DsbA-L Alleviates Endoplasmic Reticulum Stress�CInduced Adiponectin Downregulation

OBJECTIVE

Obesity impairs adiponectin expression, assembly, and secretion, yet the underlying mechanisms remain elusive. The aims of this study were 1) to determine the molecular mechanisms by which obesity impairs adiponectin multimerization and stability, and 2) to determine the potential role of disulfide-bond-A oxidoreductase-like protein (DsbA-L), a recently identified adiponectin interactive protein that promotes adiponectin multimerization and stability in obesity-induced endoplasmic reticulum (ER) stress and adiponectin downregulation.

RESEARCH DESIGN AND METHODS

Tauroursodeoxycholic acid (TUDCA), a chemical chaperone that alleviates ER stress, was used to study the mechanism underlying obesity-induced adiponectin downregulation in db/db mice, high-fat diet-induced obese mice, and in ER-stressed 3T3-L1 adipocytes. The cellular levels of DsbA-L were altered by RNAi-mediated suppression or adenovirus-mediated overexpression. The protective role of DsbA-L in obesity- and ER stress–induced adiponectin downregulation was characterized.

RESULTS

Treating db/db mice and diet-induced obese mice with TUDCA increased the cellular and serum levels of adiponectin. In addition, inducing ER stress is sufficient to downregulate adiponectin levels in 3T3-L1 adipocytes, which could be protected by treating cells with the autophagy inhibitor 3-methyladenine or by overexpression of DsbA-L.

CONCLUSIONS

ER stress plays a key role in obesity-induced adiponectin downregulation. In addition, DsbA-L facilitates adiponectin folding and assembly and provides a protective effect against ER stress–mediated adiponectin downregulation in obesity.Adiponectin is an insulin sensitizer that plays a versatile role in the regulation of energy homeostasis and insulin sensitivity. The serum adiponectin levels are significantly reduced under the conditions of obesity, insulin resistance, and type 2 diabetes (1), yet the precise underlying mechanisms remain largely unknown.Adiponectin is synthesized as a single polypeptide of 30 kDa and is then assembled in the endoplasmic reticulum (ER), primarily into three species: trimer, hexamer, and high molecular weight (HMW) multimer (2–5). The different forms of adiponectin have been found to play distinct roles in the regulation of energy homeostasis (2–4,6). Several ER-associated proteins, including Ero1-lα, ERp44, and GPR94, have recently been found to be involved in the assembly and secretion of higher-order adiponectin complexes (5,7,8). Impairment in adiponectin multimerization affects both secretion and function of this adipokine, and is associated with diabetes and hypoadiponectinemia (2,4).ER is a eukaryotic organelle responsible for several specialized and important functions such as protein translation, folding, and transport of membrane or secreted proteins. Numerous protein chaperones are present in the ER lumen that yield an oxidizing environment necessary for correct folding and assembly of various membrane and secretive proteins such as adiponectin. In obesity, increased demand on ER function leads to ER stress and the unfolded protein response (UPR) to ensure that normal cell function and viability are maintained (9). However, whether and how ER stress plays a role in obesity-induced adiponectin downregulation remain to be established.We recently identified an adiponectin-interacting protein named disulfide-bond A oxidoreductase-like protein (DsbA-L) (10). DsbA-L is expressed in various mouse tissues such as liver, kidney, pancreas, and heart, but the highest expression of this protein is detected in adipose tissue where adiponectin is synthesized and secreted (10). The cellular levels of DsbA-L are significantly reduced in adipose tissues of obese mice and human subjects. Overexpression of DsbA-L promotes adiponectin multimerization while reducing DsbA-L expression by RNAi markedly, and selectively reduces adiponectin levels and secretion in 3T3-L1 adipocytes (10). However, how DsbA-L regulates adiponectin multimerization and stability remains unknown.In the present study, we show that alleviating ER stress enhances the cellular and plasma levels of adiponectin in both db/db mice and diet-induced obese mice. In addition, we demonstrate that inducing ER stress is sufficient to downregulate the cellular levels and secretion of adiponectin in 3T3-L1 adipocytes. Furthermore, overexpression of DsbA-L suppresses the inhibitory effect of ER stress on adiponectin multimerization and stability. Our studies demonstrate that ER stress is a key factor in obesity-induced downregulation of adiponectin and that increasing the cellular levels of DsbA-L improves adiponectin assembly and stability by suppressing the negative effect of ER stress.     

Stimulation of Autophagy Improves Endoplasmic Reticulum Stress–Induced Diabetes

Accumulation of misfolded proinsulin in the β-cell leads to dysfunction induced by endoplasmic reticulum (ER) stress, with diabetes as a consequence. Autophagy helps cellular adaptation to stress via clearance of misfolded proteins and damaged organelles. We studied the effects of proinsulin misfolding on autophagy and the impact of stimulating autophagy on diabetes progression in Akita mice, which carry a mutation in proinsulin, leading to its severe misfolding. Treatment of female diabetic Akita mice with rapamycin improved diabetes, increased pancreatic insulin content, and prevented β-cell apoptosis. In vitro, autophagic flux was increased in Akita β-cells. Treatment with rapamycin further stimulated autophagy, evidenced by increased autophagosome formation and enhancement of autophagosome–lysosome fusion. This was associated with attenuation of cellular stress and apoptosis. The mammalian target of rapamycin (mTOR) kinase inhibitor Torin1 mimicked the rapamycin effects on autophagy and stress, indicating that the beneficial effects of rapamycin are indeed mediated via inhibition of mTOR. Finally, inhibition of autophagy exacerbated stress and abolished the anti-ER stress effects of rapamycin. In conclusion, rapamycin reduces ER stress induced by accumulation of misfolded proinsulin, thereby improving diabetes and preventing β-cell apoptosis. The beneficial effects of rapamycin in this context strictly depend on autophagy; therefore, stimulating autophagy may become a therapeutic approach for diabetes.In eukaryotic cells, secreted proteins undergo cotranslational folding in the endoplasmic reticulum (ER) lumen. The β-cell ER faces a high protein-folding burden due to the high proinsulin biosynthesis rate: proinsulin mRNA may reach 20% of total mRNA (1) and proinsulin production 50% of total protein synthesis in stimulated β-cells (2). Furthermore, correct folding of proinsulin is difficult due to its complex tertiary structure, containing three disulfide bonds that depend on the redox state of the ER, which can be altered by the inflammation and oxidative stress of nutrient overload and obesity (3,4). Indeed, several reports showed that ER stress is linked to β-cell dysfunction in type 2 diabetes (4–7).The causality between proinsulin misfolding and β-cell failure is epitomized in the mutant INS gene–induced diabetes of youth syndrome, in which mutations in proinsulin trigger irreparable misfolding (8,9). As an example, the C(A7)Y proinsulin mutation results in severe congenital diabetes in man and in the Akita mouse. The pathophysiology of β-cell failure in Akita is complex and involves trapping of nonmutant proinsulin in the ER, leading to impaired β-cell function, stress, and apoptosis (10–12). Notably, a subset of Akita β-cells can compensate for proinsulin misfolding, thereby avoiding diabetes (13). Therefore, unraveling the adaptive mechanisms that operate in stressed β-cells may have important implications for diabetes treatment.Accumulation of misfolded proteins in the ER stimulates the unfolded protein response (UPR), an adaptive homeostatic signaling pathway aimed to reduce stress. The UPR increases the expression of ER chaperones and oxireductases, inhibits mRNA translation, and stimulates ER-associated degradation, thus reducing ER protein load and enhancing folding capacity and clearance of misfolded proteins. However, if ER stress is not subdued, its continuous activation results in apoptosis. ER stress induces autophagy to eliminate damaged organelles and protein aggregates, thus improving cell function and survival (14). This comprises the transport of cytosolic portions and entire organelles to lysosomes via double-membrane vesicles called autophagosomes. Lysosomal degradation recycles amino and fatty acids for energy production in starvation, but also serves an important homeostatic function in response to stress in nutrient abundance (15). Transgenic mice with impaired β-cell autophagy exhibited decreased insulin secretion, glucose intolerance, and islet degeneration, indicating that basal autophagy is required for β-cell well being (16,17). The nutrient-sensing kinase mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of autophagy (18–20). Under nutrient availability, mTORC1 phosphorylates Atg13, which prevents binding to Atg1 (ULK1 in mammals) and hence reduced formation of the Atg1–Atg13–Atg17 complex (21). Conversely, mTORC1 inhibition during starvation or by rapamycin administration stimulates initiation of autophagosome budding.In this study, we studied in Akita β-cells the effects of proinsulin misfolding on autophagy and whether stimulating autophagy using mTORC1 inhibitors attenuates stress and prevents diabetes progression in Akita mice in vivo.     

Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and ��-Cell Death

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca²⁺ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP₃Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS—Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca²⁺ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca²⁺ in ER and mitochondria.RESULTS—Neither RyR nor IP₃R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca²⁺ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP₃Rs and RyRs. Conversely, stimulation of ER Ca²⁺ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.CONCLUSIONS—This study demonstrates that the activity of ER Ca²⁺ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca²⁺ homeostasis and ER stress.Inappropriate activation of cell death pathways in the pancreatic β-cell is involved in the pathogenesis of type 1 diabetes, type 2 diabetes, and rare diabetic disorders such as maturity-onset diabetes of the young, Wolcott-Rallison syndrome, and Wolfram syndrome (1–5). β-Cell apoptosis also hampers clinical islet transplantation (6). The endoplasmic reticulum (ER) plays a key role in multiple programmed cell death pathways (7–9). Apoptosis caused by ER stress has been associated with diabetes (1,2,5,10) and can be induced by the accumulation of unfolded proteins resulting from disrupted Ca²⁺-dependent chaperone function in the ER (1,11). Both thapsigargin, a potent and specific inhibitor of sarco/endoplasmic reticulum ATPase (SERCA), and endogenous factors that downregulate SERCA, evoke ER stress and apoptosis in β-cells (12,13). However, the detailed mechanisms underlying Ca²⁺-dependent apoptosis and the roles played by specific β-cell ER Ca²⁺ channels and pumps in ER stress remain unclear.In addition to multiple SERCA isoforms (14), the β-cell ER expresses several classes of intracellular Ca²⁺-releasing channels, including the inositol trisphosphate receptors (IP₃Rs) and the ryanodine receptors (RyRs) (15–19). In the diabetic state, the expression of these receptors is known to be modulated in several cell types, including β-cells (15,20–22). We have previously shown that long-term inhibition of RyR2 in low glucose leads to programmed β-cell death involving calpain-10, but not caspase-3; conversely, RyR inhibition protected islets under conditions of chronic hyperglycemia (17). We have also shown that RyR inhibition significantly reduces the ratio of ATP to ADP in MIN6 β-cells (23), an event that could conceivably activate ER stress (24,25). Furthermore, studies of other cells types have suggested that ER stress–associated damage can be affected by inhibitors of RyRs (26) or IP₃Rs (27). Despite these important questions and links, studies on the roles of RyRs and IP₃Rs in β-cell ER stress have not been published to date.In the present study, we investigated whether disrupting β-cell ER Ca²⁺ homeostasis by blocking Ca²⁺ release through IP₃Rs and RyRs is sufficient to induce ER stress. We also tested the hypothesis that stimulating or inhibiting these channels would alter ER stress or apoptosis triggered by ER Ca²⁺ depletion following SERCA inhibition. Our results demonstrate that while blocking ER Ca²⁺ release channels does not induce a major ER stress response, Ca²⁺ flux from both RyRs and IP₃Rs can modulate β-cell apoptosis and ER stress resulting from impaired SERCA function.     

Ask1 Gene Deletion Blocks Maternal Diabetes–Induced Endoplasmic Reticulum Stress in the Developing Embryo by Disrupting the Unfolded Protein Response Signalosome

Apoptosis signal–regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose– or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis.     

High Glucose–Repressed CITED2 Expression Through miR-200b Triggers the Unfolded Protein Response and Endoplasmic Reticulum Stress

High glucose in vivo and in vitro induces neural tube defects (NTDs). CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) is essential for neural tube closure. We explored the regulatory mechanism underlying CITED2 expression and its relationship with miRNA and endoplasmic reticulum (ER) stress. miR-200b levels were increased by maternal diabetes or high glucose in vitro, and this increase was abrogated by transgenic overexpression of superoxide dismutase 1 (SOD1) or an SOD1 mimetic. CITED2 was the target of miR-200b and was downregulated by high glucose. Two miR-200b binding sites in the 3′-untranslated region of the CITED2 mRNA were required for inhibiting CITED2 expression. The miR-200b mimic and a CITED2 knockdown mimicked the stimulative effect of high glucose on unfolded protein response (UPR) and ER stress, whereas the miR-200b inhibitor and CITED2 overexpression abolished high glucose–induced UPR signaling, ER stress, and apoptosis. The ER stress inhibitor, 4-phenylbutyrate, blocked CITED2 knockdown–induced apoptosis. Furthermore, the miR-200b inhibitor reversed high glucose–induced CITED2 downregulation, ER stress, and NTDs in cultured embryos. Thus, we showed a novel function of miR-200b and CITED2 in high glucose–induced UPR and ER stress, suggesting that miR-200b and CITED2 are critical for ER homeostasis and NTD formation in the developing embryo.     

Cinacalcet Improves Bone Parameters Through Regulation of Osteoclast Endoplasmic Reticulum Stress,Autophagy, and Apoptotic Pathways in Chronic Kidney Disease–Mineral and Bone Disorder

The possible mechanisms underlying the quantitative and qualitative effects of cinacalcet on bone were explored in a chronic kidney disease–mineral and bone disorder (CKD-MBD) mouse model in relation to the influence of the interactions among the osteoclast (OC) endoplasmic reticulum (ER) stress, autophagy and apoptosis pathways on OC differentiation. Body weight and biochemical parameters improved significantly in the CKD + cinacalcet groups compared to the CKD group. Micro–computed tomography (μCT) revealed both cortical and trabecular parameters deteriorated significantly in the CKD group and were reversed by cinacalcet in a dose-dependent manner. Nanoindentation analysis of bone quality proved that both cortical hardness and elastic modulus improved significantly with high dose cinacalcet treatment. In vitro studies revealed that cinacalcet inhibited receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK)–induced OC differentiation in a concentration-dependent manner through a close interaction between activation of caspase-related apoptosis, reversal of OC autophagy through the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and adenosine monophosphate–activated protein kinase (AMPK) pathways, and attenuation of the OC ER stress/CREBH/NFATc1 signaling pathway. Cinacalcet improves both bone quantity and bone quality in CKD mouse model and inhibits OC differentiation through regulation of the interactions among the apoptosis, ER stress, and autophagy pathways within OCs. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Endoplasmic Reticulum Stress in Diabetic Hearts Abolishes Erythropoietin-Induced Myocardial Protection by Impairment of Phospho�CGlycogen Synthase Kinase-3�¨CMediated Suppression of Mitochondrial Permeability Transition

OBJECTIVE

Alteration in endoplasmic reticulum (ER) stress in diabetic hearts and its effect on cytoprotective signaling are unclear. Here, we examine the hypothesis that ER stress in diabetic hearts impairs phospho–glycogen synthase kinase (GSK)-3β–mediated suppression of mitochondrial permeability transition pore (mPTP) opening, compromising myocardial response to cytoprotective signaling.

RESEARCH DESIGN AND METHODS

A rat model of type 2 diabetes (OLETF) and its control (LETO) were treated with tauroursodeoxycholic acid (TUDCA) (100 mg · kg⁻¹ · day⁻¹ for 7 days), an ER stress modulator. Infarction was induced by 20-min coronary occlusion and 2-h reperfusion.

RESULTS

Levels of ER chaperones (GRP78 and GRP94) in the myocardium and level of nonphoshopho–GSK-3β in the mitochondria were significantly higher in OLETF than in LETO rats. TUDCA normalized levels of GRP78 and GRP94 and mitochondrial GSK-3β in OLETF rats. Administration of erythropoietin (EPO) induced phosphorylation of Akt and GSK-3β and reduced infarct size (% risk area) from 47.4 ± 5.2% to 23.9 ± 3.5% in LETO hearts. However, neither phosphorylation of Akt and GSK-3β nor infarct size limitation was induced by EPO in OLETF rats. The threshold for mPTP opening was significantly lower in mitochondria from EPO-treated OLETF rats than in those from EPO-treated LETO rats. TUDCA restored responses of GSK-3β, mPTP opening threshold, and infarct size to EPO receptor activation in OLETF rats. There was a significant correlation between mPTP opening threshold and phospho–GSK-3β–to–total GSK-3β ratio in the mitochondrial fraction.

CONCLUSIONS

Disruption of protective signals leading to GSK-3β phosphorylation and increase in mitochondrial GSK-3β are dual mechanisms by which increased ER stress inhibits EPO-induced suppression of mPTP opening and cardioprotection in diabetic hearts.Despite recent progress in coronary intervention strategies, diabetes is associated with higher mortality after acute myocardial infarction due to more extensive atherosclerotic lesions and hypertrophied and dysfunctional left ventricle (1–3). Therefore, diabetic patients with coronary artery diseases are patients who most require novel protective strategies against myocardial ischemia reperfusion injury. However, diabetes is known to impair responses of the myocardium to protective interventions. Protection afforded by preinfarct angina is lost in diabetic patients (4). In animal models, ischemic preconditioning (IPC) and some pharmacological agents failed to reduce infarct size in diabetic hearts (5–8). Recently, Gross et al. (7) have reported that responses of Akt, extracellular signal–related kinase (ERK), and glycogen synthase kinase (GSK)-3β to opioid receptor stimulation were blunted in streptozotocin-induced diabetes. GSK-3β has been shown to regulate a variety of cellular functions (9,10), and recent studies (10–14) have indicated that inactivation of GSK-3β by phosphorylation at Ser9 enhances myocardial tolerance against ischemia reperfusion injury. Furthermore, accumulating evidence indicates that phospho–GSK-3β–mediated cytoprotection is achieved by elevation of the threshold for opening of the mitochondrial permeability transition pore (mPTP), a probable final common step in stress-induced cell necrosis (11,15–17). However, derangements in GSK-3β regulation and its downstream targets in type 2 diabetes have not yet been clarified.The endoplasmic reticulum (ER) has received much attention recently for its role in signal transduction relevant to cell survival and death. Various pathophysiological conditions induce Ca²⁺ overload and/or accumulation of unfolding or misfolding proteins within the ER, a condition referred to as ER stress (18). ER stress induces multiple responses, including adaptive changes in translation, protein folding, secretion, and degradation. Prolonged ER stress can trigger apoptosis by induction of C/EBP homologous protein (CHOP), activation of c-JUN NH₂-terminal kinase (JNK), or caspase 12–dependent pathways. ER stress has been reported to be involved in the pathogenesis of diabetes (18–20), neurodegenerative disease, immune response, atherosclerosis, ischemia reperfusion injury, and heart failure (18,21–25).We hypothesized that ER stress is increased in the diabetic myocardium and that increased ER stress in diabetic hearts impairs phospho–GSK-3β–mediated suppression of mPTP opening, leading to loss of myocardial response to cytoprotective signaling. The rationale for this hypothesis is threefold. First, increased ER stress has been observed in epididymal fat tissue in obese diabetic mice (26). Second, an increase in GSK-3β activity induced by ER stress through dephosphorylation of phospho-Ser9 has been reported in noncardiac cells (27). Finally, elevated levels of GSK-3 protein and activity were observed in skeletal muscle of type 2 diabetic patients (28). To test our hypothesis, we investigated changes in anti-infarct tolerance, myocardial ER stress, cytoprotective signaling, and mPTP opening threshold in a rat model of type 2 diabetes. ER stress modulators, sodium tauroursodeoxycholic acid (TUDCA), and 4-phenylbutyric acid (4-PBA) (29) were used to suppress ER stress. Erythropoietin (EPO) was used to induce GSK-3β phosphorylation in this study, since we have characterized signaling pathways from the EPO receptor leading to myocardial protection and modification of the pathways by concurrent pathological conditions (13,30,31).     

Tacrolimus-Induced Apoptosis is Mediated by Endoplasmic Reticulum–derived Calcium-dependent Caspases-3,-12 in Jurkat Cells

Apoptotic signal pathways are delivered to caspase-3, caspase-9, or both in different cells via the death receptor pathway, mitochondrial pathway, or by the endoplasmic reticulum (ER) pathway through initiators of caspase-3, -8, -9, or -12. Tacrolimus (Tac)–induced apoptosis was characterized by nuclear fragmentation and caspase-3 activation. We examined the effect of tacrolimus on ER-derived calcium and caspase-3,-12–mediated apoptosis on Jurkat human T lymphocyte. Tac decreased the viability of Jurkat cells in a dose-dependent manner. Tac also increased continuously intracellular concentration of calcium from 24 hours to 72 hours. We did not find intracellular calcium changes on the treatment of calcium ionorpore (A23187) regardless of 1 nmol/L Tac concentration level. However, calcium adenosine triphosphatase inhibitor (thapsigargin) increased intracellular calcium accumulation and co-treating 1 nmol/L Tac further induced intracellular calcium accumulation. Interestingly, we found that 1 nmol/L Tac treatment induced activation of caspase-12 protease as well as the catalytic activity of caspase-3 but not catalytic activation of caspase-6, -8, and -9 proteases in Jurkat cells. These data advance our understanding of Tac-induced apoptosis is ER-derived calcium and caspases-3,-12– mediated apoptosis in human Jurkat cell line.     

Glucokinase Activation Ameliorates ER Stress–Induced Apoptosis in Pancreatic β-Cells

The derangement of endoplasmic reticulum (ER) homeostasis triggers β-cell apoptosis, leading to diabetes. Glucokinase upregulates insulin receptor substrate 2 (IRS-2) expression in β-cells, but the role of glucokinase and IRS-2 in ER stress has been unclear. In this study, we investigated the impact of glucokinase activation by glucokinase activator (GKA) on ER stress in β-cells. GKA administration improved β-cell apoptosis in Akita mice, a model of ER stress–mediated diabetes. GKA increased the expression of IRS-2 in β-cells, even under ER stress. Both glucokinase-deficient Akita mice and IRS-2–deficient Akita mice exhibited an increase in β-cell apoptosis, compared with Akita mice. β-cell–specific IRS-2–overexpressing (βIRS-2-Tg) Akita mice showed less β-cell apoptosis than Akita mice. IRS-2–deficient islets were vulnerable, but βIRS-2-Tg islets were resistant to ER stress–induced apoptosis. Meanwhile, GKA regulated the expressions of C/EBP homologous protein (CHOP) and other ER stress–related genes in an IRS-2–independent fashion in islets. GKA suppressed the expressions of CHOP and Bcl2-associated X protein (Bax) and protected against β-cell apoptosis under ER stress in an ERK1/2-dependent, IRS-2–independent manner. Taken together, GKA ameliorated ER stress–mediated apoptosis by harmonizing IRS-2 upregulation and the IRS-2–independent control of apoptosis in β-cells.The decline in β-cell mass as a result of increased apoptosis is an important property of type 2 diabetes (1,2). Endoplasmic reticulum (ER) stress is a key mediator of β-cell apoptosis (3,4). Hence, the development of therapeutic strategies to safeguard residual β-cells against ER stress–induced apoptosis is needed for the adequate care and cure of type 2 diabetes.Glucokinase, a member of the hexokinase family, is mainly expressed in hepatocytes, pancreatic β-cells, and certain subgroups of hypothalamic neurons, forming a key component of the main glucose sensor in β-cells (5–8). Glucokinase also mediates the glucose signal–induced upregulation of insulin receptor substrate 2 (IRS-2) expression in β-cells through calcineurin or CREB (9–12). IRS-2 is required for the maintenance of the β-cell mass and plays an important role in compensatory β-cell expansion against peripheral insulin resistance and in β-cell survival, preventing diabetes (9,13–15). Mice that were heterozygous for β-cell glucokinase (βGck^+/−) and that were fed a diet rich in linoleic acid and sucrose exhibited increased ER stress and apoptosis in β-cells, compared with wild-type (WT) mice (16). Consequently, we speculated that glucokinase is also involved in the regulation of ER stress–induced apoptosis in β-cells.Glucokinase activators (GKAs) have been shown to reduce blood glucose levels in several diabetic animal models and type 2 diabetic patients (10,11,17–19). GKAs promote β-cell proliferation, which is driven by the increased expression of IRS-2 and the activation of its downstream signaling pathway (11,20,21). However, the physiological advantage of GKA-mediated signaling during β-cell apoptosis has been obscure (22,23), and the effect of GKAs on ER stress in β-cells remains unknown.These conditions inspired us to undertake a detailed investigation of the impact of GKA on ER stress and apoptosis in β-cells. Here, we report the protective effects of GKA against ER stress–induced apoptosis in β-cells, the nature of this mechanism, and the significance of glucokinase and IRS-2 in the regulation of ER stress in β-cells.     

Nuclear Translocation of β-Catenin Protein but Absence of β-Catenin and APC Mutation in Gastrointestinal Carcinoid Tumor

Background Carcinoid tumors are a group of heterogeneous tumors with neuroendocrine differentiation and are mainly located in the gastrointestinal tract. A high frequency of cytoplasmic accumulation and/or nuclear translocation of β-catenin with frequent mutations of exon 3 of β-catenin gene in gastrointestinal carcinoid tumor has been previously described, but the role of Wnt/β-catenin/APC pathway in the genesis of carcinoid tumor remains largely unknown.Methods To further characterize the role of Wnt/β-catenin/APC pathway, we investigated 91 gastrointestinal carcinoid tumors and, for comparison, 26 extragastrointestinal carcinoid tumors by immunohistochemical detection of β-catenin protein and direct sequencing of exon 3 of the β-catenin gene and exon 15 of the APC gene.Results Cytoplasmic accumulation and/or nuclear translocation of β-catenin were found in 27 gastrointestinal carcinoid tumors (29.7%) but not in any extragastrointestinal carcinoid tumors. Interestingly, neither β-catenin nor APC gene mutation was detected in all of the cases with nuclear expression of β-catenin.Conclusions Our results indicate that the role β-catenin plays in the genesis of gastrointestinal and extragastrointestinal carcinoid tumors is different. Nuclear expression of β-catenin does not occur in extragastrointestinal carcinoid tumors, and mutation of exon 3 of β-catenin gene and exon 15 of APC gene does not contribute to the activation of Wnt/β-catenin/APC pathway in gastrointestinal carcinoid tumors.