Voltage-gated Ca2+ influx and insulin secretion in human and mouse β-cells are impaired by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157

Luciani, Dan S.; Ao, Peter; Hu, Xiaoke; Warnock, Garth L.; Johnson, James D.

doi:10.1016/j.ejphar.2007.07.055

Cited by 39 publications

(34 citation statements)

References 36 publications

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“…This analysis argues against any heterogeneous effect of CGP-37157 on distinct targeting sites and thus indicates that endogenous and overexpressed NCLX share similar sensitivity to GCP-37157. Moreover, the concentration of CGP-37157 required for inhibition of Ca 2+ efflux in NCLX-overexpressing SHSY-5Y cells was similar to that reported for inhibition of the mitochondrial exchanger in intact cells (16), and is considerably lower than that reported for partial inhibition of other members of the NCX family (17,18). Hence, the results of this set of experiments point to NCLX as being the molecular moiety catalytically linked to mitochondrial Ca 2+ efflux, and its sensitivity to CGP-37157 provides additional evidence that NCLX is the mitochondrial Na + / Ca 2+ exchanger.…”

Section: Resultssupporting

confidence: 54%

NCLX is an essential component of mitochondrial Na ⁺ /Ca ²⁺ exchange

Palty

Silverman

Hershfinkel

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Section: Resultssupporting

confidence: 54%

NCLX is an essential component of mitochondrial Na ⁺ /Ca ²⁺ exchange

Palty

Silverman

Hershfinkel

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

709

560

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“…Single-cell imaging was performed in Ringer's solution containing (in mmol/l): 5.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 20 HEPES, 141 NaCl, and 3 glucose. Cytosolic Ca 2ϩ was imaged in fura-2-AM-loaded cells as described previously (4,30). Preheated solutions were applied by stable perifusion at 1 ml/min, and complete solution changes were achieved in Ͻ30 s.…”

Section: Methodsmentioning

confidence: 99%

“…CGP-37157, a drug that has been reported to indirectly interfere with ER Ca 2ϩ uptake by blocking mitochondrial Na ϩ /Ca 2ϩ exchange (30,44), had little effect on ER stress or caspase-3 activation. It should be noted, however, that we have previously demonstrated that CGP-37157 also inhibits voltage-gated Ca 2ϩ entry in ␤-cells (30). There was a tendency for caspase-3 cleavage to be reduced by changing the glucose concentration from 5 to 25 mmol/l (Fig.…”

Section: Cytosolic and Er Camentioning

confidence: 99%

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

et al. 2009

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OBJECTIVE-Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca 2ϩ 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 (IP 3 Rs) and the ryanodine receptors (RyRs) on the induction of ␤-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS-Kinetics of ␤-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 Ca 2ϩ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca 2ϩ in ER and mitochondria. RESULTS-NeitherRyR nor IP 3 R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca 2ϩ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2␣ (eIF2␣), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP 3 Rs and RyRs. Conversely, stimulation of ER Ca 2ϩ 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 Ca 2ϩ channels regulates the susceptibility of ␤-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in ␤-cell apoptosis associated with dysfunctional ␤-cell ER Ca 2ϩ homeostasis and ER stress. Diabetes 58:422-432, 2009

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“…Human islets were cultured in RPMI-1640 medium (Invitrogen) with 10% FBS and 2% penicillin-streptomycin in humidified 5% CO 2-95% air (vol/vol) at 37°C. Primary human islets were dispersed onto sterile coverslips 2 days before experiments with a previously described technique (33).…”

Section: Methodsmentioning

confidence: 99%

“…Single-cell imaging was performed in HEPESbuffered Ringer solution containing (in mM) 144 NaCl, 5.5 KCl, 1 MgCl2, 2 CaCl2, 20 HEPES, and 3 glucose, unless otherwise indicated (25). Cytosolic Ca 2ϩ was imaged in fura-2 AM-loaded cells as described previously (25,33) or in D3cpv-expressing cells (46). ER luminal Ca 2ϩ was imaged with the FRET-based D1ER cameleon (34,45,46).…”

Section: Methodsmentioning

confidence: 99%

Effects of palmitate on ER and cytosolic Ca²⁺homeostasis in β-cells

Gwiazda¹,

Yang²,

Lin³

et al. 2009

American Journal of Physiology-Endocrinology and Metabolism

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There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular Ca2+, and in particular the role of the endoplasmic reticulum (ER). In human β-cells and MIN6 cells, palmitate rapidly increased cytosolic Ca2+ through a combination of Ca2+ store release and extracellular Ca2+ influx. Palmitate caused a reversible lowering of ER Ca2+, measured directly with the fluorescent protein-based ER Ca2+ sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic Ca2+ in palmitate-treated cells. In keeping with this observed ER Ca2+ depletion, palmitate induced rapid phosphorylation of the ER Ca2+ sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and β-cell death. We detected little palmitate-induced insulin secretion, suggesting that these Ca2+ signals are poorly coupled to exocytosis. In summary, we have characterized Ca2+-dependent mechanisms involved in altered β-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER Ca2+ and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes.

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Voltage-gated Ca2+ influx and insulin secretion in human and mouse β-cells are impaired by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157

Cited by 39 publications

References 36 publications

NCLX is an essential component of mitochondrial Na ⁺ /Ca ²⁺ exchange

NCLX is an essential component of mitochondrial Na ⁺ /Ca ²⁺ exchange

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

Effects of palmitate on ER and cytosolic Ca²⁺homeostasis in β-cells

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Voltage-gated Ca2+ influx and insulin secretion in human and mouse β-cells are impaired by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157

Cited by 39 publications

References 36 publications

NCLX is an essential component of mitochondrial Na + /Ca 2+ exchange

NCLX is an essential component of mitochondrial Na + /Ca 2+ exchange

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

Effects of palmitate on ER and cytosolic Ca2+homeostasis in β-cells

Contact Info

Product

Resources

About

NCLX is an essential component of mitochondrial Na ⁺ /Ca ²⁺ exchange

NCLX is an essential component of mitochondrial Na ⁺ /Ca ²⁺ exchange

Effects of palmitate on ER and cytosolic Ca²⁺homeostasis in β-cells