Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice

Lemaire, Katleen; Ravier, Magalie A.; Schraenen, Anica; Creemers, John W.M.; Plas, Raf Van de; Granvik, Mikaela; Lommel, Leentje Van; Waelkens, Etienne; Chimienti, Fabrice; Rutter, Guy A.; Gilon, Patrick; Veld, Pieter In 'T; Schuit, Frans

doi:10.1073/pnas.0906587106

Cited by 306 publications

(381 citation statements)

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“…As OCM is sensitive to changes in refractive index and because pancreatic beta cells have a high zinc content [27], we hypothesised that the origin of the strong OCM signal is dominantly caused by the zinc-insulin crystals in the pancreatic beta cells. To test this hypothesis, thick pancreas sections of ZnT8-KO mice, which lack zinc-insulin crystals in the secretory granules of beta cells [24,27], were subjected to immunohistochemistry (IHC) using antiinsulin antibodies and analysed simultaneously by OCM and confocal fluorescence microscopy. In order to suppress the specular reflection from the cover slip, we used a darkfield OCM (dfOCM) configuration combined with a confocal fluorescence channel (ESM Fig.…”

Section: Resultsmentioning

confidence: 99%

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Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging

et al. 2015

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Aims/hypothesis It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation. Methods NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation. Results We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet. Conclusions/interpretation FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted Diabetologia (2016) 59:550-559 DOI 10.1007 Corinne Berclaz, Anja Schmidt-Christensen and Daniel Szlag contributed equally to this study. Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-015-3819-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

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Section: Resultsmentioning

confidence: 99%

“…Guinea pigs were purchased from Harlan Laboratories (Indianapolis, IN, USA). Mice with ZnT8 (also known as Slc30a8) knockout (KO) and littermates were generated as previously described [24]. The Ethics Committee at Lund University and the Swiss cantonal veterinary authorities approved all experimental animal procedures.…”

Section: Methodsmentioning

confidence: 99%

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging

et al. 2015

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“…Striking granule abnormalities were observed by electron microscopy in ZnT8-depleted β cells, notably that granules lacked the characteristic insulin dense cores, whilst many ISGs contained a "rod-like" structure [60,77,80]. Moreover, islet and β cell free Zn 2+ was decreased in both global and β cell specific ZnT8 null animals [60,77,81].…”

Section: Znt8 Studies In Vivomentioning

confidence: 99%

Zinc and diabetes

Chabosseau

Rutter

2016

Archives of Biochemistry and Biophysics

149

104

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Zn 2+ ions are essential for the normal processing and storage of insulin and altered pancreatic insulin content is associated with all forms of diabetes mellitus. Work of the past decade has identified variants in the human SLC30A8 gene, encoding the zinc transporter ZnT8 which is expressed highly selectively on the secretory granule of pancreatic islet β and α cells, as affecting the risk of Type 2 Diabetes. Here, we review the regulation and roles of Zn 2+ ions in islet cells, the mechanisms through which SLC30A8 variants might affect glucose homeostasis and diabetes risk, and the novel technologies including recombinant targeted zinc probes and knockout mice which have been developed to explore these questions. Abbreviation ISG: Insulin Secreting Granules; T2D: Type 2 Diabetes; T1D: Type 1 Diabetes; 2 Diabetes mellitus is a common metabolic disease, affecting approximately 9% of the adult population worldwide. It is characterized by high circulating glucose levels over a prolonged period of time which leads to long-term health complications [1]. Type 1 Diabetes (T1D) involves the autoimmune destruction of insulin-secreting β cells while Type 2 Diabetes (T2D) is linked to both a decrease of insulin release and deficient hormone action on targeted organs [2].The links among Zn 2+ , diabetes and insulin were established in the 1930s, a decade after the discovery of the hormone, when a study showed crystallized insulin contains Zn 2+ and that Zn 2+ , along with other metal ions, could reversibly trigger insulin crystallization [3]. Zinc was later demonstrated to prolong insulin action when co-injected with the hormone [4].These early studies, as well as much more recent findings showing association between T2D risk and the inheritance of gene variants encoding a critical β cell Zn 2+ transporter, ZnT8[5] have generated considerable interest in the role of Zn 2+ in diabetes aetiology and as a possible therapeutic target [6]. Zinc and the diabetic patientScott and Fisher were the first to report a direct link between zinc and diabetes in patients.While assessing the insulin content in the pancreas of diabetic patients compared to nondiabetic cadavers, they showed that in the former group zinc content was reduced by 75 % [7].Epidemiological studies also demonstrated that diabetes and whole body zinc status are associated [8][9][10][11]. In T1D and T2D patients, serum zinc levels are significantly decreased [9][10][11], this being associated with an increased zinc urinary loss [8].Zinc may have important anti-oxidant properties, as it acts as a cofactor of the superoxide dismutase enzyme which regulates the detoxification of reactive oxygen species, regulating the expression and protecting against the oxidative stress induced by chronic hyperglycaemia (for review, [12]). Furthermore, zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration suggesting that Zn 2+ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen [13].Zinc supplementation h...

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“…However, because FluoZin-3 or other Zn 2+ sensors are applied to the extracellular bath, the sensitivity of detecting local Zn 2+ release near the plasma membrane is compromised by the background fluorescence from the bulk solution. Consequently, total internal reflection of fluorescence (TIRF) microscopy has been applied to FluoZin-3 or RhodZin-3 imaging to reject bulk fluorescence signal and to study secretion at the interface between a cell and the underlying glass coverslip (10,16). More sensitive imaging probes that are compatible with wide-field epifluorescence detection or confocal laser scanning microscopy (CLSM) would be extremely valuable for following the pattern or the site of insulin release in 3D in cell populations over time.…”

mentioning

confidence: 99%

“…This phenomenon has been exploited to develop experiments using the fluorescent Zn 2+ sensors Zinquin (11), FluoZin-3 (12), RhodZin-3 or Newport Green DCF (10), and ZnAF-2 (13) as a surrogate for measuring insulin release (14)(15)(16). However, because FluoZin-3 or other Zn 2+ sensors are applied to the extracellular bath, the sensitivity of detecting local Zn 2+ release near the plasma membrane is compromised by the background fluorescence from the bulk solution.…”

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confidence: 99%

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

Chen

Bellomo

et al. 2011

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

145

176

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Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn 2+ , and that Zn 2+ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn 2+ chelation and bound Zn 2+ with high selectivity against Ca 2+ and Mg 2+ . When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn 2+ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn 2+ /insulin release occurred largely in small groups of adjacent β cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca 2+ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca 2+ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn 2+ -containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.probe development | zinc imaging | hormone secretion assay

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Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice

Cited by 306 publications

References 36 publications

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging

Longitudinal three-dimensional visualisation of autoimmune diabetes by functional optical coherence imaging

Zinc and diabetes

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

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