIP-eIF2---translation PERK/BIP-eIF2---translation attenuationattenuation ---ATF4 ---ATF4

Pancrea ER kinase. or Pancreatic enriched kinasePancrea ER kinase. or Pancreatic enriched kinase ，， PEKPEK PERK is a type I transmembrane serine/threonine kinase localized in PERK is a type I transmembrane serine/threonine kinase localized in

the ER. Like IRE1α, activation of PERK is triggered by the dissociatithe ER. Like IRE1α, activation of PERK is triggered by the dissociation of Bip from its ER lumenal domain and this leads to oligomerizatioon of Bip from its ER lumenal domain and this leads to oligomerization and trans-autophosphorylationn and trans-autophosphorylation..

The protein kinase PERK couples protein folding in the endoplasmic The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpreticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. The exocrine aenuating translation initiation in response to ER stress. The exocrine and endocrine pancreas developed normally in Perk-/- mice. These findnd endocrine pancreas developed normally in Perk-/- mice. These findings suggest a special role for translational control in protecting secretings suggest a special role for translational control in protecting secretory cells from ER stress.ory cells from ER stress.

Wolcott-Rallison syndrome Wolcott-Rallison syndrome 是一种婴儿是一种婴儿糖尿病，伴有胰腺发育不全，骨营养不良，糖尿病，伴有胰腺发育不全，骨营养不良，由由 PERKPERK 基因突变引起。小鼠中基因突变引起。小鼠中 PERKPERK 基基因敲除也有类似的表现。出生时具有几乎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，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。糖环境下，胰岛素分泌能力较野生型更强。

PERK(-/-)PERK(-/-)的细胞更易受到内质网应激的伤害。的细胞更易受到内质网应激的伤害。

eIF2a51eIF2a51氨基酸的突变小鼠出生后氨基酸的突变小鼠出生后 88小时死于低小时死于低血糖。血糖。

eIF2aeIF2a的磷酸化可以由的磷酸化可以由 PERKPERK等激酶调节。而通等激酶调节。而通常情况下由于相当数量的常情况下由于相当数量的 BIPBIP与与 PERKPERK在内质网在内质网膜上结合，导致膜上结合，导致 PERKPERK活性较低。当活性较低。当 ERSERS时，翻时，翻译受到抑制，但是这种抑制也是特异性的，因为译受到抑制，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，如同时也有蛋白受到诱导表达，如 ATF4ATF4。但是能。但是能引起引起 ATF4ATF4表达增高的还有其他的因素，如上游表达增高的还有其他的因素，如上游的其他能磷酸化的其他能磷酸化 eIF2aeIF2a的激酶，如的激酶，如 GCNGCN等。等。 eIeIF2aF2a的的 5151位丝氨酸磷酸化位点的突变会导致胰腺位丝氨酸磷酸化位点的突变会导致胰腺的发育障碍。这证明除了的发育障碍。这证明除了 PERKPERK之外，其他的之外，其他的 eIeIF2aF2a激酶可能会影响胰腺的发育。激酶可能会影响胰腺的发育。

Hand 3-----ATF6 Hand 3-----ATF6 p50p50ATF6ATF6

Moriand colleaguesMoriand colleagues 发现在发现在 ERSERS 条件下，条件下，两种类似的转录因子两种类似的转录因子 ATF-6aATF-6a 和和 ATF-4bATF-4b ，，可以通过可以通过 ERER 膜蛋白水解激活。在膜蛋白水解激活。在 unstressunstressed ed 细胞中，细胞中， ATF6ATF6 在内质网膜上处于一种在内质网膜上处于一种失活的状态，失活的状态， ERSERS 时，蛋白与时，蛋白与 ERER 结合的氮结合的氮端被水解，之后转移到细胞核激活端被水解，之后转移到细胞核激活 ERSERS 的基的基因表达。水解因表达。水解 ATF6ATF6 的蛋白水解酶与修剪的蛋白水解酶与修剪 SRSREBP-1EBP-1 和和 -2-2 的蛋白酶同源，均为的蛋白酶同源，均为 site-1,site-site-1,site-2 protease.2 protease.

S1PS1P

S2PS2P

WFS1,WFS1, 是位于内质网的一种蛋白，与是位于内质网的一种蛋白，与 SEL-SEL-11 和和 Hrd3pHrd3p 具有相似的一段序列，该基因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，可以造成折叠的蛋白有关，可以造成Wolfram synWolfram syndrome,drome, 一种特殊类型的糖尿病。一种特殊类型的糖尿病。

Hand 3----ERADHand 3----ERAD

IRE1IRE1蛋白属于蛋白属于 type1 ER resident proteintype1 ER resident protein ，具有与，具有与 PEPERKRK相似的结构。相似的结构。 IRE1IRE1也是激酶，而且也受到也是激酶，而且也受到 BIPBIP的负的负向调节，向调节， ERSERS时可以自我激活，对底物时可以自我激活，对底物 mRNAmRNA的特异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。位点进行特异剪切，去除内含子，之后通过连接酶连接。这些通过剪切的这些通过剪切的 mRNAmRNA可以编码为转录因子继而启动可以编码为转录因子继而启动 EERSRS的相关基因表达。在哺乳动物和酵母中这些基因不同。的相关基因表达。在哺乳动物和酵母中这些基因不同。在前者在前者 IRE1IRE1激活后激活后 X-box binding protein1 mRNAX-box binding protein1 mRNA 受受到剪接，并导致到剪接，并导致 XBP-1XBP-1蛋白的表达。蛋白的表达。 XBP-1XBP-1是一种在具是一种在具有分泌功能细胞中高表达的转录因子。与有分泌功能细胞中高表达的转录因子。与 BB细胞转换为细胞转换为浆细胞过程中内质网的分泌功能调节有关。浆细胞过程中内质网的分泌功能调节有关。

目前目前 IRE1a-/-IRE1a-/- 和和 XBP-1-/-XBP-1-/- 都是致死性的，所以其功能还都是致死性的，所以其功能还有待条件敲除的进一步验证。有待条件敲除的进一步验证。

Hand 4-----IRE1/BIP--XBP-1Hand 4-----IRE1/BIP--XBP-1 --TRA2--- --TRA2---JNKJNK --- ---Caspase12Caspase12

哺乳动物中，哺乳动物中， IRE1IRE1 蛋白有两种亚型，其中蛋白有两种亚型，其中 aa广泛表达，而广泛表达，而 bb只选择性的表达于小肠上皮。只选择性的表达于小肠上皮。

caspase-12caspase-12 主要分布于内质网膜上，在主要分布于内质网膜上，在 ERSERS时被剪切激活。属于时被剪切激活。属于 caspase-1caspase-1 家族上游，有家族上游，有文献报道，文献报道， m-calpain m-calpain 可以剪切可以剪切 caspase-12caspase-12 。。而而 caspase-7caspase-7 也可能具有这个作用。也可能具有这个作用。 caspascaspase-12-/-e-12-/- 的小鼠和细胞在暴露于内质网应激诱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。导剂时死亡显著减少。

CHOP-/-CHOP-/- 的细胞对的细胞对 ERSERS 具有抵抗，而过表达具有抵抗，而过表达CHOPCHOP 则会导致细胞死亡。则会导致细胞死亡。

Akita mouseAkita mouse 自发性糖尿病，由于自发性糖尿病，由于 INS2INS2的的 9696位半胱氨酸突变位半胱氨酸突变为了酪氨酸，使胰岛素原为了酪氨酸，使胰岛素原 22的两个二硫键之一形的两个二硫键之一形成障碍，滞留于内质网。但是敲除成障碍，滞留于内质网。但是敲除 INS2INS2的小鼠的小鼠没有代谢异常。没有代谢异常。

AkitaAkita小鼠出生时有形态正常的胰岛，但随着生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长会有高血糖，低胰岛素和进展性的 betabeta细胞细胞massmass减少。电镜下内质网形态肿胀，发现减少。电镜下内质网形态肿胀，发现 bipbip和和 chopchop都表达增加。当都表达增加。当 AktiaAktia小鼠被导入小鼠被导入 CHOCHOP-/-P-/-背景后，糖尿病发病延迟，而且减少胰岛细背景后，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。胞死亡。但是并不能完全阻止发病。

目前被公认的目前被公认的 ERS ERS 动物模型。动物模型。

IRE1alpha phosphorylation is coupled to insulin biosynthesis iIRE1alpha phosphorylation is coupled to insulin biosynthesis in response to transient exposure to high glucose; inactivation on response to transient exposure to high glucose; inactivation of IRE1alpha signaling by siRNA or inhibition of IRE1alpha phf IRE1alpha signaling by siRNA or inhibition of IRE1alpha phosphorylation hinders insulin biosynthesis. osphorylation hinders insulin biosynthesis. IRE1 activation by IRE1 activation by high glucose does not accompany XBP-1 splicing and BiP disshigh glucose does not accompany XBP-1 splicing and BiP dissociation but upregulates its target genes such as WFS1ociation but upregulates its target genes such as WFS1. Thus, I. Thus, IRE1 signaling activated by transient exposure to high glucose RE1 signaling activated by transient exposure to high glucose uses a unique subset of downstream components and has a benuses a unique subset of downstream components and has a beneficial effect on pancreatic beta cells. In contrast, chronic expoeficial effect on pancreatic beta cells. In contrast, chronic exposure of beta cells to high glucose causes ER stress and hyperacsure of beta cells to high glucose causes ER stress and hyperactivation of IRE1, leading to the suppression of insulin gene extivation of IRE1, leading to the suppression of insulin gene expression. IRE1 signaling is therefore a potential target for therpression. IRE1 signaling is therefore a potential target for therapeutic regulation of insulin biosynthesis.apeutic 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. TObesity causes endoplasmic reticulum (ER) stress. This stress in turn leads to suppression of insulin recehis stress in turn leads to suppression of insulin receptor signaling through hyperactivation of c-Jun N-terptor signaling through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphoryminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). Mice delation of insulin receptor substrate-1 (IRS-1). Mice deficient in X-box-binding protein-1 (XBP-1), a transcripficient in X-box-binding protein-1 (XBP-1), a transcription factor that modulates the ER stress response, detion factor that modulates the ER stress response, develop insulin resistance. These findings demonstrate velop insulin resistance. These findings demonstrate that ER stress is a central feature of peripheral insulithat ER stress is a central feature of peripheral insulin resistance and type 2 diabetes at the molecular, celln resistance and type 2 diabetes at the molecular, cellular, and organismal levels. Pharmacologic manipulaular, and organismal levels. Pharmacologic manipulation of this pathway may offer novel opportunities fotion of this pathway may offer novel opportunities for treating these common diseasesr treating these common diseases

Ozcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticOzcan U, Cao Q, Yilmaz E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type ulum stress links obesity, insulin action, and type 2 diabetes. Science, 2004,306:457-61. 2 diabetes. Science, 2004,306:457-61.

Chop-promoter luciferase assay in the Chop-promoter luciferase assay in the presence of cotransfected Foxo1 suppopresence of cotransfected Foxo1 support a putative target, Chop.rt 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. .

最近对最近对 ERSERS 参与胰岛素抵抗又有了新的证据参与胰岛素抵抗又有了新的证据1111 ，在，在 3T33T3 细胞中，内质网应激可以导致细胞中，内质网应激可以导致 GGlut4lut4 转录水平的降低。这可能是因为转录水平的降低。这可能是因为 CHOP10, CHOP10,

an inhibitor of the activity and expression of C/EBP an inhibitor of the activity and expression of C/EBP ,, 可以可以引起引起 C/EBPC/EBP的活性和表达减少，而后者则的活性和表达减少，而后者则被认为可以增加被认为可以增加 Glut4Glut4 的表达。的表达。