Electrophysiological and Metabolic Characterization of Single β-Cells and Islets From Diabetic GK Rats

Hughes, Simon; Faehling, Martin; Thorneley, C W; Proks, Peter; Ashcroft, Frances M.; Smith, Paul A.

doi:10.2337/diab.47.1.73

Cited by 43 publications

(37 citation statements)

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“…On single-channel recording, the glucose sensitivity of the beta cell K ATP channel is remarkably reduced in GK rats, while the inhibitory effect of ATP on channel activity is not significantly different in control and GK rats [5]. The intracellular ATP elevation induced by high glucose is impaired in GK rats [44] as well as in patients with type 2 diabetes [45]. Thus, the impaired insulinotrophic action of glucose in beta cells of GK rats may be attributable to insufficient closure of the K ATP channel because of deficient ATP production derived from impaired glucose metabolism.…”

Section: Discussionmentioning

confidence: 96%

“…Since depletion of mitochondrial DNA abolishes the glucose-induced ATP elevation, mitochondria clearly are a major source of ATP production in pancreatic beta cells [2,3]. Glucose-induced insulin secretion from beta cells is often impaired by exposure to high concentrations of fuels including glucose, NEFAs and ketone bodies, and by administration of diabetogenic pharmacological agents, all of which involve impaired glucose-induced ATP elevation in beta cells [4][5][6][7][8][9][10][11]. Thus, reduced mitochondrial ATP production plays an important role in impaired glucoseinduced insulin secretion.…”

Section: Introductionmentioning

confidence: 99%

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Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

et al. 2008

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Aims/hypothesis Na + /K + -ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na + /K + -ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined. Methods Isolated islets from Wistar rats and diabetic GotoKakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed. Results Ouabain caused a rapid Tyr 418 phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets. Conclusions/interpretation These results indicate that Src plays a role in the signal-transducing function of Na + /K + -ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucoseinduced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

et al. 2008

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“…ATP/ADP ratios (less than 3) were inexplicably low even in control rats (10). Hughes et al (51) reported that ATP rose somewhat less after exposure of GK islets to 10 mmol/l glucose than what occurred in control islets; however, AT P / A D P ratios were not reported. In contrast, Zong-Chao et al (48) reported that isolated mitochondria from GK islets synthesize ATP essentially normally.…”

Section: Pancreatic Islet Function In Gk Ratsmentioning

confidence: 91%

A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats.

et al. 1999

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A widely accepted genetically determined rodent model for human type 2 diabetes is the Goto-Kakizaki (GK) rat; however, the lesion(s) in the pancreatic islets of these rats has not been identified. Herein, intact islets from GK rats (aged 8-14 weeks) were studied, both immediately after isolation and after 18 h in tissue culture. Despite intact contents of insulin and protein, GK islets had markedly deficient insulin release in response to glucose, as well as to pure mitochondrial fuels or a non-nutrient membrane-depolarizing stimulus (40 mmol/l K+). In contrast, mastoparan (which activates GTP-binding proteins [GBPs]) completely circumvented any secretory defect. Basal and stimulated levels of adenine and guanine nucleotides, the activation of phospholipase C by Ca2+ or glucose, the secretory response to pertussis toxin, and the activation of selected low-molecular weight GBPs were not impaired. Defects were found, however, in the autophosphorylation and catalytic activity of cytosolic nucleoside diphosphokinase (NDPK), which may provide compartmentalized GTP pools to activate G-proteins; a deficient content of phosphoinositides was also detected. These studies identify novel, heretofore unappreciated, defects late in signal transduction in the islets of our colony of GK rats, possibly occurring at the site of activation by NDPK of a mastoparan-sensitive G-protein-dependent step in exocytosis.

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“…Taking into account cell capacitance, this corresponds to a K ATP channel density of ~4 pA/pF. In a similar study conducted using rat ␤-cells, Hughes et al (42) reported K ATP channel densities of 4.6 pA/pF. This suggests that K ATP channel density is not significantly different between the two species and is therefore in agreement with our finding that diazoxide brings the resting membrane potential of rat ␤-cells to a level close to that of mouse ␤-cells.…”

Section: +mentioning

confidence: 95%

Differential patterns of glucose-induced electrical activity and intracellular calcium responses in single mouse and rat pancreatic islets.

et al. 2000

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Although isolated rat islets are widely used to study in vitro insulin secretion and the underlying metabolic and ionic processes, knowledge on the properties of glucose-induced electrical activity (GIEA), a key step in glucose-response coupling, has been gathered almost exclusively from microdissected mouse islets. Using a modified intracellular recording technique, we have now compared the patterns of GIEA in collagenase-isolated rat and mouse islets. Resting membrane potentials of rat and mouse ␤-cells were approximately -50 and -60 mV, respectively. Both rat and mouse ␤-cells displayed prompt membrane depolarizations in response to glucose. However, whereas the latter exhibited a bursting pattern consisting of alternating hyperpolarized and depolarized active phases, rat ␤-cells fired action potentials from a nonoscillating membrane potential at all glucose concentrations (8.4-22.0 mmol/l). This was mirrored by changes in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which was oscillatory in mouse and nonoscillatory in rat islets. Stimulated rat ␤-cells were strongly hyperpolarized by diazoxide, an activator of ATP-dependent K + channels. Glucose evoked dosedependent depolarizations and [Ca 2+ ] i increases in both rat (EC 50 5.9-6.9 mmol/l) and mouse islets (EC 50 8.3-9.5 mmol/l), although it did not affect the burst plateau potential in the latter case. We conclude that there are important differences between ␤-cells from both species with respect to early steps in the stimulussecretion coupling cascade based on the following findings: 1) mouse ␤-cells have a larger resting K + conductance in 2 mmol/l glucose, 2) rat ␤-cells lack the compensatory mechanism responsible for generating membrane potential oscillations and holding the depolarized plateau potential in mouse ␤-cells, and 3) the electrical and [Ca 2+ ] i dose-response curves in rat ␤-cells are shifted toward lower glucose concentrations. Exploring the molecular basis of these differences may clarify several a priori assumptions on the electrophysiological properties of rat ␤-cells, which could foster the development of new working models of pancreatic ␤-cell function.

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Electrophysiological and Metabolic Characterization of Single β-Cells and Islets From Diabetic GK Rats

Cited by 43 publications

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Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats.

Differential patterns of glucose-induced electrical activity and intracellular calcium responses in single mouse and rat pancreatic islets.

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