Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism

Rountree, Austin M.; Neal, Adam; Lisowski, Mark; Rizzo, Norma O.; Radtke, Jared; White, Sarah; Luciani, Dan S.; Kim, Francis; Hampe, Christiane S.; Sweet, Ian R.

doi:10.1074/jbc.m114.556050

Cited by 21 publications

(27 citation statements)

References 48 publications

(23 reference statements)

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“…A common factor amongst this class of secretagogues is the ability to increase the reductive state of cytochrome c, without which activation of insulin secretion does not occur. 32,34 A second class of secretagogues such as acetylcholine and GLP-1, are unable to stimulate ISR independently, but can potentiate nutrient-stimulated ISR. The results of this study place PA in the latter category.…”

Section: Pa Can Amplify But Not Trigger Insulin Secretionmentioning

confidence: 99%

“…The multi-parametric, realtime approach enables optimal comparison between parameters reflecting different stages of stimulussecretion coupling and has been used for many mechanistically based studies of islet physiology. 28,[32][33][34] To take advantage of real-time methods, we utilized acute and pharmacologic approaches to assess rapid mechanisms mediating metabolic signaling and insulin secretion. Overall, based on the lack of effect of PA on ISR at low glucose, we predicted that PA would not be utilized significantly as a fuel to increase ATP production, nor increase Ca 2+ influx via voltage-dependent Ca 2+ channels.…”

Section: Introductionmentioning

confidence: 99%

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Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum

Kuok

Rountree

Jung

et al. 2019

Islets

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The aim of the study was to determine the acute contribution of fuel oxidation in mediating the increase in insulin secretion rate (ISR) in response to fatty acids. Measures of mitochondrial metabolism, as reflected by oxygen consumption rate (OCR) and cytochrome c reduction, calcium signaling, and ISR by rat islets were used to evaluate processes stimulated by acute exposure to palmitic acid (PA). The contribution of mitochondrial oxidation of PA was determined in the presence and absence of a blocker of mitochondrial transport of fatty acids (etomoxir) at different glucose concentrations. Subsequent to increasing glucose from 3 to 20 mM, PA caused small increases in OCR and cytosolic calcium (about 20% of the effect of glucose). In contrast, the effect of PA on ISR was almost 3 times that by glucose, suggesting that the metabolism of PA is not the dominant mechanism mediating PA's effect on ISR. This was further supported by lack of inhibition of PA-stimulated OCR and ISR when blocking entry of PA into mitochondria (with etomoxir), and PA's lack of stimulation of reduced cytochrome c in the presence of high glucose. Consistent with the lack of metabolic stimulation by PA, an inhibitor of calcium release from the endoplasmic reticulum, but not a blocker of L-type calcium channels, abolished the PA-induced elevation of cytosolic calcium. Notably, ISR was unaffected by thapsigargin showing the dissociation of endoplasmic reticulum calcium release and second phase insulin secretion. In conclusion, stimulation of ISR by PA was mediated by mechanisms largely independent of the oxidation of the fuel. ARTICLE HISTORY

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Section: Pa Can Amplify But Not Trigger Insulin Secretionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum

Kuok

Rountree

Jung

et al. 2019

Islets

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“…The normalization of the NAD(P)H signal was as a percent of RFU FCCP and RFU KCN , defined as 0 and 100% respectively for each cell31. The imaging of calcium, after loading cells with the calcium-sensitive dye FURA-1, was carried out as previously described32.…”

Section: Methodsmentioning

confidence: 99%

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

Neal

Rountree

Radtke

et al. 2016

Sci Rep

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Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.

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“…To establish the 4 genotypes of mice used for these studies we first bred Bax flox/flox :Bak -/mice (Stock number 006329, The Jackson Laboratory) with Pdx1-CreER mice (33) to create littermate mice with Bak single knockout (Bak SKO; Bax flox/flox :Bak -/-) and Bax-Bak double knockout (Bax-Bak DKO; Bax flox/flox :Bak -/-:Pdx1-CreER + ) (34,35). We further mated Bax flox/flox :Bak +/progeny to re-introduce the wild-type Bak allele and obtain a parallel colony of Bax single knockout (Bax SKO; Bax flox/flox :Bak +/+ :Pdx1-CreER + ) and littermate wild-type mice (WT; Bax flox/flox :Bak +/+ ).…”

Section: Experimental Procedures Mice and In Vivo Studiesmentioning

confidence: 99%

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress

White

Zhang

Yang

et al. 2019

Preprint

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ER stress and apoptosis contribute to the loss of pancreatic β-cells under the pro-diabetic conditions of glucolipotoxicity. Although activation of the canonical pathway of intrinsic apoptosis is known to require Bax and Bak, their individual and combined involvement in glucolipotoxic β-cell death have not been demonstrated. It has also remained an open question if Bax and Bak in β-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined β-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic β-cell death in vitro happens in sequential stages; first via non-apoptotic mechanisms and later by apoptosis, which Bax and Bak were redundant in triggering. In contrast, they had non-redundant roles in mediating staurosporine-induced β-cell apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated β-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that Bax and Bak together dampen the unfolded protein response in β-cells during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings identify novel roles of the canonical apoptosis machinery in modulating stress signals that are important for the pathobiology of β-cells in diabetes.

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Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism

Cited by 21 publications

References 48 publications

Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum

Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress

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