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Endoplasmic reticulum stress-induced apoptosis in the development of diabetes: is there a role for adipose tissue and liver?

Diabetes mellitus (DM) is a multifactorial chronic metabolic disease characterized by hyperglycaemia. Several different mechanisms have been implicated in the development of the disease, including endoplasmic reticulum (ER) stress. ER stress is increasingly acknowledged as an important mechanism in the development of DM, not only for β-cell loss but also for insulin resistance. Accumulating evidence suggests that ER stress-induced apoptosis may be an important mode of β-cell loss and therefore important in the development of diabetes. Recent data also suggest a role of ER stress-induced apoptosis in liver and adipose tissue in relation to diabetes, but more extensive studies on human adipocyte and hepatocyte (patho)physiology and ER stress are needed to identify the exact interactions between environmental signals, ER stress and apoptosis in these organs.

Introduction
Diabetes mellitus (DM) is a multifactorial chronic metabolic disease characterized by hyperglycaemia. Several different mechanisms have been implicated in the development of the disease. Although the precise molecular events underlying the different forms of diabetes still remain unclear, it is generally accepted that the underlying defects include decreased secretion of insulin, its impaired signalling or both. Type 1 diabetes (T1DM) is known to result from an excessive loss of pancreatic b-cells, leading to insulin deficiency. Among other important causes, autoimmune and inflammatory processes have been reported to disrupt b-cells, cause insulin deficiency and hyperglycaemia and subsequently T1DM. Type 2 diabetes (T2DM), the most common form of diabetes, is characterized by impaired insulin action (insulin resistance) paralleled by impaired insulin secretion and a progressive decline in b-cell function. Insulin resistance, often associated with obesity and physical inactivity, is a major factor in the progression of T2DM. Obesity is a well-known risk factor for the development of T2DM. Importantly, obesity is not only associated with lipid accumulation in adipose tissue, but also in non-adipose tissues, such as liver and muscle. Lipid accumulation in non-adipose tissue, also known as ectopic lipid accumulation, has also been associated with the development of insulin resistance. Therefore, muscle, adipose tissue and liver are, beside pancreas, crucial tissues contributing to the development of insulin resistance and thus to the development of T2DM. A relatively new player in the DM field is endoplasmic reticulum (ER) stress. ER stress and/or ER stress-induced apoptosis are increasingly acknowledged as important mechanisms in the development of DM, not only for b-cell loss but also for insulin resistance. Since the last decade, it has been generally accepted that ER stress plays an important role in b-cell function and loss [1]. This is for instance illustrated in Akita mice [2,3], and the Wolcott-Rallison syndrome [4,5]. Akita mice spontaneously develop diabetes with significant early loss of pancreatic b-cell mass resulting from a missense mutation (Cys96Tyr) in the insulin 2 gene that disrupts a disulfide bond between A and B chains of insulin [6]. The Wolcott-Rallison syndrome is a rare autosomal-recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, and growth retardation, and other variable multisystemic clinical manifestations. The gene responsible for this syndrome is EIF2AK3 (PERK), the pancreatic eukaryotic initiation factor 2 (eIF2) kinase [4,5]. More recently, it was acknowledged that high fatand obesity-induced insulin resistance is also associated with ER stress in adipose tissue and liver [7,8]. Remarkably, until now no studies have demonstrated a role for ER stress in skeletal muscle in relation to (the development of) obesity or diabetes [7,9]. Besides, the role of ER stress in the development of diabetes that will be discussed in this paper, there is also evidence that diabetes can induce or aggravate ER stress and thereby affect the complications of diabetes, such as renal disease, retinopathy and vascular abnormalities [10][11][12].
In this review an overview of ER stress, the unfolded protein response (UPR), and ER stress induced apoptosis is given (see also refs [13][14][15][16][17][18][19][20]) with a further focus on the possible role of ER stress-induced apoptosis in the liver and adipose tissue in the onset of diabetes.
Endoplasmic reticulum stress-unfolded protein response The endoplasmic reticulum (ER) is an important organelle required for cell survival and normal cellular function. In the ER, nascent proteins are folded with the assistance of ER chaperones (i.e. molecular chaperones and folding enzymes). Subsequently, correctly folded proteins are transported to the Golgi apparatus. Unfolded and misfolded proteins, on the other hand, are retained in the ER, retrotranslocated to the cytoplasm by the machinery of ER associated degradation (ERAD), and degraded by the proteasome. As a major intracellular calcium storage compartment, the ER also plays a critical role towards maintenance of cellular calcium homeostasis. In addition, the ER also has a role in lipid biosynthesis, e.g. lipid membrane synthesis and controlling the synthesis of cholesterol and other membrane lipid components. ER stress is caused by perturbations of any of the three homeostatic functions of the ER, i.e. functioning as a site for protein folding, for synthesis of unsaturated fatty acids (FA), sterols, and phospholipids and for intracellular Ca 2? storage. ER stressors include: (1) disturbances in cellular redox regulation caused by hypoxia, oxidants, or reducing agents, which interfere with disulfide bonding of proteins in the lumen of the ER, (2) glucose deprivation, probably by interfering with N-linked protein glycosylation in the ER, (3) disruption of Ca 2? metabolism causing impaired functions of Ca 2? dependent chaperones such as Grp78, Grp94 and calreticulin, (4) viral infections, which overload the ER with virus encoded proteins, (5) high fat diet, and (6) protein mutations that hamper adequate folding [17,18]. The consequence of ER stress is an overwhelmed or compromised ability of the ER to properly fold proteins.
Accumulation of unfolded and/or misfolded proteins in the ER lumen is a hallmark of perturbation of any of the three functions of the ER and results in activation of the unfolded protein response (UPR). The UPR is a complex and coordinated adaptive signalling mechanism to re-establish homeostasis of ER functions (Fig. 1). ER stress sensors [IRE1 (inositol requiring 1), ATF6 (activated transcription factor 6) and PERK (ER-resident PKR-like eIF2a kinase)] detect the accumulation of unfolded and/or misfolded protein at the onset of ER stress and initiate the UPR. To re-establish homeostasis and normal ER function, the UPR initiates a global decrease in protein synthesis while increasing the production of ER chaperone proteins and ER-associated degradation (ERAD).
The mammalian UPR with its signalling components is complex, diverse and flexible as has been described in great detail in recent reviews [16,20]. In short, UPR signals through three pathways, that each utilizes one of the three ER stress sensors IRE1, ATF6 and PERK (Fig. 2). IRE1 is a transmembrane kinase/endoribonuclease (RNAse). Activation of IRE1 initiates the nonconventional splicing of Xbp-1 mRNA. Spliced Xbp-1 mRNA encodes a transcription activator that drives transcription of genes such as ER chaperones, whose products directly participate in ER protein folding. PERK is a transmembrane kinase that phosphorylates the eukaryotic translation initiation factor 2 subunit (eIF2). This leads to a reduced protein synthesis, which counteracts ER protein overload. ATF6 is an ER-resident transmembrane protein. Upon activation, the cytoplasmic domain of ATF6 is released from its membrane anchor by regulated proteolysis. The cleaved N-terminal fragment migrates to the nucleus, acts as an active transcription factor, and increases the expression of the genes encoding proteins that function to augment the ER protein folding capacity. The exact mechanism of UPR activation is unknown. One of the most described models is the competition model, in which the ER chaperone protein glucose regulated protein (Grp)78/BiP, is an UPR regulator and plays an essential role in the activation of IRE1, PERK and ATF6. In the inactive state, i.e. in resting cells, all three ER stress sensors (IRE-1, PERK and ATF6) are maintained in an inactive state through their association with the ER chaperone BiP (Fig. 2a). When the ER homeostasis is perturbed, i.e. upon ER stress, BiP is sequestered by unfolded and/or misfolded proteins that accumulate in the ER lumen (Fig. 2b, c). Dissociation of BiP from de ER stress sensors triggers the activation of IRE1, PERK and ATF-6 ( Fig. 2d). Other models of UPR activation are the ligand-binding model in which unfolded and/or misfolded proteins directly interact with the ER stress-sensing domains of the ER stress sensors, and the probing model, in which newly synthesized stress-sensing proteins probe the efficiency of the ER-resident protein-folding machinery by presenting themselves as substrates to the folding machinery [20].

Endoplasmic reticulum stress-apoptosis
Under conditions of severe and prolonged ER stress, the UPR is unable to restore normal cellular function. Subsequently, cell death, usually occurring by apoptosis, is triggered ( Fig. 1). Cell death results in loss of cell/tissue function and may be the primary reason for the manifestation of disease in several ER stress-related disorders. Indeed, cell death induced by ER stress has been implicated in a wide variety of diseases including ischemic injury (stroke, myocardial infarction), heart failure, several neurodegenerative diseases, diabetes and bipolar disorder [17,18]. The mechanisms of apoptosis are highly complex, involving an energy-dependent cascade of molecular events. There are two main apoptotic pathways: the extrinsic or death receptor Fig. 1 The relation between ER stress and ER stress induced apoptosis in the development of diabetes pathway and the intrinsic or mitochondrial pathway [21]. Current evidence suggests that these two pathways are linked and that molecules in one pathway can influence the other [22]. The extrinsic signalling pathways act via transmembrane receptor-mediated interactions. These involve death receptors that are members of the tumor necrosis factor (TNF) receptor gene superfamily [23]. The intrinsic signalling pathways involve a diverse array of non-receptormediated stimuli that produce intracellular signals that act directly on targets within the cell and are mitochondrialinitiated events. These non-receptor stimuli include radiation, toxins, hypoxia, hyperthermia, viral infections, and free radicals but also the absence of certain growth factors, hormones and cytokines [21].
Signalling through the ER stress sensors can trigger pro-apoptotic signals during prolonged ER stress. However, the ER stress sensors do not directly cause cell death but rather initiate the activation of downstream molecules such as CHOP or JNK, which further push the cell down the path of death. This results in caspase activation, the execution phase of ER stress-induced apoptosis, and finally in the ordered and sequential dismantling of the cell. Caspases are cysteine proteases that exist within the cell as inactive pro-forms or zymogens and are cleaved to form active enzymes following the induction of apoptosis. ER stress activates both intrinsic and extrinsic apoptotic pathways [13,14]. Currently, three main pathways of ER stress-induced apoptosis are identified (Fig. 3): (1) the proapoptotic pathway of CHOP/GADD153 transcription factor which is mainly induced via PERK/eIF2, (2) IRE1mediated activation of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun NH 2 -terminal kinase (JNK), and (3) activation of the ER localized cysteine protease, caspase 12 [15,18,19].   [19,24]. We now addressed the most relevant data of the last year, with a focus on ER stress and apoptosis in the pancreas, adipose tissue and the liver. Several studies show evidence for a role of ER stress in b cell failure. Mutations in the primary sensors of the UPR or mutations that affect chaperone functions of the UPR, e.g. EIF2AK3, IRE1, P58 IPK (DNAJC3) and EIF2a, impair b cell health and function [4,[25][26][27][28]. Moreover, mutations in proinsulin causing disruption of disulfide bond pairing result in misfolding and accumulation of proinsulin in the ER lumen of b cells. This accumulation can cause b cell failure [6,29,30]. In vivo data show that also pathological conditions like high glucose, free fatty acids, cytokines, and nitric oxide induce UPR gene expression and compromise b cell function [25,[31][32][33]. Moreover, in islets of T2DM patients, ER stress has been demonstrated by increased staining for ER chaperones and CHOP along with increased ER size [34][35][36][37].
However, the exact molecular mechanisms for the ER stress-induced apoptosis in b cells are not entirely clear. The most recent data support that the PERK-ATF4-CHOP stress signalling pathway is important in b-cell apoptosis (Fig. 3). This pathway plays a role in b-cell injury induced by oxidative stress and saturated fatty acids [38][39][40][41][42]. This is confirmed by the finding that CHOP deletion reduces oxidative stress, improves b cell function, and promotes cell survival in multiple mouse models of diabetes [39]. However, the PERK-ATF4-CHOP pathway is not the only pathway inducing apoptosis in b-cells. In contrast to apoptosis by high lipids, the PERK-ATF4-CHOP ER stresssignalling pathway is not necessary for cytokine-induced b-cell death [42]. Other data show that also the IRE1-JNK pathway is associated with the apoptosis in b cells [41] (Fig. 3). This pathway is also involved in ER stress-induced apoptosis caused by chronic high glucose, fatty acids, and Il-1b induced depletion of Ca 2? [41,[43][44][45].

ER stress, UPR and apoptosis in adipose tissue
The prevalence of obesity is increasing rapidly worldwide, especially in developing countries. An important consequence of obesity is an increased risk of developing impaired glucose tolerance and T2DM. Indeed, along with the increase in obesity, a parallel increase in the prevalence of T2DM, impaired glucose tolerance has occurred [46,47]. The metabolic complications of obesity, usually referred to as the metabolic syndrome, consist of insulin resistance (often culminating in b-cell failure, impaired glucose tolerance and T2DM), dyslipidemia, hypertension, and premature heart disease. Our understanding of the role of adipose tissue in metabolic syndrome has continued to evolve with the identification of adipose tissue as a potent endocrine organ. Adipose tissue secretes large amounts of adipocyte-generated factors, such as adipokines, cytokines and complement components. Cells that are specialized for a high secretory capacity, such as mature B lymphocytes, liver cells and pancreatic b-cells, are known to expand and adopt their ER capabilities to meet an increased demand of protein synthesis [48]. It is, therefore, likely that ER stress plays a role in adipose tissue dysfunction and most probably also in cell death.
Although apoptosis of (pre)adipocytes has not been extensively studied, there is growing evidence that, under  Fig. 3 ER stress induced apoptosis. Three main pathways of ER stress-induced apoptosis are identified: (1) the proapoptotic pathway of CHOP/GADD153 transcription factor which is mainly induced via PERK/eIF2. CHOP down-regulates the anti-apoptotic factor B cell lymphoma-2 (Bcl-2), but also upregulates Ero-1, a thiol oxidase that promotes protein folding in the ER but also generates reactive oxygen species (ROS), and thereby promotes apoptosis, (2) IRE1-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun NH2terminal kinase (JNK). IRE1 interacts with TRAF2 (TNF receptorassociated factor-2) and ASK1. This leads to activation of ASK1 and JNK, followed by apoptosis, and (3) activation of the ER localized cysteine protease, caspase 12. Caspase 12 is activated by m-Calpain in the cytoplasm. Activation of m-Calpain is a consequence Ca 2? efflux out of the ER upon ER stress. These three pathways all end in caspase cascade activation, the execution phase of ER stress-induced apoptosis specific circumstances, decreases in adipose tissue mass in humans could result from a loss of fat cells through programmed cell death. The general idea is that in a normal healthy situation adipocyte number is relatively stable when the energy intake is less than the energy output. In this case, the adipose tissue mass only decreases as a result hypotrophy via mobilization of triglycerides [49]. On the other hand, conditions of pathological fat wasting can involve loss of adipocytes through apoptotic mechanisms. For example, apoptotic events were observed in fat tissue of patients with tumor cachexia and in the fat remodelling processes associated with highly active antiretroviral therapy, e.g. ritonavir, in HIV infected patients with lipodystrophy [50][51][52]. Ritonavir not only induces apoptosis and inhibits adipocyte differentiation, but also affects inflammatory mediators, ER stress and oxidative stress, as shown by gene profiling [53,54]. Recent data suggest that ER stress may, via several mechanisms, also be involved in apoptosis of (pre)adipocytes in relation to the development of obesity/diabetes. In obese individuals, adipose tissue is poorly oxygenated [55,56], which may lead to local hypoxia in adipose tissue. ER stress may form a link between hypoxia and apoptosis. Disturbances in cellular redox regulation caused by hypoxia interfere with disulphide bonding in the lumen of the ER, leading to unfolded and misfolded proteins. In 3T3-L1 adipocytes, hypoxia is associated with ER stress, as shown by increased levels of GRP78 and CHOP [57]. Yin et al. [58] described that hypoxia induces cell death, promotes free FA release and inhibits glucose uptake in adipocytes by inhibition of insulin signalling pathway. These metabolic effects of hypoxia may also add to the generation of ER stress, e.g. in addition to hypoxia itself, palmitate, a saturated fatty acid (FA), also activated UPR and induced apoptosis in preadipocytes. CHOP was one of the proteins that were influenced [59]. Moreover, very recently, three papers for the first time show ER stress in human adipose tissue [60][61][62]. Although none of these papers show direct evidence for a relation between obesity and ER stressinduced apoptosis, the results of Sharma et al. [61], are very suggestive for this. They used ATF4, GADD43 and ATF3 as markers of apoptosis pathways, and show a relation with obesity. Thus, although the data strongly suggest a role for ER stress in apoptosis of adipose tissue, experiments are needed to fully explore this pathway.
For all studies performed with adipose tissue biopsies, it should be emphasized that the precise identity of cells within adipose tissue that show ER stress, and possibly related apoptosis, is not clear. Adipocytes generally account for only 50% of the total number of cells in adipose tissue. Other cells within adipose tissue, e.g. preadipocytes, macrophages and vascular cells, can also secrete an extensive range of protein signals and factors linked to inflammatory response and may therefore also be sensitive for ER stress. This is of special interest since adipose tissue is more and more recognized as a tissue containing a molecular network that connects obesity, adipokine secretion, chronic inflammation and insulin resistance. Inflammation of adipose tissue is often observed in obesity and diabetes and is associated with the infiltration of macrophages into adipose tissue, which may be triggered by adipocyte death, adipokine secretion e.g. TNF-alpha and IL-6, and elaboration of chemokines by adipocyte e.g. monocyte chemo-attractant protein (MCP)-1 [63][64][65]. The mechanism via which adipocyte death stimulates macrophage infiltration has been proposed to occur via an alternative death pathway that share features of both necrosis and apoptosis [66]. This possibility is supported by the finding that macrophages are located around dead adipocytes in the adipose tissue [67]. Apoptosis of macrophages in adipose tissue may also be linked to diabetes. It has been suggested that macrophage cell death in adipose tissue is an important effect of pioglitazone treatment and this may play an essential role in the management of diabetes mellitus and metabolic syndrome [68]. Hypoxia and hypoxia related ER stress may also play a role in apoptosis of macrophages in adipose tissue. Hypoxia does not only stimulate the inflammatory response of macrophages [69,70], but also induced apoptosis and cell cycle arrest at G0/G1 phase, via AKT and JNK [71]. To our knowledge no studies have been published on adipose tissue histology showing ER stress related apoptosis in a specific cell type.
ER stress, UPR and apoptosis in the liver ER stress has been recognized in various models of liver injury and human liver diseases (as reviewed in [72]). The liver plays essential roles in metabolism, biosynthesis, excretion, secretion and detoxification. Comparable to adipose tissue, the liver contains a range of different cell types. The three main liver cell types are hepatocytes, resident macrophages (i.e. Kupffer cells), and endothelial cells. Apoptosis in the liver occurs in many forms of liver injury, e.g. chronic viral liver disease, nonalcoholic and alcoholic steatohepatitis [73][74][75][76].
Nonalcoholic fatty liver disease (NAFLD) results from metabolic hepatic dysregulation in metabolic syndrome and T2DM. NAFLD refers to a wide spectrum of liver disease ranging from simple fatty liver (steatosis), to nonalcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). Several studies have shown that NAFLD predicts future development of T2DM (reviewed in [77]). The pathogenesis of NAFLD is thought to be a multiple-hit process involving insulin resistance, oxidative stress, apoptosis, and adipokines. In NASH, inflammation of the liver is associated with the accumulation of fat in the liver and additionally to different degrees of scarring, which may lead to severe liver scarring and cirrhosis. The general idea is that as consequence of both hepatic and peripheral insulin resistance, the hepatocellular accumulation of triglycerides, initially leads to an altered metabolism of glucose and free fatty acids in the liver. Increased expression of death receptors in response to this altered hepatic metabolism enhances the hepatocytes' susceptibility for pro-apoptotic stimuli, thus eliciting excessive hepatocyte apoptosis and inflammation. Interestingly, hepatocyte apoptosis is significantly increased in patients with NASH and correlates with disease severity [75,78].
Evidence is mounting for an important role for ER stress-induced apoptosis in NAFLD. In relation to the onset of diabetes, most in vivo and in vitro studies on the relation between ER stress-induced apoptosis and fatty liver focus on saturated FA. Saturated FA induce ER stress and apoptosis at physiologic concentrations and with a relatively rapid time course in H4IIE liver cells [79,80], as illustrated by the induction of ER stress response genes and apoptosis which occurred after 4 h and between 6 and 16 h, respectively [79]. Ozcan et al. [7] showed that chronic excessive nutrient intake activated the UPR both in liver and in adipose tissue. A very recent study used transgenic mice carrying the XBP-1-delta-DBD-venus expression gene, which acts as an ER stress-activated indicator (ERAI). In these transgenic mice, the gene encoding venus, a variant of green fluorescent protein, is fused as a reporter downstream of a partial sequence of human XBP-1 including the ER stress-specific intron. The XBP-1/venus fusion protein is produced in cells under ER stress conditions, and cells under ER stress can be detected by monitoring the generation of fluorescence. They showed in the liver of the ERAI transgenic mice that ERAI fluorescence was observed as early as 4 weeks after treatment with a high fat, high sucrose (HF/HS) diet, whereas it was not detected in the fat and muscle, even after 12 weeks of HF/HS diet treatment [9]. It is important to realize that not all FA activate the UPR. Only livers and hepatocytes from rats on a high saturated fat diet, but not high polyunsaturated fat diet, were characterized by the presence of spliced XBP-1 mRNA and increased GRP78 and CHOP protein [81]. This not only suggests that the UPR may sense and respond to the fatty acid environment but also that the ratio of saturated to unsaturated FA may be an important determinant of hepatic ER homeostasis. Although not directly shown in hepatocytes, several mechanisms have been proposed for fatty acid induced ER stress. One possible mechanism is the rapid incorporation of palmitate into lipid components of the rough ER followed by disruption of ER structure and function [82]. Another mechanism of palmitate-induced ER stress is the generation of reactive oxygen species (ROS). ROS by itself can induce ER stress. Prolonged or severe ER stress, which may occur in the presence of excess palmitate, can lead to further ROS accumulation, potentially amplifying the apoptotic/cell death response [83]. Alternatively, as described in b cells, palmitate can lead to an early and sustained depletion of ER Ca 2? stores, which may trigger ER stress via impaired protein folding [41].
ER stress-UPR-insulin resistance ER stress and the UPR are not only associated with apoptosis in of b-cells, hepatocytes and adipocytes but also with metabolic derangements, especially with insulin resistance. In adipose tissue and liver, the relation of ER stress with insulin resistance is actually more evident than its relation with apoptosis. The general idea is that ER stress interferes with the signalling of the insulin receptor via JNK (Fig. 4). Therefore JNK can not only be a link between ER stress and apoptosis ( Fig. 3) but also between ER stress and insulin resistance. A major site of regulation of insulin signalling, both positive and negative, is phosphorylation of the important insulin receptor docking protein insulin receptor protein-1 (IRS-1), whereby phosphorylation of the tyrosine (Tyr) residues in IRS-1 induces phosphorylation of the serine (Ser) residues in IRS-1 and hampers insulin signal transduction (reviewed in [84]). Although the exact mechanisms that lead to Ser phosphorylation of IRS-1 are not yet known, it is apparent that several intracellular serine kinases, e.g. IjB kinase (IKK) and JNK, mTOR and PKC-h are involved. A wide variety of factors, including nutrients such as FA and amino acids, have been found to induce insulin resistance at least in part through inhibitory IRS-1 Ser phosphorylation. Insulin resistant states (e.g obesity, T2DM) are associated with activation of JNK and/or IKK leading to Ser phosphorylation of IRS1 and hence induction of insulin resistance [85][86][87][88]. Activation of JNK in obesity may be a particular consequence of ER stress since IRE-1 has, apart from endoribonuclease activity, also kinase activity that activates JNK (Fig. 4). The liver and adipose tissue of genetic and high-fat diet-induced mouse models of obesity demonstrated increased levels of several ER stress markers as well as induction of insulin resistance via increased Ser phosphorylation/decreased Tyr phosporylation IRS-1. It is of interest that JNK and IKK are also potential links between ER stress and inflammation [89]. Other evidence for a link between ER stress and insulin resistance comes from studies using chaperones, such as 4-phenyl butyric acid (PBA), trimethylamine N-oxide dihydrate (TMAO), and dimethyl sulfoxide or oxygen regulated protein 150kD (ORP150). These chaperones protect cells from ER stress, e.g via stabilization of protein conformation, improvement of ER folding capacity, and therefore enhance the adaptive capacity of the ER. Introduction of chaperones increased insulin sensitivity in the liver of obese diabetic mice [8,90]. Moreover, an in vitro model of hepatocytes (HepG2) shows that triglycerides induce the expression of endogenous ER stress markers, including GRP 78, IRE-1alpha, XBP-1, p-eIF2alpha, CHOP, and p-JNK. ER stress, in turn, leads to the suppression of insulin receptor signalling through increase in serine phosphorylation and decrease of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), and therefore insulin resistance [91]. More evidence for a link between insulin resistance and ER stress is shown in a study using a mouse model that is hypersensitive to insulin (i.e. liver-specific-PTP1B deficient mice). The livers of these mice are both insulin sensitive and protected against a high fat diet-induced ER stress response [92].

Conclusion
Taken together, these data indicate that ER stress plays a role in diabetes by affecting at least two major events: b-cell failure and generation of insulin resistance. Although most of the current understanding of the known mediators of the ER stress pathway comes from other experimental systems, it is clear that ER stress-induced apoptosis of b cells plays a role in the development of diabetes. Data obtained in liver and adipose tissue suggest that also ER stress-induced apoptosis in these tissues is important in the development of diabetes. In contrast to apoptosis of b cells, which will primarily affect insulin production, ER induced apoptosis in liver and adipose tissue will rather lead to increased insulin resistance. More extensive studies with human adipocytes and hepatocytes are needed to identify the exact interactions between environmental signals and ER stress.
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extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry Psychology

text:
Alpha adrenergic modulation on effects of norepinephrine transporter inhibitor reboxetine in five-choice serial reaction time task

The study examined the effects of a norepinephrine transporter (NET) inhibitor reboxetine (RBX) on an attentional performance test. Adult SD rats trained with five-choice serial reaction time task (5-CSRTT) were administered with RBX (0, 3.0 and 10 mg/kg) in the testing day. Alpha-1 adrenergic receptor antagonist PRA and alpha-2 adrenergic receptor antagonist RX821002 were used to clarify the RBX effect. Results revealed that rat received RBX at 10 mg/kg had an increase in the percentage of the correct response and decreases in the numbers of premature response. Alpha-1 adrenergic receptor antagonist Prazosin (PRA) at 0.1 mg/kg reversed the RBX augmented correct responding rate. However, alpha-2 adrenergic receptor antagonist RX821002 at 0.05 and 0.1 mg/kg dose dependently reversed the RBX reduced impulsive responding. Our results suggested that RBX as a norepinephrine transporter inhibitor can be beneficial in both attentional accuracy and response control and alpha-1 and alpha-2 adrenergic receptors might be involved differently.

Background
The action of norepinephrine (NE) can be terminated not only by enzymes that destroy NE but also by a transport pump for NE, i.e., removing it from the synapse without destroying it. The transport pump that terminates the synaptic action of NE is called NE "transporter" or abbreviated as NET. NET locates in the pre-synaptic terminal and acts to remove NE out of the synaptic cleft thus stops its action [1]. In terms many issues of the selectivity and mechanisms of action remained unresolved, selective NET inhibitor has now been developed to treat a variety of brain-related disorders, including depression, attention deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD) and cocaine dependence [1][2][3][4]. Reboxetine (RBX) is the first potent, selective and specific NE transporter inhibitor that has been marketed as an antidepressant [5]. Most of the previous studies focused on the antidepressant activity of RBX in rodents [6] in which RBX exhibited an exceptional antidepressant effect [7]. Nevertheless there is relatively lack of study examining the effect of RBX on the aspect of response control and attentional function. The latter is worth to address given the fact the disturbed amount of synaptic NE generally associated with the symptoms such as impaired attention, problems concentrating, slowness of information processing and poor response control [8,9]. The present experiment therefore aimed to examine the RBX-induced attentional changes by employing a recognized behavioral test, named five-choice serial reaction time task (5-CSRTT).
The 5-CSRTT was modeled after Leonard's five-choice serial task [10], which was commonly used to assess the behavioral effects of different forms of arousal in humans [11]. In the 5-CSRTT, animals are required to discriminate, spatially, a short visual stimulus occurring randomly in one of five locations after a scheduled waiting period. The test requires that the rat must be able to attend to the array of openings in a specially designed apparatus in order to detect the discriminative stimulus and respond correctly to it. Accurate responding requires attention both in the temporal and spatial domains, thus, providing a high degree of parametric flexibility and the potential for independent assessments of the spatial and temporal components of attention [12]. Furthermore, the 5-CSRTT provides measures of 'premature' responding, as the animals need to wait for the imperative visual stimulus prior to making a discriminative response [13]. The 5-CSRTT could be widely used to separate animals with deficits in their attentional processes from other behavioral or cognitive features, for example, locomotor activity when adding with a pair of infra-red beams in the testing box, foodrelated motivation in terms of the speed in collecting earned food pellets [14] and motoric impulsivity as recording the numbers of premature response prior to the occurrence of visual signal [15,16].
In the present study, the 5-CSRTT was aimed to manifest drug-induced attention/impulsivity modifications by clarifying whether RBX treated rats could exhibit less premature responding or better accuracy of detecting visual target stimuli as they did when applied to humans in the recovery from depressive state. The authors designed a series of experiments to investigate the effects of RBX on the performance of the 5-CSRTT in rats in order to assess the benefits of RBX concerning the function of visual attention and the capacity of response control. The performance of 5-CSRTT was examined in rats following the administration of RBX, PRA and RX821002. The latter two were selective agents to antagonize alpha-1 and alpha-2 adrenergic receptors, respectively, and thus were used to test the possibility of reversing the RBX effects on the performance of the 5-CSRTT. The results obtained from this study may contribute to the understanding of the functional role of NET inhibitor and be helpful for further clinical implementations.

Subjects
A total of 57 male Sprague-Dawley rats (National Laboratory Animal Center, Taiwan R.O.C.) aged approximately 4 months and weighted 300350 gm at the start of the experiment were selected. All rats were housed in a temperature-humidity-controlled holding facility with 12-hour light/dark cycles (light on from 07:00 to 19:00). All rates received ad libitum water. However, food was restricted to that earned during the test sessions [maximum of 100 × 45 mg purified rodent pellets (Labdiet, USA) and 20 gm/ rat standard rodent chow (TSE, Taiwan) at the end of the test. The test took place between 08:00 and 18:00 with all rats being tested at the same time every day where possible. All experiment procedures were evaluated and permitted to perform by the animal care committee of National Defense Medical Center with the permission code number IACUC-04-156. All efforts were made to reduce the numbers of animals used and to minimize animal suffering during the experiment.

Apparatus
The apparatus (TSE, Germany) used in this study was very similar to those described in other studies [17,18]. It consisted of four 25 × 25 cm aluminium operant chambers without levers and each of them accompanied with a curved rear wall. Set into the curved wall were five, 2.5 cm square holes, 4 cm deep and 2.5 cm above a wire mesh floor. Each hole had an infrared photocell beam to monitor its entrance. Illumination of each hole was provided by a 3W bulb located at the rear of the hole. Reinforcement was provided by a food-pellet dispenser to deliver pellets into a magazine at the front of the chamber. The chamber was illuminated by a 3W house-light mounted in the centre of the ceiling. The rats were introduced to the chamber through a hinged Perspex flap in the top half of the front wall. The chamber was contained within a sound-attenuating wooden box. Ventilation was supplied by means of a fan, providing a low level of background noise that masked extraneous sound. On-line control of the apparatus and data collection were performed by using a microcomputer.

Training procedure Food deprivation
Rats were instructed to the restricted feeding regimen one week prior to training programs with standard rodent chow, and gradually reached 90% of their free feeding weight.

Magazine training
Initial shaping consisted of a magazine training, whereby the rats learned that food was available in the magazine in which the hinged metal panel was wedged open with surplus pellets. At this stage, rats were exposed to the boxes for 15 minutes per session. Once all rats reliably ate all food pellets within a session, the rats were moved onto a fixed interval schedule.
Fixed interval schedule From the following 3 days, rats were placed in the dark chamber 20 minutes per day with a fixed time schedule, in which a food pellet was delivered every 20 seconds into the magazine.

Five-choice serial reaction time task (5-CSRTT)
Every rat was consistently placed in the same chamber and well-trained in the 5-CSRTT. A session terminated after a maximum of 100 completed trials or after 30 minutes. A trial started with the illumination of magazine until rats to response with a nose-poke response. After a delay (the intertrial interval, ITI), the stimulus light was presented over the hole in pseudorandom order. If the rat then broke a photo-beam crossing by nose-poke within a limited hold of 5 s, a food pellet was delivered into the magazine. An ITI began if the food pellet was retrieved and the next stimulus light presented after the ITI. A period of time-out (i.e., a period of darkness for 5 seconds) would be followed by an incorrect response or an error of omission with the house-light extinguished, and during which no stimuli were presented. After a time-out, the rats started a new ITI by nose poking to magazine. Figure 1 provided a schematic illustration of 5-CSRTT used in the present study.
The acquisition of the 5-CSRTT was divided into several sub-stages based on a scheduled stepped descending sequence of stimulus duration at 60, 32, 16, 8, 4, 2, 1.5 and finally 1.0 seconds. For these sub-stages, rats were required to meet all of the following criteria in continuous 3 sessions before passing on to the next training stage: (1) More than 80 trials were carried out within a period of 30 minutes; (2) Omission rate was less than 20%; (3) An accuracy rate of 80% or more was achieved; and (4) The numbers of premature response were less than 30.

Measures and analysis
Accuracy of performance was measured by the calculation of the percentage of correct response [correct responses/ (correct responses + incorrect responses)]. An error of omission was scored if the rat failed to respond sufficiently soon after the stimulus onset. A premature response was scored if the rat responded in any hole during the ITI. To assess speed of responding, correct latency was measured as time elapsing between the occurrence of visual stimulus and a nose poke in the correct hole. Magazine latency was measured as time elapsing between a nose poke in the correct hole and retrieving reward in the magazine.
Obtained data was analyzed using SPSS (version 12.0) for variables of interest. In experiment 1, one-way ANOVA was conducted with dose as a non-repeated factor. In experiment 2, one-way ANOVA was conducted with dose as a repeated factor. In experiment 3, two-way ANOVA was conducted with both treatment and dose as repeated factors. Further analyses and post-hoc comparisons were performed if necessary. A p value < 0.05 was considered statistically significant.

Drugs and experimental design Drugs
The performance of 5-CSRTT was examined after the administration of vehicle (0.9% saline solution) or the following drugs: RBX (sponsored by Pfizer Pharmaceutics, USA), PRA (Sigma, USA) and RX821002 (Sigma, USA). All drugs were administered in a volume of 2 ml/kg and were diluted in isotonic saline. RBX was administered intraperitoneally 60 minutes prior to the test whereas for PRA or RX8212002, 10 minutes before the test. The doses used were 10 mg/kg for RBX, 0.1 and 0.3 mg/kg for PRA, and 0.05 and 0.1 mg/kg for RX821002.

Experiment 1: dose-response study of RBX in the 5-CSRTT
Twenty-four rats reached the acquisition criteria level of 5-CSRTT (i.e., 1.0 sec of stimulus duration) were used. Rats were tested their 5-CSRTT performance after the administration of RBX (0, 3 and 10 mg/kg). The number of animal was 8 for each dose. RBX was injected intraperitoneally 60 minutes prior to the test.

Experiment 2: dose-response study of PRA and RX821002 in the 5CSRTT
Twenty-three rats were examined their 5-CSRTT performance 10 min later following PRA (N = 11) or RX821002 (N = 12) administration. Each rat received three dose conditions (for PRA, 0, 0.1 and 0.3 mg/kg; for RX821002, 0, 0.05 and 0.1 mg/kg). The order of injection condition was based on a Latin Square design. Each test session was preceded by two control sessions in which saline vehicle was administered.

Experiment 3: The interaction of RBX with PRA or RX in the 5-CSRTT
Ten rats were examined their 5-CSRTT performance under the following 4 conditions (vehicle + vehicle, RBX + vehicle, vehicle + PRA, RBX + PRA). The dose for RBX was 10 mg/kg and for PRA, 0.3 mg/kg. Another 10 rats were examined their 5-CSRTT performance under the following 4 conditions (vehicle + vehicle, RBX + vehicle, vehicle + RX821002, RBX + RX821002). The dose for RBX was 10 mg/kg and for RX821002, 0.1 mg/kg. The order of injection condition was based on a Latin Square design. Each test session was preceded by two control sessions in which saline vehicle was administered.

Effect of RBX on 5-CSRTT
Effects of RBX on performance of the 5-CSRTT were shown in Figure 2. RBX was found to altered rats' 5-CSRTT performance in all aspects. (correct response, F(2,21) = 3.89, p < 0.05; premature response, F(2,21) = 6.33, p < 0.01; omission rate, F(2,21) = 4.24, p < 0.05; correct latency, F(2,21) = 6.86, p < 0.01 and magazine latency, F(2,21)= 7.01, p < 0.01). Further analyses by Dunnett's test revealed that the difference was mainly contributed from the condition between vehicle and 10 mg/kg RBX. A trial started with the illumination of magazine until rat made a nose-poke response. After a delay (the intertrial interval, ITI), the stimulus light was presented over the hole in pseudorandom order. If an animal respond with a nose-poke into the illuminated hole within a limited hold of 5 s, a pellet was delivered into the magazine. An ITI began when the food pellet was retrieved, with the next stimulus light presented after the ITI. A period of time-out would be followed by an incorrect response with the house-light on for 5 seconds, during which no stimuli were presented. After a timeout, the rat could start a new ITI by nose-poking to magazine. Analyses of measures were based on the formulations as indexed.

Effects of RBX on 5-CSRTT following pretreatment with PRA
Effects of RBX on performance of the 5-CSRTT in the condition of pretreatment of alpha-1 adrenergic receptor antagonist PRA (0.3 mg/kg) were examined (see Figure 5). There was a significant effect of RBX × PRA in correct response [F (1,9)

Effects of RBX on 5-CSRTT following pretreatment with RX821002
Effects of RBX on performance of the 5-CSRTT in the condition of pretreatment of alpha-2 adrenergic receptor antagonist RX821002 (0.1 mg/kg) were examined (see Figure 6). In correct response, there was little effect on RBX, RX821002 or RBX × RX821002. In premature response, There was a significant effect of RBX × RX821002 [F (1,9)

Discussion
The present study described NET inhibitor RBX could improve attentional function and the ability of response control in a task requiring visual accuracy and the management of impulsiveness. The contributions of alpha-1 and alpha-2 adrenoceptors in the RBX effect were exam-ined. While the alpha-1 effect was mainly responsible for attentional accuracy, Alpha-2 adrenergic receptors contributed to the adjustment of impulsiveness. Interpretations of these findings were discussed as the following.
For protocol used in the present study, behavior was mainly controlled by the rats' attentional function and their motor impulsiveness. For example, rats made their correct response largely depending on their sustained and divided attentional functions. At the same time, rats also had to restrain their impetuous nature for not nose-poking hastily, which was representative of their ability to control motor impulsiveness. Attentional impairment has been considered for a long time as one of the major cognitive psychopathologies of major depression [19][20][21]. The beneficial effect of NET inhibitor reboxetine found in this study was in line with the concept that the attentional and executive disturbance in major depressive patients could be alleviated following the treatment of antidepressant medications [22].
In general it takes al least two weeks for antidepressants to attain their clinical efficacy. Accordingly in the present study, we demonstrated RBX treatment for unremittingly two weeks appeared to produce different effects on attention and impulsiveness. As their impulsiveness remained unaffected, rats under RBX treatment became less attentive. Attentional performance and impulse control had been assumed based on distinctive neurobiological mechanisms and they both depended largely on the integrity of prefrontal cortex, particularly dorsa-lateral prefrontal cortex. The present experiment demonstrated that when RBX was combined with alpha-2 antagonist RX821002, the latter successfully reversed the RBX improved impulse control in 5-CSRTT. The result supported the idea that when analyzing the improvement of rats' attentional performance, selection response and impulse control might be involved in a dissociated manner [18]. The present study continued to extend the idea and found alpha-1 and alpha-2 adrenoceptors might contribute in a different way to accuracy and vigor responding.
Central noradrenergic system was considered significantly being involved in the regulation of attentional function. For the knowledge to date, alpha-1 adrenoceptor revealed a greater extent than alpha-2 adrenoceptor in modulating response accuracy in the task concentrating subject's attention [23]. Systemic administration of alpha-1 agonist St-587 improved the response accuracy and this effect could be blocked by alpha-1 receptor antagonist prozasin [24]. Activation of alpha-1 adrenergic receptor manifested importantly in conditions requiring attention. For example, alpha-1 adrenergic activation facilitated cognitive performance of rats in situation requiring a variety of arousal levels, such as attentional set-shifting task [25]. The ** * * present study was in the other way to demonstrate that the alpha-1 adrenergic receptor was crucial for both sustained and divided attentional functions [23,24,26,27]. Apparently in 5-CSRTT of the present study, the action of motor impulsiveness occurred prior to the emergency of any corresponding visual signal, therefore it inherited with anticipatory character. Alpha-1 antagonist PRA when given alone had a minor effect on the anticipatory response in 5-CSRTT. Previously PRA at the dose of 0.1 mg/kg was found unable to reverse the effect of alpha-1 agonist St-587 in reducing premature responses [24]. This was consistent with what happened in the present experiment as PRA at the same dose could not abolish the RBX induced reduction of premature response.
Taken together it appeared that the alpha-1 adrenoceptor contributed little in controlling anticipatory actions in situation requiring sustained and divided attentions. On the other hand, the role of alpha-2 adrenoceptor in the regulations of motor impulse control might be more important. Our results demonstrated that although alpha-2 adrenoceptor antagonist per se was little to do with premature responding as revealed before [4], it may potentially help to adjust the effect of NET inhibitor. Possible mechanisms might be related to either an increase of norepinephrine availability following presynaptic autoregulation [28], or the postsynaptic mechanism through which the alpha-2 adrenoceptor antagonist abolished the subsequent effect following RBX induced norepinephrine surge. The former seemed less plausible due to the following considerations. First, the effects of an alpha 2-adrenoceptor agonist dexmedetomidine on anticipatory responses were found no difference between controls and rats with central NA depleted by N-(2-chloroethyl)-Nethyl-2-bromobenzy-lamine (DSP-4). Second, alpha-2 adrenoceptor may function as a terminal presynaptic autoreceptor to restrain NE release [29]; or heteroreceptor at 5HT terminal to hold back 5HT release [29,30]. In the present study if the enhanced ability of impulse control by RBX was attributable to the acceleration of 5HT neuronal activity via alpha-1 adrenoceptor at 5HT cell body, the activation of alpha-2 autoreceptor located on NA terminal on the other hand would hold back the release of NE, that in turn attenuated alpha-1 activation therefore rats should be more impulsive.
In attentional task as 5-CSRTT, measures in fact provided valuable information other than visual accuracy and impulse control. For example, correct latency generally refers to the reaction time elapsed from coming out visual signal to the rats' nose-poking the correct hole. Increase of correct latency suggests a longer motor readiness or hypoactivity [31]. Compared to correct latency, magazine latency refers to the reaction time elapsed from coming out the food pallet to the collection of that reward, nor-mally reflecting animals' motivational state. In the present experiment, correct latency was found longer in both treatment conditions, RBX alone and RBX combined with PRA or RX821002. Magazine latency was also longer following RBX treatment. The similar impacts over correct response time and food collecting time suggested the reaction time was prolonged, no matter the target stimulus was reward itself or its antecedent. However, the cognitive benefit of RBX in reducing the premature response was unlikely caused by the prolonged reaction time since the elapse duration of correct reaction time was around 200 ms (i.e., 0.2 second), far from the time scale of impulsiveness indexed by the premature response counted within the scale of 5 sec ITI.
Along with many other antidepressants, the peripheral effect of RBX is well worth considering in which autonomic side effects such as dry mouth and adjusted sympathetic vasomotor tone had been reported previously [32,33]. This might elicit the concerning that the result obtained form the present experiment might be contributed to a degree by peripheral effects of RBX. Although this issue was worth taking into account; it was not much likely to confound the interpretation of the result. In an experimental design such as 5CSRTT, it was the level of motor function more responsible for influencing the result of 5-CSRTT. In the present study, animals' motor function was preserved well since both correct and magazine latencies did not prolong following the administration of RBX.
In summary, the present study demonstrated NET inhibitor RBX may exhibit some potential benefit in the aspects of both response accuracy and behavioral inhibition. Future studies would suggest validating the argument obtained from the present study in a more comprehensive way, for example, examining the effect of RBX on signal transduction pathways mediated by alpha-1 and -2 adrenergic receptors.