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Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional β cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory β cell mass expansion is largely unknown. We found in ents that β cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in β cells using specific anti-miR molecules mimicked gene expression changes occurring during13 cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory0 cell mass expansion occurring under different insulin resistance states., Introduction Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia resulting from defective function and/or loss of insulin-secreting β cells (1), (2). These cells, located within the islets of [...]

Alteration of pancreatic [beta]-cell survival and Preproinsulin gene expression by prolonged hyperglycemia may result from increased c-MYC expression. However, it is unclear whether c-MYC effects on [beta]-cell function are compatible with its proposed role in glucotoxicity. We therefore tested the effects of short-term c-MYC activation on key [beta]-cell stimulus-secretion coupling events in islets isolated from mice expressing a tamoxifenswitchable form of c-MYC in [beta]-cells (MycER) and their wild-type littermates. Tamoxifen treatment of wild-type islets did not affect their cell survival, Preproinsulin gene expression, and glucose stimulussecretion coupling. In contrast, tamoxifen-mediated c-MYC activation for 2-3 days triggered cell apoptosis and decreased Preproinsulin gene expression in MycER islets. These effects were accompanied by mitochondrial membrane hyperpolarization at all glucose concentrations, a higher resting intracellular calcium concentration ([[[Ca.sup.2+].sub.i]], and lower glucose-induced [[[Ca.sup.2+]].sub.i] rise and islet insulin content, leading to a strong reduction of glucose-induced insulin secretion. Compared with these effects, 1-wk culture in 30 mmol/l glucose increased the islet sensitivity to glucose stimulation without reducing the maximal glucose effectiveness or the insulin content. In contrast, overnight exposure to a low [H.sub.2][O.sub.2] concentration increased the islet resting [[[Ca.sup.2+]].sub.i] and reduced the amplitude of the maximal glucose response as in tamoxifen-treated MycER islets. In conclusion, c-MYC activation rapidly stimulates apoptosis, reduces Preproinsulin gene expression and insulin content, and triggers functional alterations of [beta]-cells that are better mimicked by overnight exposure to a low [H.sub.2][O.sub.2] concentration than by prolonged culture in high glucose. [beta]-cell mass; apoptosis; cytosolic calcium concentration; insulin secretion; mitochondrial membrane potential

Stimulatory concentrations of glucose induce two patterns of cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]) oscillations in mouse islets: simple or mixed. In the mixed pattern, rapid oscillations are superimposed on slow ones. In the present study, we examined the role of the membrane potential in the mixed pattern and the impact of this pattern on insulin release. Simultaneous measurement of [[[Ca.sup.2+]].sub.c] and insulin release from single islets revealed that mixed [[[Ca.sup.2+]].sub.c] oscillations triggered synchronous oscillations of insulin secretion. Simultaneous recordings of membrane potential in a single [beta]-cell within an islet and of [[[Ca.sup.2+]].sub.c] in the whole islet demonstrated that the mixed pattern resulted from compound bursting (i.e., clusters of membrane potential oscillations separated by prolonged silent intervals) that was synchronized in most [beta]-cells of the islet. Each slow [[[Ca.sup.2+]].sub.c] increase during mixed oscillations was due to a progressive summation of rapid oscillations. Digital image analysis confirmed the good synchrony between subregions of an islet. By contrast, islets from sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase isoform 3 (SERCA3)-knockout mice did not display typical mixed [[[Ca.sup.2+]].sub.c] oscillations in response to glucose. This results from a lack of progressive summation of rapid oscillations and from altered spontaneous electrical activity, i.e., lack of compound bursting, and membrane potential oscillations characterized by lower-frequency but larger-depolarization phases than observed in SERCA[3.sup.+/+] [beta]-cells. We conclude that glucose-induced mixed [[[Ca.sup.2+]].sub.c] oscillations result from compound bursting in all [beta]-cells of the islet. Disruption of SERCA3 abolishes mixed [[[Ca.sup.2+]].sub.c] oscillations and augments [beta]-cell depolarization. This latter observation indicates that the endoplasmic reticulum participates in the control of the [beta]-cell membrane potential during glucose stimulation. electrical activity; insulin-secreting cell; thapsigargin

Help for hypertrophy: One way that cardiomyocytes respond to injury or stress is by increasing in size (hypertrophy). Montiel et al. studied the role of oxidative stress in myocardial hypertrophy. They saw that the water channel aquaporin-1 (AQP1) was increased in mouse and human hypertrophic heart tissue and that AQP1 regulated hydrogen peroxide transport within myocytes. Deletion or pharmacological blockade of AQP1 abrogated hypertrophy in response to different stressors in cells and in a mouse model of angiotensin II–induced cardiac hypertrophy, identifying a potential therapeutic target for hypertrophic cardiomyopathy. Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2. Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell–derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies. [ABSTRACT FROM AUTHOR]

This work reports on synchrotron X-ray fluorescence (SR-XRF) imaging of vitrified islets of Langerhans in two-dimensional and computed tomography (CT) mode and serves as a proof-of-principle for the preparation and SR-XRF imaging of cryoloops in a cryogenic environment. Selection of suited cryoloops and cryoprotectant solution enabled vitrification of the islets. The SR-XRF experimental setup was adapted with a cryogenic gas stream, enabling analysis of cryofrozen islets and a dual silicon drift detector (SDD) configuration, providing an increased solid angle for XRF collection. Element distributions of K, Ca, Fe and Zn within single, cryofrozen islets could be obtained with 500 nm spatial resolution using X-ray compound refractive lenses (CRLs) with an adaptive phase plate. Titanium, present as a trace element in the cryoloops, was used as a marker signal to efficiently define the XRF scan area. Preliminary 2D cryo-analysis of wild type (n = 1) and Mt1-Mt2-knock-out (n = 1) islets under low/high glucose conditions did not reveal changes in the distribution and concentration of Ca, Fe and Zn. Cryotomography XRF scans indicated aspherical islets within the cryoloop, likely due to capillary effects in the cryoloop before plunge freezing. The presence of diffracting ice crystals hampered CT reconstruction, indicating the need for further development of a routine workflow ensuring perfect vitrification. Collectively, this novel multidisciplinary approach proved to be valid and can be used for future studies to assess elemental compositions of islets of Langerhans or other cell clusters/tissues under different pathophysiological conditions. [ABSTRACT FROM AUTHOR]

Insulin-secreting pancreatic β-cells play a critical role in blood glucose homeostasis and the development of type 2 diabetes (T2D) in the context of insulin resistance. Based on data obtained at the whole cell level using poorly specific chemical probes, reactive oxygen species (ROS) such as superoxide and hydrogen peroxide have been proposed to contribute to the stimulation of insulin secretion by nutrients (positive role) and to the alterations of cell survival and secretory function in T2D (negative role). This raised the controversial hypothesis that any attempt to decrease β-cell oxidative stress and apoptosis in T2D would further impair insulin secretion. Over the last decade, the development of genetically-encoded redox probes that can be targeted to cellular compartments of interest and are specific of redox couples allowed the evaluation of short- and long-term effects of nutrients on β-cell redox changes at the subcellular level. The data indicated that the nutrient regulation of β-cell redox signaling and ROS toxicity is far more complex than previously thought and that the subcellular compartmentation of these processes cannot be neglected when evaluating the mechanisms of ROS production or the efficacy of antioxidant enzymes and antioxidant drugs under glucolipotoxic conditions and in T2D. In this review, we present what is currently known about the compartmentation of redox homeostatic systems and tools to investigate it. We then review data about the effects of nutrients on β-cell subcellular redox state under normal conditions and in the context of T2D and discuss challenges and opportunities in the field. Image 1 • New genetically-encoded redox probes allow the study of changes in cellular redox state in a compartment-specific manner. • Nutrient-induced changes in pancreatic β-cell redox state are highly compartmentalized. • The impact of acute glucose stimulation on cytosolic and mitochondrial redox probe oxidation remains controversial. • The cytosol, mitochondria, ER and peroxisomes could play a role in β-cell oxidative stress under nutrient toxic conditions. • Intravital microscopy may improve the study of subcellular oxidative stress in insulin resistance and type 2 diabetes. We are aware, and agree, that the term "oxidants" may be more adequate than "reactive oxygen species" (ROS) to collectively refer to hydrogen peroxide (H 2 O 2), superoxide (O 2 −), and other oxidants when the specific molecule involved is not known [1]. However, to avoid disturbing the reader from the β-cell field, and as both terms are equally vague, we chose to use "ROS" rather than "oxidants" throughout this review. [ABSTRACT FROM AUTHOR]

The mechanisms of control of glucagon secretion are largely debated. In particular, the paracrine role of somatostatin (SST) is unclear. We studied its role in the control of glucagon secretion by glucose and KATP channel blockers, using perifused islets and the in situ perfused pancreas. The involvement of SST was evaluated by comparing glucagon release of control tissue or tissue without paracrine influence of SST (pertussis toxin-treated islets, or islets or pancreas from Sst-/- mice). We show that removal of the paracrine influence of SST suppresses the ability of KATP channel blockers or KATP channel ablation to inhibit glucagon release, suggesting that in control islets, the glucagonostatic effect of KATP channel blockers/ablation is fully mediated by SST. By contrast, the glucagonostatic effect of glucose in control islets is mainly independent of SST for low glucose concentrations (0-7 mmol/L) but starts to involve SST for high concentrations of the sugar (15-30 mmol/L). This demonstrates that the glucagonostatic effect of glucose only partially depends on SST. Real-time quantitative PCR and pharmacological experiments indicate that the glucagonostatic effect of SST is mediated by two types of SST receptors, SSTR2 and SSTR3. These results suggest that alterations of the paracrine influence of SST will affect glucagon release. [ABSTRACT FROM AUTHOR]

Like all the cells of an organism, pancreatic ß-cells originate from embryonic stem cells through a complex cellular process termed differentiation. Differentiation involves the coordinated and tightly controlled activation/repression of specific effectors and gene clusters in a time-dependent fashion thereby giving rise to particular morphological and functional cellular features. Interestingly, cellular differentiation is not a unidirectional process. Indeed, growing evidence suggests that under certain conditions, mature ß-cells can lose, to various degrees, their differentiated phenotype and cellular identity and regress to a less differentiated or a precursor-like state. This concept is termed dedifferentiation and has been proposed, besides cell death, as a contributing factor to the loss of functional ß-cell mass in diabetes. ß-cell dedifferentiation involves: (1) the downregulation of ß-cell-enriched genes, including key transcription factors, insulin, glucose metabolism genes, protein processing and secretory pathway genes; (2) the concomitant upregulation of genes suppressed or expressed at very low levels in normal ß-cells, the ß-cell forbidden genes; and (3) the likely upregulation of progenitor cell genes. These alterations lead to phenotypic reconfiguration of ß-cells and ultimately defective insulin secretion. While the major role of glucotoxicity in ß-cell dedifferentiation is well established, the precise mechanisms involved are still under investigation. This review highlights the identified molecular mechanisms implicated in ß-cell dedifferentiation including oxidative stress, endoplasmic reticulum (ER) stress, inflammation and hypoxia. It discusses the role of Foxo1, Myc and inhibitor of differentiation proteins and underscores the emerging role of non-coding RNAs. Finally, it proposes a novel hypothesis of ß-cell dedifferentiation as a potential adaptive mechanism to escape cell death under stress conditions. [ABSTRACT FROM AUTHOR]

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators. [ABSTRACT FROM AUTHOR]

In rat pancreatic islets, β-cell gene expression, survival, and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here, we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. Rat pancreatic islets were cultured 1-7 days at G5, G10, or G30 with or without 3 µM of the GK activator Ro 28-0450 (Ro). After culture, (β-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H auto-fluorescence, intracellular calcium concentration, and insulin secretion were tested in the absence or presence of Ro. Prolonged culture of rat islets at G5 or G30 instead of G10 triggered (β-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected (β-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to (β-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10 but failed at restoring (β-cell glucose responsiveness after culture at G5 or G30. We conclude that pharmacological GK activation prevents the alteration of (β-cell survival and function by long-term culture at G5 but mimics gluco-toxicity when added to G10. The complex effects of glucose on the β-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se. from an effect of glucose per se. [ABSTRACT FROM AUTHOR]

The glucose stimulation of insulin secretion by pancreatic ß- cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in ß-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in ß-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280mVin the absence of glucose to -299mVin 30 mM glucose. These changes were largely independent from changes in intracellular Ca2+ concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic ß- cells under control conditions. [ABSTRACT FROM AUTHOR]