A switch from life to death in endoplasmic reticulum stressed β-cells

β-Cell death is an important pathogenic component of both type 1 and type 2 diabetes. Recent findings indicate that cell signalling pathways emanating from the endoplasmic reticulum (ER) play an important role in the regulation of β-cell death during the progression of diabetes. Homeostasis within the ER must be maintained to produce properly folded secretory proteins, such as insulin, in response to the body's need for them. However, the sensitive protein-folding environment in the ER can be perturbed by genetic and environmental factors leading to ER stress. To counteract ER stress, β-cells activate cell signalling pathways termed the unfolded protein response (UPR). The UPR functions as a binary switch between life and death, regulating both survival and death effectors. The outcome of this switch depends on the nature of the ER stress condition, the regulation of UPR activation and the expression and activation of survival and death components. This review discusses the mechanisms and the components in this switch and highlights the roles of this UPR's balancing act between life and death in β-cells.     

Z α-1 antitrypsin deficiency and the endoplasmic reticulum stress response

The serine proteinase inhibitor α-1 antitrypsin (AAT) is produced principally by the liver at the rate of 2 g/d. It is secreted into the circulation and provides an antiprotease protective screen throughout the body but most importantly in the lung, where it can neutralise the activity of the serine protease neutrophil elastase. Mutations leading to deficiency in AAT are associated with liver and lung disease. The most notable is the Z AAT mutation, which encodes a misfolded variant of the AAT protein in which the glutamic acid at position 342 is replaced by a lysine. More than 95% of all individuals with AAT deficiency carry at least one Z allele. ZAAT protein is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum (ER) of hepatocytes and other AAT-producing cells. This results in a loss of function associated with decreased circulating and intrapulmonary levels of AAT. However, the misfolded protein acquires a toxic gain of function that impacts on the ER. A major function of the ER is to ensure correct protein folding. ZAAT interferes with this function and promotes ER stress responses and inflammation. Here the signalling pathways activated during ER stress in response to accumulation of ZAAT are described and therapeutic strategies that can potentially relieve ER stress are discussed.     

Metabolic syndrome enhances endoplasmic reticulum,oxidative stress and leukocyte–endothelium interactions in PCOS

ObjectivePolycystic ovary syndrome (PCOS) is associated with insulin resistance, which can lead to metabolic syndrome (MetS). Oxidative stress and leukocyte–endothelium interactions are related to PCOS. Our aim was to evaluate whether the presence of MetS in PCOS patients can influence endoplasmic reticulum (ER) and oxidative stress and leukocyte–endothelium interactions.Material and MethodsThis was a prospective controlled study conducted in an academic medical center. The study population consisted of 148 PCOS women (116 without/32 with MetS) and 112 control subjects (87 without / 25 with MetS). Metabolic parameters, reactive oxygen species (ROS) production, ER stress markers (GRP78, sXBP1, ATF6), leukocyte–endothelium interactions, adhesion molecules (VCAM-1, ICAM-1, E-Selectin), TNF-α and IL-6 were determined.ResultsTotal ROS, inflammatory parameters and adhesion molecules were enhanced in the presence of MetS (p < 0.05), and the PCOS + MetS group showed higher levels of IL-6 and ICAM-1 than controls (p < 0.05). Increased adhesion and leukocyte rolling flux were observed in PCOS and PCOS + MetS groups vs their respective controls (p < 0.05). GRP78 protein expression was higher in the PCOS groups (p < 0.05 vs controls) and sXBP1 was associated with the presence of MetS (p < 0.05 vs controls without MetS). Furthermore, PCOS + MetS patients exhibited higher GRP78 and ATF6 levels than controls and PCOS patients without MetS (p < 0.05). In PCOS women, HOMA-IR was positively correlated with ICAM-1 (r = 0.501; p < 0.01), ROS (r = 0.604; p < 0.01), rolling flux (r = 0.455;p < 0.05) and GRP78 (r = 0.574; p < 0.001).ConclusionOur findings support the hypothesis of an association between altered metabolic status, increased ROS production, ER stress and leukocyte–endothelium interactions in PCOS, all of which are related to vascular complications.     

Causes and cures for endoplasmic reticulum stress in lipotoxic β-cell dysfunction

Pancreatic β-cell dysfunction is central to the pathogenesis of type 2 diabetes, and the loss of functional β-cell mass in type 2 diabetes is at least in part secondary to increased β-cell apoptosis. Accumulating evidence suggests that endoplasmic reticulum (ER) stress is present in β-cells in type 2 diabetes. Free fatty acids (FFAs) cause ER stress and are putative mediators of β-cell dysfunction and death. In this review, we discuss the molecular mechanisms underlying ER stress induced by saturated and unsaturated FFAs. Oleate and palmitate trigger ER stress through ER Ca(2+) depletion and build-up of unfolded proteins in the secretory pathway. Saturated and unsaturated FFAs elicit a differential signal transduction in the three branches of the ER stress response, resulting in different survival/apoptosis outcomes. The protection of β-cells against FFAs through the interference with ER stress signalling has opened novel therapeutic perspectives for type 2 diabetes. Chemical chaperones, salubrinal and glucagon-like peptide-1 (GLP-1) analogues have been used to protect β-cells from lipotoxic ER stress. Importantly, the pro- and antiapoptotic effects of these compounds are cell and context dependent.     

Uterine endoplasmic reticulum stress-unfolded protein response regulation of gestational length is caspase-3 and -7–dependent

We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that “normal” gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic.Although the rates of preterm birth (PTB) continue to decrease in the United States, there has been a steady rise in prevalence globally over the past decade (1). Multiple risk factors have been associated with preterm labor (2); however, the events that precede and elicit the signals allowing for the onset of premature uterine contractions and labor remain unclear. Thus, PTB continues to pose an acute risk for neurodevelopmental and respiratory complications that adversely effect neonatal health (3, 4). In this study, we demonstrate that the pregnant rodent uterus uses an integrated unfolded protein response (UPR)-endoplasmic reticulum stress response (ERSR) pathway to maintain steady levels of activated caspase-3 (CASP3) and caspase-7 (CASP7), which preserve uterine quiescence across gestation. We also demonstrate that an increase in the adaptive UPR limits CASP3 and 7 activation to allow the induction of both term and preterm labor mediated through increased levels of gap junction protein, alpha 1 (GJA1).We have identified that CASP3 and 7 play compensatory roles in regulating uterine myocyte quiescence. Previous investigations from our laboratory and others have identified a gestationally regulated activation of nonapoptotic uterine CASP3 during pregnancy (5–7). Furthermore, we have proposed that activation of CASP3 during pregnancy occurs as a result of gestationally regulated increases in uterine ERSR (8).The endoplasmic reticulum (ER) is the organelle that facilitates protein folding and transport (9), misfolded protein ubiquitination, and proteasomal degradation. Functional irregularities at the ER level cause the accumulation of misfolded proteins, leading to initiation of an ERSR (10). A prolonged and/or excessive ERSR has been implicated in potentiating increased CASP3 and 7 activation (11). In every pregnancy, the uterus experiences physiological and biochemical stimuli that in other biological systems trigger an ERSR, including stretch (12), inflammation (13), hormone fluctuations (14, 15), hypoxia (16), hyperplasia (17), hypertrophy (18), and demand for metabolic fuels (19).We propose that the pregnant uterus also may use the ERSR to activate and harness the tocolytic potential of CASP3 and 7, allowing it to retain its quiescent phenotype during these periods of adaptation. Furthermore, we propose that an inappropriate ERSR or UPR mismanagement may modulate uterine CASP3 and 7 activity, thereby influencing gestational length. We tested this hypothesis by manipulating the uterine ERSR and UPR in the pregnant mouse and monitoring the levels of active CASP3 and 7 together with the timing of labor. We found that excessive/prolonged potentiation of uterine ERSR fails to maintain uterine CASP3 and 7 levels, owing to the unexpected triggering of a precociously heightened adaptive UPR, which ultimately leads to the onset of PTB. Coadministration of 4-phenylbutrate (PB) and tunicamycin (TM) allows for the maintenance of uterine CASP3 and 7 levels, which reverses and rescues the TM-induced PTB phenotype. We have identified GJA1, known to play an essential role in myometrial gap junction intercellular communication (20–22), integrating the signals for active contraction during labor in the pregnant uterus, as a target of uterine CASP3 and 7 activity both in vitro and in vivo.     

Inhibition of macrophage fatty acid β-oxidation exacerbates palmitate-induced inflammatory and endoplasmic reticulum stress responses

Aims/hypothesis

Saturated fatty acids (SFAs) such as palmitate activate inflammatory pathways and elicit an endoplasmic reticulum (ER) stress response in macrophages, thereby contributing to the development of insulin resistance linked to the metabolic syndrome. This study addressed the question of whether or not mitochondrial fatty acid β-oxidation (FAO) affects macrophage responses to SFA.

Methods

We modulated the activity of carnitine palmitoyl transferase 1A (CPT1A) in macrophage-differentiated THP-1 monocytic cells using genetic or pharmacological approaches, treated the cells with palmitate and analysed the proinflammatory and ER stress signatures.

Results

To inhibit FAO, we created THP-1 cells with a stable knockdown (KD) of CPT1A and differentiated them to macrophages. Consequently, in CPT1A-silenced cells FAO was reduced. CPT1A KD in THP-1 macrophages increased proinflammatory signalling, cytokine expression and ER stress responses after palmitate treatment. In addition, in human primary macrophages CPT1A KD elevated palmitate-induced inflammatory gene expression. Pharmacological inhibition of FAO with etomoxir recapitulated the CPT1A KD phenotype. Conversely, overexpression of a malonyl-CoA-insensitive CPT1A M593S mutant reduced inflammatory and ER stress responses to palmitate in THP-1 macrophages. Macrophages with a CPT1A KD accumulated diacylglycerols and triacylglycerols after palmitate treatment, while ceramide accumulation remained unaltered. Moreover, lipidomic analysis of ER phospholipids revealed increased palmitate incorporation into phosphatidylethanolamine and phosphatidylserine classes associated with the CPT1A KD.

Conclusions/interpretation

Our data indicate that FAO attenuates inflammatory and ER stress responses in SFA-exposed macrophages, suggesting an anti-inflammatory impact of drugs that activate FAO.     

The role of endoplasmic reticulum stress in angiotensinⅡinduced apoptosis in cultured neonatal rat cardiomyocytes

Background AngiotensinⅡ(AngⅡ) plays a critical role in the pathophysiology of cardiovascular diseases. Recently,studies have shown that Endoplasmic Reticulum (ER) stress was activated in failure hearts.This study was designed to examine whether ER stress participates in the pathologic process of AngⅡ-induced cardiomyocytes apoptosis. Methods Neonatal rat cardiomyocytes were incubated with concentrations of AngⅡ(0,1,10,100 nmol/L) for 24 hours.Confocal fluorescence microscopy with double staining of TUNEL and CHOP detected the percentage of apoptotic cells.Levels of GRP78,JNK,p-JNK,CHOP and caspase-12 were analyzed by western blot.Telmisartan(10- ~6mol/L) was used to test the effects of ATI receptor on AngⅡ- induced cell apoptosis,ER stress chaperones and signaling molecules.Results Treatment with AngⅡat 1,10, and 100 nmol/L for 24 hours stimulated GRP78,JNK,p-JNK and CHOP protein production,and increased apoptosis of myocytes.The protein expression and the number of apoptotic cells were depedent on AngⅡconcentration.About 60%of apoptotic cells were CHOP positive at 10 and 100nmol/L AngⅡtreatment,while no CHOP positive apoptotic cells were found at myocytes under physiological condition and 1 nmo/L AngⅡtreatment.Telmisartan decreased signaling molecules expression and abolished ER stress-mediated apoptosis induced by 100 nmol/L AngⅡ.Conclusions These results indicate that ER stress may be involved in the mechanisms of AngⅡ-induced cardiomyocytes apoptosis.JNK, caspase12 and CHOP all participate in the pathologic process.     

Mild endoplasmic reticulum stress augments the proinflammatory effect of IL-1β in pancreatic rat β-cells via the IRE1α/XBP1s pathway

The prevalence of obesity and type 1 diabetes in children is increasing worldwide. Insulin resistance and augmented circulating free fatty acids associated with obesity may cause pancreatic β-cell endoplasmic reticulum (ER) stress. We tested the hypothesis that mild ER stress predisposes β-cells to an exacerbated inflammatory response when exposed to IL-1β or TNF-α, cytokines that contribute to the pathogenesis of type 1 diabetes. INS-1E cells or primary rat β-cells were exposed to a low dose of the ER stressor cyclopiazonic acid (CPA) or free fatty acids, followed by low-dose IL-1β or TNF-α. ER stress signaling was inhibited by small interfering RNA. Cells were evaluated for proinflammatory gene expression by RT-PCR and ELISA, gene reporter activity, p65 activation by immunofluorescence, and apoptosis. CPA pretreatment enhanced IL-1β- induced, but not TNF-α-induced, expression of chemokine (C-C motif) ligand 2, chemokine (C-X-C motif) ligand 1, inducible nitric oxide synthase, and Fas via augmented nuclear factor κB (NF-κB) activation. X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1, but not CCAAT/enhancer binding protein homologous protein, knockdown prevented the CPA-induced exacerbation of NF-κB-dependent genes and decreased IL-1β-induced NF-κB promoter activity. XBP1 modulated NF-κB activity via forkhead box O1 inhibition. In conclusion, rat β-cells facing mild ER stress are sensitized to IL-1β, generating a more intense and protracted inflammatory response through inositol-requiring enzyme 1/XBP1 activation. These observations link β-cell ER stress to the triggering of exacerbated local inflammation.     

Investigating the involvement of the ATF6α pathway of the unfolded protein response in adipogenesis

The unfolded protein response (UPR) is activated by endoplasmic reticulum stress resulting from an accumulation of unfolded or mis-folded proteins. The UPR is divided into three arms, involving the activation of ATF-6, PERK and IRE-1, that together act to restrict new protein synthesis and increase the production of chaperones. Recent studies have implicated the PERK and IRE-1 components of the UPR in adipocyte differentiation. In this study, we investigate the importance of ATF6α during adipogenesis using stable knockdown of this protein in the model adipogenic cell line, C3H10T1/2. Reduction of ATF6α expression by >70% resulted in impaired expression of key adipogenic genes and reduced lipid accumulation following the induction of adipogenesis. In contrast, loss of ATF6α did not impair the ability of cells to undergo osteogenic differentiation. Overall, our data indicate that all three arms of the UPR, including ATF6α, must be intact to permit adipogenesis to occur.     

Estrogen receptor α inhibitor activates the unfolded protein response,blocks protein synthesis,and induces tumor regression

Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen–ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen–ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα⁺ cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP₃), which opens EnR IP₃R calcium channels, rapidly depleting EnR Ca²⁺ stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca²⁺ levels, but the open EnR IP₃R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI’s novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration.Estrogens, acting via estrogen receptor α (ERα), stimulate tumor growth (1–3). Approximately 70% of breast cancers are ERα-positive and most deaths due to breast cancer are in patients with ERα⁺ tumors (2, 4). Endocrine therapy using aromatase inhibitors to block estrogen production, or tamoxifen and other competitor antiestrogens, often results in selection and outgrowth of resistant tumors. Although 30–70% of epithelial ovarian tumors are ERα-positive (1), endocrine therapy is largely ineffective (5–7). After several cycles of chemotherapy, tumors recur as resistant ovarian cancer (5), and most patients die within 5 years (8).Noncompetitive ERα inhibitors targeting this unmet therapeutic need, including DIBA, TPBM, TPSF, and LRH-1 inhibitors that reduce ERα levels, show limited specificity, require high concentrations (>5 μM), and usually have not advanced through preclinical development (9–12). These noncompetitive ERα inhibitors and competitor antiestrogens are primarily cytostatic and act by preventing estrogen–ERα action; therefore, they are largely ineffective in therapy-resistant ERα containing cancer cells that no longer require estrogens and ERα for growth.To target the estrogen–ERα axis in therapy-resistant cancer cells, we developed (13) and implemented an unbiased pathway-directed screen of ∼150,000 small molecules. We identified ∼2,000 small molecule biomodulators of 17β-estradiol (E₂)–ERα-induced gene expression, evaluated these biomodulators for inhibition of E₂–ERα-induced cell proliferation, and performed simple follow-on assays to identify inhibitors with a novel mode of action. Here, we describe 3,3-bis(4-hydroxyphenyl)-7-methyl-1,3-dihydro-2H-indol-2-one (BHPI), our most promising small molecule ERα biomodulator.In response to stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). We recently showed that as an essential component of the E₂–ERα proliferation program, estrogen induces a different mode of UPR activation, a weak anticipatory activation of the UPR before increased protein folding loads that accompany cell proliferation. This weak and transient E₂–ERα-mediated UPR activation is protective (14). BHPI distorts this normal action of E₂–ERα and induces a massive and sustained ERα-dependent activation of the UPR, converting UPR activation from cytoprotective to cytotoxic. Moreover, independent of its effect on the UPR and protein synthesis, BHPI rapidly suppresses E₂–ERα-regulated gene expression.     

Phosphorylation of eIF2α mitigates endoplasmic reticulum stress and hepatocyte necroptosis in acute liver injury

Introduction and objectivesNecroptosis and endoplasmic reticulum (ER) stress has been implicated in acute and chronic liver injury. Activated eukaryotic initiation factor 2 alpha (eIF2α) attenuates protein synthesis and relieves the load of protein folding in the ER. In this study, we aimed to analyze the impact of eIF2α phosphorylation on hepatocyte necroptosis in acute liver injury.Materials and methodsMale BALB/c mice were injected with tunicamycin or d-galactosamine, and LO2 cells were incubated with tunicamycin to induce acute liver injury. 4-Phenylbutyric acid (PBA) and salubrinal were used to inhibit ER stress and eIF2α dephosphorylation, respectively. We analyzed the eIF2α phosphorylation, ER stress, and hepatocyte necroptosis in mice and cells model.ResultsTunicamycin or d-galactosamine significantly induced ER stress and necroptosis, as well as eIF2α phosphorylation, in mice and LO2 cells (p < 0.05). ER stress aggravated tunicamycin-induced hepatocyte necroptosis in mice and LO2 cells (p < 0.05). Elevated eIF2α phosphorylation significantly mitigated hepatocyte ER stress (p < 0.05) and hepatocyte necroptosis in mice (34.37 ± 3.39% vs 22.53 ± 2.18%; p < 0.05) and LO2 cells (1 ± 0.11 vs 0.33 ± 0.05; p < 0.05). Interestingly, tumor necrosis factor receptor (TNFR) 1 protein levels were not completely synchronized with necroptosis. TNFR1 expression was reduced in d-galactosamine-treated mice (p < 0.05) and cells incubated with tunicamycin for 12 and 24 h (p < 0.05). ER stress partially restored TNFR1 expression and increased necroptosis in tunicamycin-incubated cells (p < 0.05).ConclusionsThese results imply that ER stress can mediate hepatocyte necroptosis independent of TNFR1 signaling and elevated eIF2α phosphorylation can mitigate ER stress during acute liver injury.     

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency

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Vitamin C modulates the metabolic and cytokine profiles,alleviates hepatic endoplasmic reticulum stress,and increases the life span of Gulo?/? mice

Suboptimal intake of dietary vitamin C (ascorbate) increases the risk of several chronic diseases but the exact metabolic pathways affected are still unknown. In this study, we examined the metabolic profile of mice lacking the enzyme gulonolactone oxidase (Gulo) required for the biosynthesis of ascorbate. Gulo^−/− mice were supplemented with 0%, 0.01%, and 0.4% ascorbate (w/v) in drinking water and serum was collected for metabolite measurements by targeted mass spectrometry. We also quantified 42 serum cytokines and examined the levels of different stress markers in liver. The metabolic profiles of Gulo^−/− mice treated with ascorbate were different from untreated Gulo^−/− and normal wild type mice. The cytokine profiles of Gulo^−/− mice, in return, overlapped the profile of wild type animals upon 0.01% or 0.4% vitamin C supplementation. The life span of Gulo^−/− mice increased with the amount of ascorbate in drinking water. It also correlated significantly with the ratios of serum arginine/lysine, tyrosine/phenylalanine, and the ratio of specific species of saturated/unsaturated phosphatidylcholines. Finally, levels of hepatic phosphorylated endoplasmic reticulum associated stress markers IRE1α and eIF2α correlated inversely with serum ascorbate and life span suggesting that vitamin C modulates endoplasmic reticulum stress response and longevity in Gulo^−/− mice.     

Activation of the AMP-activated protein kinase enhances glucose-stimulated insulin secretion in mouse β-cells

The AMP-activated protein kinase (AMPK) is one of the key players in cellular energy regulation adapting cellular demands to nutritional and metabolic variations. Oral antidiabetic drugs like metformin and glitazones (thiazolidinediones) are known to stimulate this enzyme. Besides their established action on peripheral organs including liver and muscles, it has been claimed that these drugs may affect β-cell function. However, it is still a matter of debate whether pharmacological AMPK stimulation increases or decreases insulin secretion. To study this point and to reveal mechanisms underlying changes in insulin secretion we used the specific AMPK activator AICAR and investigated its effects on stimulus-secretion coupling. Membrane potential and currents were measured by the patch-clamp technique, [Ca (2+)]c, mitochondrial membrane potential, and NAD(P)H by fluorescence techniques and insulin secretion by a radioimmunoassay. AICAR enhanced glucose-stimulated insulin release, an effect that can be attributed to the augmentation of electrical activity and [Ca (2+)]c resulting from an AICAR-evoked inhibition of the KATP current. This latter effect was not due to a direct interaction of AICAR with the K[ATP] channels but was dependent on cell metabolism. AICAR did not affect mitochondrial membrane potential or NAD(P)H autofluorescence. Metformin mimicked the action of AICAR on electrical activity, [Ca (2+) ]c, and K[ATP] current. However, compared to AICAR the effects were less pronounced and not sufficient to stimulate insulin secretion. In conclusion, activation of AMPK augments nutrient-induced insulin secretion. Thus, targeting AMPK of β-cells may be an appropriate strategy for the treatment of disturbed glucose homeostasis..