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Targeting endoplasmic reticulum stress in insulin resistance Laia Salvado´1,2*, Xavier Palomer1,2*, Emma Barroso1,2, and Manuel Va´zquez-Carrera1,2 1

Department of Pharmacology and Therapeutic Chemistry, Faculty of Pharmacy, University of Barcelona, Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain 2 Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Diseases (CIBERDEM)-Instituto de Salud Carlos III, Madrid, Spain

The endoplasmic reticulum (ER) is involved in the development of insulin resistance and progression to type 2 diabetes mellitus (T2DM). Disruption of ER homeostasis leads to ER stress, which activates the unfolded protein response (UPR). This response is linked to different processes involved in the development of insulin resistance (IR) and T2DM, including inflammation, lipid accumulation, insulin biosynthesis, and b-cell apoptosis. Understanding the mechanisms by which disruption of ER homeostasis leads to IR and its progression to T2DM may offer new pharmacological targets for the treatment and prevention of these diseases. Here, we examine ER stress, the UPR, and downstream pathways in insulin sensitive tissues, and in IR, and offer insights towards therapeutic strategies. ER stress and UPR The underlying causes of IR are numerous and recent evidence suggests that the ER is involved in both the development of IR and its progression to T2DM (for review, see [1]). The ER performs important functions related to the synthesis, folding and transport of proteins [2], and also plays a critical role in lipid synthesis and Ca2+ homeostasis [3]. Therefore, one of the major ER responsibilities is to ensure that proteins achieve the structure that confers their activity. When the unfolded or misfolded protein load increases, these proteins are directed to the cytoplasm and degraded by the ER-associated degradation (ERAD) process (see Glossary). However, when the folding capacity of the ER cannot cope with the high load of unfolded and misfolded proteins, ER homeostasis is disturbed and ER stress is promoted. Several stimuli can trigger this situation, such as metabolic and calcium homeostasis disturbances, inflammation, or oxidative stress. To ameliorate ER stress and ensure correct protein folding, the ER activates the UPR (Box 1) to reduce the protein load [4]. This temporary adaptation is achieved by reducing Corresponding author: Va´zquez-Carrera, M. ([email protected]). Keywords: apoptosis; endoplasmic reticulum stress; inflammation; insulin resistance; type 2 diabetes mellitus; unfolded protein response. * These authors equally contributed to this review. 1043-2760/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2015.05.007

the rate of protein synthesis. A longer-term adaptation involves transcriptional activation of UPR target genes, including chaperones that participate in ER protein folding and proteins from the ERAD pathway. Finally, if ER homeostasis cannot be restored, cell death is induced. Unresolved ER stress and sustained activation of UPR contribute to IR The UPR is considered an acute mechanism that re-establishes cellular homeostasis; but, if sustained chronically, it can lead to different diseases: obesity, T2DM, atherosclerosis, heart and liver diseases, and neurodegenerative disorders, among others [5]. ER stress in obese mice was first observed in 2006, and it was proposed as a risk factor for IR [6]. As with many other signal transduction systems, the signal elicited by the UPR depends on the cell type and the ER stress trigger. Adipose tissue Obesity results in chronic ER stress in adipose tissue. Indeed, increased activation of inositol-requiring enzyme 1 alpha (IRE-1a) and c-Jun N-terminal kinase (JNK), and upregulation of X-box binding protein 1 (XBP1s) expression is observed in adipose tissue from obese patients Glossary Autophagy: a homeostatic process that involves cell degradation of unnecessary or dysfunctional cytoplasmic components ranging from protein aggregates to whole organelles through the action of lysosomes. Chaperone: a molecule that prevents protein misfolding by stabilizing folding intermediates and preventing their aggregation in the cell. It can be an endogenous protein, such as BiP/GRP78 or ORP150 (oxygen-regulated protein 150)] or pharmacological compounds, such as PBA or TUDCA. ER-associated degradation (ERAD): process whereby misfolded or unassembled proteins are eliminated from the ER. ER stress: a consequence of any perturbation that results in a high load of unfolded and misfolded proteins in the ER. The ER stress activates the UPR. 4-phenyl butyric acid (PBA): chemical chaperone that inhibits ER stress by improving ER protein folding capacity Thapsigargin: ER stressor which induces ER stress by inhibiting SERCA and, consequently, blocking the calcium entry into the ER lumen. Tauroursodeoxycholic acid (TUDCA): bile acid derivative that acts as a chemical chaperone to enhance protein folding and ameliorate ER stress. Tunicamycin: ER stressor that disrupts glycosylation of newly synthesized proteins resulting in ER stress. Unfolded protein response (UPR): adaptive (defensive) ER stress response that involves the activation of a signaling pathway with the purpose to restore the folding capacity. If ER homeostasis cannot be restored apoptosis is induced. Trends in Endocrinology and Metabolism xx (2015) 1–11

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Box 1. UPR signal transduction The UPR involves activation of three transmembrane proteins: IRE-1, which exists in a and b forms, ATF6, and PERK (Figure I). Under normal conditions, these proteins are associated with the luminal BiP [also known as 78-kDa glucose-regulated protein (GRP78)], and thus remain inactive. Under stress conditions, BiP is released and joins the increased unfolded or misfolded proteins in the ER lumen, where it acts as a chaperone, participating in protein folding. Consequently, a complex signaling cascade, involving IRE-1, ATF6, and PERK, is activated [5]. There are other proposed UPR sensors and mechanisms besides BiP, such as IRE-1b itself [82], which may directly interact through its luminal domain with unfolded proteins rather than BiP; the glycosylation status of ATF6 [83], which is greatly compromised during ER stress; or thioredoxin-interacting protein (TXNIP) [19], which regulates ER stress through protein disulfide isomerase (PDI) activation. IRE-1 is a transmembrane protein with an ER luminal domain that senses unfolded or misfolded proteins, and a cytoplasmic domain with both intrinsic kinase activity and ribonuclease (RNase) activity [5]. In homeostasis, IRE-1a is associated with BiP, whereas under stress conditions, IRE-1a is released from BiP, forming homodimers or oligomers, and subsequently trans-autophosphorylates at Ser724 [5]. Phosphorylation activates IRE-1a RNase activity, which removes 26 nucleotides from the X-box binding protein 1 (XBP1) mRNA. Consequently, the unspliced form (XBP1u) mRNA is converted to the shorter spliced XBP1 (XBP1s), which is translated to the transcription factor XBP1s. XBP1s increases the transcription of UPR genes [4], together with genes involved in adipogenesis, lipid metabolism and

inflammation [5]. In addition, via its endoribonuclease activity, IRE-1 produces either adaptive or death signals through regulated IRE-1-dependent decay of mRNA (RIDD), whose mRNA cleavage substrates include many genes belonging to the lipid metabolic pathways [84] or specific miRNAs that de-repress translation of proapoptotic genes [85]. ATF6 consists of a cytoplasmic domain and an ER luminal domain that senses protein-folding status. During ER stress, ATF6 is released from BiP and translocates to the Golgi where it is processed by site-1 protease (S1P) and S2P. The cytoplasmic part is liberated and acts as a transcription factor that regulates expression of XBP1 and other genes involved in ERAD processes and protein folding, maturation and secretion [4]. In normal conditions, BiP is also associated with the PERK monomer; under ER stress, PERK is released, homodimerizes and autophosphorylates, leading to activation of its cytoplasmic kinase domain. Activated PERK phosphorylates the a subunit of eIF2a at Ser51, which inhibits the guanine nucleotide exchange factor eIF2B. Thus, eIF2a phosphorylation results in minor translation initiation, thereby reducing the load of newly synthesized proteins [5]. Paradoxically, the PERK–eIF2a pathway facilitates translation and the consequent increase in ATF4 activity, which upregulates the expression of C/ EBP homologous protein (CHOP) and growth arrest and DNA damageinducible gene 34 (GADD34) [4]. PERK also phosphorylates and activates the antioxidant transcription factor nuclear factor erythroid 2 related factor 2 (Nrf2). It is accepted that the ATF6 and PERK pathways trigger apoptosis by increasing CHOP transcription.

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Figure I. Unfolded protein response signal transduction machinery. Illustrations showing the factors and pathways that function in the unfolded protein response induced by nonesterified fatty acid and hyperglycemia that drives the induction of genes involved in apoptosis. For a detailed description, see Box 1. Abbreviations: ATF, activating transcription factor; BiP, immunoglobulin binding protein; CHOP, C/EBP homologous protein; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GADD34, growth arrest and DNA damage-inducible gene 34; IRE-1, inositol-requiring enzyme 1; NEFAs, nonesterified fatty acids; Nrf2, nuclear factor erythroid 2 related factor 2; PDI, protein disulfide isomerase; PERK, protein kinase R (PKR)-like ER kinase; RIDD, regulated IRE-1-dependent decay; S1P, site-1 protease; TXNIP, thioredoxin-interacting protein; XBP1, X-box binding protein 1; XBP1s, spliced XBP1; XBP1u, unspliced XBP1.

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Figure 1. Model recapitulating the interplay between ER stress and IR in adipose tissue. During obesity, the release of NEFAs from visceral adipose tissue is an important factor that triggers ER stress, which induces inflammation through a PERK–eIF2a-dependent mechanism. The resultant increase in proinflammatory cytokine expression and secretion (e.g. TNF-a and IL-6) may induce ER stress in a loop manner perpetuating this process and the development of IR. Exposure of human adipocytes to LPS or high glucose levels potentiates the ATF6- and IRE-1a-dependent chaperone expression. In addition, Perilipin A is inhibited by ER stress, resulting in the activation of TG lipolysis in adipocytes and subsequent intracellular FA increase. Finally, ER stress induces TXNIP expression, which acts as a key negative feedback regulatory mechanism of the unfolded protein response to decrease XBP1s levels via direct PDI activation. Abbreviations: ATF, activating transcription factor; ATGL, adipose triglyceride lipase; DAG, diacylglycerol; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; FA, fatty acid; GLUT4, glucose transporter 4; HSL, hormone sensitive lipase; IKK, IkB kinase; IL, interleukin; IR, insulin resistance; IRE-1, inositol-requiring enzyme 1; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAG, monoacylglycerol; MGL, monoacylglycerol lipase; NEFA, non-esterified fatty acid; NF-kB, nuclear factor-kB; PDI, protein disulfide isomerase; PERK, protein kinase R (PKR)like ER kinase; S, serine; S1P, site-1 protease; TG, triglyceride; TLR4, Toll-like receptor 4; TNF-a, tumor necrosis factor a; TXNIP, thioredoxin-interacting protein; XBP1, X-box binding protein 1; Y, tyrosine.

[7]. Interestingly, the loss of fat in obese patients following gastric bypass surgery reduces XBP1s and immunoglobulin binding protein (BiP) expression in white adipose tissue, as well as the phosphorylation levels of alpha subunit of eukaryotic initiation factor 2 (eIF2a) and JNK [8]; whereas exercise training decreases ER stress and IR in white adipose tissue of obese rodents [9]. In vivo experiments have demonstrated an important role for ER stress in high fat diet (HFD)-induced inflammation [10]. Release of nonesterified fatty acids (NEFAs) from the visceral adipose tissue of obese individuals can trigger ER stress [10], which increases the expression of cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-6 through a protein kinase R (PKR)-like ER kinase (PERK)-dependent mechanism (Figure 1) [11]. This increase in cytokine expression can induce positive ER stress feedback (Box 2, [12]), perpetuating the process and the development of IR. Activated PERK also modulates insulin responsiveness in adipose tissue. Thus, PERK harbors intrinsic lipid kinase that favors diacylglycerol (DAG) as a substrate to generate phosphatidic acid, which inhibits insulin action [13]. Moreover, PERK activity may

promote adipocyte differentiation and protein kinase B (PKB)/Akt activity. ER stress can also induce lipolysis by downregulating the lipid-droplet-associated protein perilipin A in adipocytes [14], suggesting that, once initiated, ER stress feeds itself. In addition to saturated NEFAs, human adipocytes exposed to lipopolysaccharide or glucose increase the expression of activating transcription factor (ATF)6- and IRE-1a-dependent chaperones [15]. The activation of IRE-1a in adipose tissue during obesity could be responsible for JNK activation, which in turn can phosphorylate insulin receptor substrate (IRS)-1 on serine residues to promote IR [10]. Studies in human beings suggest that hyperinsulinemia, which is often observed in obesity, causes ER stress in adipose tissue, possibly via enhancement of protein biosynthesis and post-translational protein modification that leads to the accumulation of unfolded/ misfolded and ubiquitinated proteins [16]. Very low-density lipoproteins (VLDLs), through the VLDL receptor (VLDLR), participate in adipocyte hypertrophy, a fact which, together with macrophage infiltration, is crucially involved in adipose tissue inflammation 3

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Box 2. Interplay between inflammatory pathways and ER stress Chronic low-grade inflammation is a major cause of obesity-induced IR and, interestingly, the three branches of the UPR are linked to inflammation (Figure I) [86]. The transcription factors activator protein-1 (AP-1) and NF-kB control the expression of most immunomodulatory genes. AP-1 is a heterodimeric transcription factor composed of members belonging to the Jun, Fos, ATFs, and Jun-dimerization partner families. Phosphorylated JNKs activate c-Jun, which upon dimerization with its principal partner c-Fos, results in the activation of AP-1. NF-kB consists of five members: p65/RelA, RelB, c-Rel, NF-kB1 and NF-kB2, which form either homodimers or heterodimers. In mammals, the most abundant form of the NF-kB family is the p65/p50 heterodimer. In resting cells, NFkB is present in the cytoplasm as an inactive heterodimer bound to an inhibitor protein subunit, IkB. After stimulation, the classical signaling pathway involves the activation of IkB kinase (IKK) complex, which phosphorylates IkBs. Phosphorylation of IkBs induces ubiquitination and its subsequent proteasome-mediated degradation, releasing NF-kB, which then translocates into the nucleus to begin the transcription machinery. During ER stress, PERK–eIF2a pathway activation and, thus, protein translation inhibition, together with the shorter half-life of IkBa compared with that of NF-kB, result in a reduction of the IkBa/NF-kB ratio,

PERK p

PKR

leading to nuclear NF-kB translocation and consequent increase in the expression of proinflammatory genes [86]. After induction by several proinflammatory stimuli (fatty acids, ceramides, and lipopolysaccharide), protein kinase R (PKR) may activate JNK and eIF2a itself to perpetuate the inflammatory pathways. Besides this, activated IRE1a recruits TNF-a-receptor-associated factor 2 (TRAF2) and this complex activates JNK and IKKb, by this means activating AP-1 and NF-kB. Moreover, XBP1s, generated by the IRE-1a RNase activity, also participates in the inflammatory response, increasing the transcription of proinflammatory cytokines, which can themselves activate ER stress and maintain the inflammatory state. The PERK pathway is also related to the inflammatory process through its regulation of ATF5 transcription and translation. ATF5 is involved in TXNIP regulation under ER stress conditions, which induces inflammation through the nucleotidebinding oligomerization domain (NOD)-like receptor (NLR) family, pyrin-domain containing 3 (NLRP3) inflammasome activation and inflammatory cytokine production [87]. Similarly, IRE-1a is also capable of activating the TXNIP–NLRP3 pathway, resulting in an inflammatory process [88]. Finally, the ATF6 branch of the UPR also induces inflammation through NF-kB activation, but the mechanisms involved are currently unknown [86].

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Figure I. Inflammatory pathways and ER stress. Illustration shows the inflammatory pathways activated during ER stress that lead to the increased expression of inflammatory markers. For a detailed description, see Box 2. Abbreviations: AP1, activator protein-1; ATF, activating transcription factor; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; IKK, IkB kinase; IL, interleukin; IRE-1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal kinase; NEFAs, nonesterified fatty acids; NF-kB, nuclear factor-kB; NLRP3, nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin-domain containing 3 inflammasome; PERK, protein kinase R (PKR)-like ER kinase; TNF, tumor necrosis factor; TRAF2, TNF receptor-associated factor 2; TXNIP, thioredoxin-interacting protein; XBP1, X-box binding protein 1; XBP1s, spliced XBP1; XBP1u, unspliced XBP1.

and metabolic dysfunction during obesity. Interestingly, recent data reveal that VLDLR deficiency strongly decreases HFD-induced lipid accumulation, inflammation, and ER stress in adipose tissue, in conjunction with reduced macrophage infiltration [17]. Another important 4

player in the interplay between IR and ER stress in adipose tissue is the thioredoxin interacting protein (TXNIP), a mediator of IR and regulator of adipogenesis that inhibits glucose uptake in fat and muscle [18]. Moreover, and as depicted above (Box 1), TXNIP regulates ER stress through

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Figure 2. Unfolded protein response promotes hepatic IR by regulating gluconeogenesis and lipogenesis. IR prevents FOXO1 phosphorylation by inducing protein kinase B/Akt activity, thereby increasing FOXO1 nuclear translocation and the subsequent increase in the transcription of gluconeogenic genes. Interestingly, PERK may oppose insulin responsiveness through phosphorylation of FOXO. CREBH, a liver-specific ATF6 homolog induced by IR, is activated by ER stress and increases the expression of the gluconeogenic genes PEPCK and glucose 6 phosphatase. By contrast, XBP1 inhibits gluconeogenesis by promoting proteasome-mediated degradation of FOXO1. ER stress also plays a key role in the regulation of lipogenesis: ER stress activates the lipogenic transcription factor SREBP-1c through the activation of PERK and XBP1s-mediated Insig inhibition. Increased accumulation of TG in liver may also occur as a result of increased expression of VLDLR through the activation of ATF4. This receptor increases the delivery of TG to the cell and modulates the extrahepatic metabolism of TG-rich lipoproteins (VLDLs). Abbreviations: ATF, activating transcription factor; CHOP, C/EBP homologous protein; CREBH, cAMP-response element-binding protein H; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; FOXO1, forkhead box O1; Insig, insulin-induced gene; IR, insulin resistance; IRE-1, inositol-requiring enzyme 1; IRS, insulin receptor substrate; PERK, protein kinase R (PKR)-like ER kinase; S, serine; SREBP1c, sterol regulatory element-binding protein 1c, TG, triglyceride; VLDL, very low-density lipoprotein; VLDLR, VLDL receptor; XBP1, X-box binding protein 1; Y, tyrosine.

protein disulfide isomerase (PDI) activation. Interestingly, changes in PDI activity are associated with protein misfolding and ER stress, and TXNIP is one of the most strongly induced proteins in diabetic patients [19]. Liver ER stress contributes to hepatic IR by activating transcription factors that regulate the expression of gluconeogenic genes (Figure 2). However, depending on which UPR branch is activated, gluconeogenesis could be potentiated or inhibited. For instance, the cAMP-response element-binding protein H (CREBH), a rather liver-specific ATF6 homolog induced by IR, is activated by ER stress and increases the expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), along with inflammatory markers such as C-reactive protein (CRP) [20]. By contrast, the XBP1 pathway induces proteosomal degradation of forkhead box (Fox)O1, resulting in reduced gluconeogenesis [21].

ER-stress-induced hepatic IR may also be the result of increased lipogenesis, which favors intracellular accumulation of intermediary complex lipids [e.g., diacylglycerol (DAG) and ceramides]. Specifically, DAG accumulation is toxic for the hepatocyte and is pivotal for ER stress to cause IR and hepatic steatosis [22]. In this regard, exposure of HepG2 cells to hyperglycemia or the saturated fatty acid palmitate, induced ER stress, activated the transcription factor sterol regulatory element binding protein (SREBP)1, and enhanced lipid accumulation [23]; all regulated by the PERK–eIF2a pathway. This pathway may also account for ER-stress-induced hepatic steatosis through the increased expression of hepatic VLDLR, which promotes lipoprotein delivery to the liver [24]. SREBP-1c, which is induced in the liver of obese rodents, is proteolytically activated during ER stress, thereby increasing the transcription of lipogenic genes including fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD)1 [25]. Strikingly, the mammalian target of rapamycin (mTOR) complex 1 5

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Figure 3. UPR contributes to skeletal muscle IR. The saturated FA palmitate induces IR in myotubes through phosphorylation of IRS proteins on serine residues by the IRE-1/ JNK pathway. By contrast, monounsaturated fatty acids (i.e., oleate) do not cause ER stress in myotubes and, indeed, prevent palmitate-induced ER stress, inflammation and IR through an AMPK-dependent mechanism. Additional mechanisms that can also contribute to the reduction in insulin sensitivity following ER stress involve TRB3 and PTP1B. The ER stress inducers thapsigargin and tunicamycin increase the activity of the protein kinase TRB3, which inhibits Akt phosphorylation and, thus, Akt activity. On the other hand, PTP1B acts as a negative regulator of insulin pathway through hydrolysis of insulin receptor b and IRS1. Moreover, PTP1B is able to dephosphorylate Akt, a fact which results in IR. The ER stress inhibitor TUDCA is able to reduce UPR markers, as well as PTP1B protein activity in skeletal muscle cells. Abbreviations: AMPK, AMP-activated protein kinase; ER, endoplasmic reticulum; FA, fatty acid; IR, insulin resistance; IRE-1, inositol-requiring enzyme 1; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP, phosphatidylinositol; PTP1B, protein-tyrosine phosphatase 1B; S, serine; TRAF2, TNF receptor-associated factor 2; TRB3, tribbles homolog 3; TUDCA, tauroursodeoxycholic acid; UPR, unfolded protein response; Y, tyrosine.

(mTORC1), a key regulator of SREBP-1 expression, activates ER stress to promote hepatic IR and lipid accumulation [23]. Additional mechanisms involved in SREBP-1c activation by ER stress include rapid degradation of its upstream inhibitor insulin-induced gene 1 (Insig1) [26] and direct transcriptional activation of the SREBP-1c gene by the IRE-1a–XBP1 pathway [27]. Of note, XBP1 is also capable of enhancing the degradation of lipogenic mRNAs through a SREBP-1c-independent mechanism, in a process known as regulated IRE-1-dependent decay (RIDD, Box 1) [28]. Notwithstanding the foregoing, some authors support the hypothesis that hepatic IR is secondary to ER stress modulation of lipogenesis [29], while others consider that fat accumulation per se does not induce IR [30]. The latter hypothesis was suggested after demonstrating that knockout mice for the C/EBP homologous protein (CHOP) gene, which is induced by ER stress, displayed normal glucose tolerance and insulin sensitivity despite marked obesity. This discrepancy was accompanied by diminished inflammation in fat and liver of knockout mice, thus reinforcing the idea that IR is not induced by fat accumulation by itself, but rather by inflammatory processes controlled by CHOP [30]. By contrast, it has been reported that PERK may oppose to insulin responsiveness through phosphorylation of the forkhead transcription factor FOXO in hepatic cells 6

[31]. Since insulin signaling, via Akt, reduces FOXO activity and promotes insulin responsiveness, it has been suggested that inhibition of PERK might improve insulin signaling in the liver. Fibroblast growth factor (FGF)21 is a hormone predominantly secreted by the liver that carries out a wide range of effects on metabolism. It is worth mentioning that recent studies indicate that ER stress [32] and autophagy deficiency [33] induce FGF21 expression via the IRE-1a–XBP1 and PERK–ATF4 pathways, respectively [32]. Moreover, obesity leads to downregulation of autophagy and reduction in the activity of the proteasome in liver, which in turn leads to activation of chronic UPR, hepatic IR, and increased glucose production [34,35]. Interestingly, administration of recombinant FGF21 or restoration of autophagy in liver of obese mice alleviated tunicamycin-induced liver ER stress and steatosis, and enhanced both hepatic insulin action and systemic glucose tolerance [32]. As FGF21 exerts beneficial effects on lipid metabolism through counteracting ER stress, it is feasible that it may display antiobesity and antidiabetic activity. Skeletal muscle Skeletal muscle plays a major role in whole-body nutrient balance since it accounts for the majority of insulin-stimulated glucose utilization and, therefore, it is the primary

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pathway is the inhibition of PKB/Akt. There is no easy explanation for this discrepancy, but differences in the fatty acid–bovine serum albumin (BSA) conjugation could be involved. Further mechanisms that could also contribute to the reduction in insulin sensitivity following ER stress involve tribbles (TRB)3 and protein tyrosine phosphatase (PTP)1B. Thapsigargin and tunicamycin increase TRB3 and impair insulin signaling and glucose uptake in myotubes and mouse skeletal muscle, and these adjustments are reversed in cells by silencing TRB3 expression and in the muscles of TRB3-deficient mice [43]. PTP1B is tethered to the ER membrane in mammalian cells and acts as a negative regulator of the insulin pathway by hydrolyzing the tyrosine phosphorylated insulin receptor b and IRS-1 [44]. Panzhinskiy et al. demonstrated that HFD induced UPR markers as well as PTP1B protein levels in skeletal muscle, and that these increases were prevented by treatment with the ER chaperone tauroursodeoxycholic acid (TUDCA) [44]. TUDCA is a taurine conjugate of ursodeoxycholic acid that reduces ER stress probably by acting as chemical chaperone, thus preventing protein misfolding and aggregation and facilitating protein trafficking and secretion in the cell. Interestingly, tunicamycin-induced phosphorylation of eIF2a and JNK2 was significantly

site of IR in obesity and T2DM. Interestingly, potent chemical ER stressors, such as tunicamycin and thapsigargin, can enhance ER-stress-induced IR in myotubes through phosphorylation of IRS proteins via the IRE-1/ JNK pathway (Figure 3) [36–38]. ER stress has been reportedly involved in the association between saturated NEFA-induced inflammation and IR. Direct exposure of myotubes to the saturated FA palmitate induces ER stress [38,39]. Likewise, feeding a HFD to rodents, or genetic models of diabetes, also show ER stress in skeletal muscle [37–39]. In contrast to palmitate, the monounsaturated FA oleate did not cause ER stress in human and murine myotubes and, indeed, co-incubation with oleate prevented palmitate-induced ER stress, inflammation and IR [38]. The protective effect of oleate in palmitate-induced ER stress was due to activation of AMP-activated protein kinase (AMPK) [38]. AMPK exhibits multiple protective effects, including inhibition of inflammation, oxidative stress, and IR, besides attenuating ER stress [40–42], thereby reducing the risk for developing obesity and T2DM. In contrast to these results, Hage-Hasan et al. [39] found that ER stress was not the molecular mechanism responsible for palmitate-induced IR in muscle cells. Instead, they proposed that the main site of action of palmitate in downregulating the insulin signaling

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Figure 4. Mechanisms by which unfolded protein response activation promote b cell failure and apoptosis. Lipotoxicity is one of the most important triggers of ER stress in pancreatic b cells. Saturated FAs (e.g., palmitate) activate the PERK and IRE-1 pathways of the UPR, thereby inducing, respectively, eIF2a phosphorylation and XBP1 splicing. By contrast, stimulation with oleate (monounsaturated FA) displays protective effects. Accumulation of misfolded proinsulin and toxic aggregates derived from misfolded hIAPP also induce ER stress, thus being involved in b cell dysfunction and apoptosis. Inflammation arising from IL-23, IL-24 and IL-33 induce oxidative and ER stress in b cells via activation of STATs and NF-B. The ER stress sensor TXNIP is another relevant pro-apoptotic b cell factor, which is induced by hyperglycemia. Likewise, ASK1, through the activation of IRE-1, contributes to b cell failure and apoptosis. Abbreviations: ASK1, apoptosis signal-regulating kinase; ATF, activating transcription factor; Bcl-2, B-cell lymphoma 2; CHOP, C/EBP homologous protein; eIF2a, eukaryotic initiation factor 2a; ER, endoplasmic reticulum; FA, fatty acids; hIAPP, human islet amyloid polypeptide; IL, interleukin; IRE-1, inositol-requiring enzyme 1; JAK, janus kinase; NEFA, non-esterified fatty acid; NF-kB, nuclear factor-kB; PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase; STATs, signal transducers and activators of transcription; TXNIP, thioredoxin-interacting protein; XBP1, X-box binding protein 1.

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Review attenuated in PTP1B-deficient mice, and treatment with TUDCA or silencing PTP1B reversed the reduction in myotube glucose uptake caused by tunicamycin [44]. These findings suggest that PTP1B is activated by ER stress and is required for full activation of ER stress pathways that mediate IR in skeletal muscle. Pancreatic b cells The involvement of ER stress in b cell apoptosis and failure is demonstrated by the fact that 4-phenylbutyrate (PBA) partially prevents lipid-induced b cell dysfunction in overweight subjects [45]. Although not completely elucidated, the mechanism of action of PBA seems to rely on its ability to act as a molecular chaperone, thus assisting in protein folding and trafficking, and preventing protein aggregation. There are many potential triggers of ER stress that may be involved in b cell dysfunction in T2DM, including lipotoxicity, glucotoxicity, inflammatory challenge, and the accumulation of proinsulin and amyloid (Figure 4). Saturated FAs are generally cytotoxic, whereas unsaturated FAs protect against b cell apoptosis by suppression of ER stress [46]. Stimulation of lipid metabolism (i.e., lipogenesis and fatty acid oxidation) by oleate, which probably causes ER stress reduction, might account for b cell defense against palmitate-induced lipotoxicity [47,48]. The ER stress sensor is another relevant proapoptotic b cell factor, induced by hyperglycemia [49], whose control may represent a novel therapeutic approach to protect b cells from apoptosis and preserve insulin-producing b cell mass in diabetic patients. The stress-activated apoptosis signalregulating kinase (ASK)1, through the activation of IRE-1, has also been recently demonstrated to contribute to b cell failure and apoptosis [50]. A recent study has identified a pro-survival role for ATF6b in pancreatic b cells by transcriptionally regulating the Wolfram syndrome 1 (WFS1) gene [51]. Depletion of ATF6b in INS-1 insulinoma cells demonstrated that this transcription factor is essential to maintain cell survival in b cells undergoing chronic ER stress. In fact, mutations in this gene cause Wolfram syndrome, a genetic form of diabetes caused by non-autoimmune loss of b cells, whereas WFS1 polymorphisms are associated with T2DM [52]. Misfolded protein (e.g., proinsulin) accumulation in the ER leads to b cell death through PERK-mediated phosphorylation of eIF2a and ensuing transcriptional induction of ATF4 and the proapoptotic gene CHOP. Insulin synthesis begins in the ER, where nascent proinsulin must fold correctly for efficient export from the ER to the Golgi, where it is packaged and subsequently cleaved to form mature insulin [53]. Under conditions of increased proinsulin synthesis, as occurs in hyperinsulinemic states before the development of overt T2DM, misfolded proinsulin accumulates in the ER of pancreatic b cells, thereby leading to the activation of UPR pathways that may culminate in b cell apoptosis [54]. The synthesis of toxic protein aggregates, called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP), which occurs in 90% of patients with T2DM, activates the UPR and is also involved in b cell dysfunction. Notably, attenuation of ER stress with different chaperones increased insulin secretion in cells expressing hIAPP [55]. 8

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ER stress also correlates with inflammation in pancreatic b cells. IL-23, IL-24, and IL-33, via activation of signal transducers and activators of transcription (STATs) and nuclear factor (NF-kB), are the most potent inflammatory cytokines that induce oxidative and ER stress in b cells [56]. By contrast, IL-22, by repressing the production and accumulation of reactive oxygen species and nitrite in b cells, prevents ER stress induced by cytokines or glucolipotoxicity. Specifically, IL-22 downregulated the expression of pro-oxidative genes, while upregulated the antioxidant genes [56]. Strategies aimed at neutralizing IL-23 or IL-24 in obese mice partially improved b cell ER stress and glucose intolerance; whereas IL-22 administration suppressed ER stress and inflammation, ameliorated insulin secretion and restored insulin sensitivity [56]. Overproduction in mice of the resident ER chaperone immunoglobulin binding protein (BiP) in pancreatic b cells protects against HFD-induced obesity, glucose intolerance, hyperinsulinemia, and IR [57]. Prolonged HFD feeding is associated with reduced levels of the b cell glucose transporter GLUT2, which is synthesized in the ER and is essential for glucose-stimulated insulin secretion. Interestingly, islet insulin content and GLUT2 levels were normalized in transgenic mice overexpressing BiP. Maintenance of proper folding of proinsulin [58] or other ERsynthesized proteins [59] might contribute to improved b cell function in these mice. Therapeutic strategies against ER stress in IR Several strategies have been proposed to target ER stress as a therapeutic approach for pharmacological intervention in IR and T2DM [60]. One of these strategies is the therapeutic use of chemical chaperones. Reducing chronic ER stress by the administration of PBA or TUDCA increased systemic insulin sensitivity, normalized hyperglycemia and reduced hepatosteatosis in murine models of obesity and T2DM [6]. In humans, PBA prevents lipidinduced IR and restores b cell functionality [45], and administration of TUDCA to obese and insulin-resistant patients ameliorated insulin sensitivity in skeletal muscle and liver [61], thus suggesting that ER stress inhibition by these drugs might be a viable therapeutic strategy towards increasing insulin sensitivity. However, since TUDCA did not reduce ER stress in obese patients [61], it is feasible that a slight, undetectable reduction in ER stress was enough to reduce IR, or that the antidiabetic effects of TUDCA are independent of ER stress inhibition [62]. In fact, PBA and TUDCA may activate peroxisome-proliferator activated receptor (PPAR)a [63] and PPARg [64], suggesting that additional mechanisms may contribute to the antidiabetic effects of these drugs. Treatments aimed at increasing BiP protein levels can also attenuate ER stress. As depicted above, overproduction of BiP in mouse pancreatic b cells protected against HFD-induced obesity, glucose intolerance, hyperinsulinemia, and IR [57]. Activation of AMPK protects against lipid-induced hepatic disorders by reducing ER stress [42], thus being another potential target to reduce ER stress in metabolic diseases. In the liver, AMPK prevents HFD-induced lipid accumulation and IR by inhibiting ER stress-induced SREBP-1 activation [23]. Metformin is one of the leading

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Review antidiabetic drugs and, although its molecular target has not yet been unequivocally elucidated, it increases AMPK activity [65]. Several studies have demonstrated that metformin attenuates ER stress as well as the subsequent apoptosis and serine phosphorylation of IRS-1 in b cells exposed to chemical ER stressors and palmitate [66]. Metformin, as well as the AMPK activator 50 -aminoimidazole4-carboxymide-1-b-d-ribofuranoside (AICAR), increased the protein levels of BiP, while reducing CHOP proapoptotic protein levels in cardiac cells [67]. Also, glucagon-like peptide-1 agonists, which are currently used in the treatment of T2DM, protect pancreatic b cells from lipotoxic ER stress through upregulation of BiP [68]. A recent study indicates that PPARb/d could also be involved in the antidiabetic effects of metformin [69]. Taking into account that AMPK complexes with PPARb/d and potentiates its transcriptional activity [70], it has been proposed that metformin prevents ER stress and oxidative stress in endothelial cells by activating the AMPK–PPARb/d complex [71]. Interestingly, part of the anti-inflammatory and antidiabetic effects following PPARb/d activation in skeletal muscle cells involved inhibition of ER stress and were dependent on AMPK [72]. This is consistent with the reported negative crosstalk between AMPK and extracellular signal-regulated kinase (ERK)1/2 and may also contribute to the effects observed, since ERK1/2 inhibition was found to improve the AMPK and Akt pathways, and to reverse ER stress-induced IR in skeletal muscle cells [73]. During ER stress, Ca2+ released from the ER leads to mitochondrial damage, with subsequent activation of the proapoptotic pathway. Disturbances in hepatic lipid composition during obesity in mice inhibit the sarcoplasmic/ER Ca2+ ATPase (SERCA), which induces ER stress and disrupts glucose homeostasis in the liver [74]. Indeed, SERCA overexpression improves glucose metabolism in obese mice [75]. On the other hand, Ca2+ channel blockers, including antihypertensive drugs (diltiazem and verapamil) or the muscle relaxant dantrolene, inhibit ER calcium efflux and induce a subset of molecular chaperones, overall enhancing ER protein folding capacity [60]. Therefore, regulators of calcium homeostasis are postulated as promising therapeutic tools for preventing ER stress during metabolic diseases. Under ER stress conditions, the UPR initially activates an adaptive program that decreases the protein load in the lumen of the ER to restore homeostasis but, under irremediable ER stress, the UPR activates a proinflammatory and pro-death program. Thus, another therapeutic approach may be to prolong the initial adaptive program of the UPR, which would include XBP1 and ATF6 transcription factors. Taking into account that ER stress hyperactivates both IRE-1a and PERK, leading to apoptosis, the inhibition of this hyperactivated state, using PERK or IRE-1a inhibitors [62,76], might be an additional therapeutic approach. Park and Ozcan proposed that IRE-1a kinase inhibition, but not its RNase activity may be a viable therapeutic strategy to reduce ER stress-induced IR [62]. IRE-1a has two catalytic domains and it is known that the kinase domain can be occupied by a kinase inhibitor called 1NM-PP1, thereby bypassing IRE-1a autophosphorylation. This triggers RNase activity, which prompts splicing of XBP1 mRNA without ER stress, leading to activation of the pro-survival XBP1

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transcription factor. Regarding PERK, since hyperactivated PERK–eIF2a–CHOP signaling may contribute to b cell death, IR and hepatosteatosis, a well-controlled modulation of this UPR branch is peremptory. PERK inhibitors (e.g., GSK2606414) [77] are a promising approach to achieve this goal. Also, there is currently in the market an anti-hypertensive a2-adrenergic receptor agonist, Guanabenz, which selectively prolongs eIF2a phosphorylation-mediated adaptive UPR signaling [78]. Autophagy plays a protective role in ER stress-mediated pancreatic b cell death and the design of agents that enhance autophagic activity in b cells might serve as a novel approach for the treatment of diabetes (for review see [79]). Interestingly, PPARb/d activation prevented palmitate- or thapsigargin-induced ER stress in a human cardiomyocyte cell line in an AMPK-independent manner, and this effect was associated with an increase in the protein levels of autophagy markers [80]. In accordance with this, PPARb/ d-deficient mice exhibited increased ER stress in the heart and displayed a reduction in autophagic markers [80]. Although pharmacological inhibition of ER stress is of great interest for diabetes treatment, it is important to consider that depending on the UPR branch affected, the inhibition may either aggravate or ameliorate metabolic dysregulation. For instance, XBP1s overexpression in liver improves glucose tolerance in obese and diabetic ob/ob mice [21]. In addition, it has been reported that phosphorylation of XBP1s on Thr48 and Ser61 residues by p38 mitogenactivated protein kinase (p38 MAPK) increases its nuclear translocation, reduces ER stress, and improves hyperglycemia in obese and diabetic mice [81]. Since hepatic p38 MAPK activity is reduced in obese mice, the authors of the study suggested that increasing its activity in liver and the subsequent XBP1s nuclear translocation might emerge as a therapeutic approach for the treatment of T2DM. In a similar way, fenofibrate, a PPARa agonist that reduces lipid accumulation in the liver, abrogated high fructose diet-induced glucose intolerance hepatic steatosis, and corrected impaired hepatic insulin signaling, in spite of both the IRE-1/XBP1 and PERK/eIF2a arms of the UPR were activated [22]. Concluding remarks and future perspectives Recent findings demonstrate that protein misfolding in the ER and the subsequent activation of UPR signaling play critical roles in the pathogenesis of IR and T2DM. Targeting protein folding and UPR signaling might be a viable therapeutic approach for the treatment of T2DM. Moreover, some of the metabolically beneficial effects of the drugs currently used in the treatment of diabetes may result from their ability to modulate ER stress or the UPR. However, more studies are needed to elucidate which arms of the ER stress process are susceptible to being targeted, since paradoxically, activation of some key proteins involved in the UPR might be beneficial in T2DM treatment. Acknowledgments We apologize for contributors to this field not cited owing to space restrictions. Funding support for the authors is from the Ministerio de Economı´a y Competitividad of the Spanish Government [SAF2012– 30708]; and CIBER de Diabetes y Enfermedades Metabo´licas Asociadas (CIBERDEM). CIBERDEM is an initiative of the Instituto de Salud 9

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Review Carlos III (ISCIII) - Ministerio de Economı´a y Competitividad. We thank the University of Barcelona’s Language Advisory Service for revising the manuscript.

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