RNA editing blood biomarkers for predicting mood alterations in HCV patients.

Treatment-emergent depression is a common complication in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin. It has recently been shown that changes in A-to-I RNA editing rates are associated with various pathologies such as inflammatory disorders, depression and suicide. Interestingly, IFN-α induces gene expression of the RNA editing enzyme ADAR1-1 (ADAR1a-p150) and alters overall RNA editing activity. In this study, we took advantage of the high prevalence of pharmacologically induced depression in patients treated with IFN-α and ribavirin to test the interest of RNA editing-related biomarkers in white blood cells of patients. In this 16-week longitudinal study, a small cohort of patients was clinically evaluated using standard assessment methods prior to and during antiviral therapy and blood samples were collected to analyse RNA editing modifications. A-I RNA editing activity on the phosphodiesterase 8A (PDE8A) gene, a previously identified RNA editing hotspot in the context of lupus erythematosus, was quantified by using an ultra-deep next-generation sequencing approach. We also monitored gene expression levels of the ADAR enzymes and the PDE8A gene during treatment by qPCR. As expected, psychiatric evaluation could track treatment-emergent depression, which occurred in 30% of HCV patients. We show that PDE8A RNA editing is increased in all patients following interferon treatment, but differently in 30% of patients. This effect was mimicked in a cellular model using SHSY-5Y neuroblastoma cells. By combining the data of A-I RNA editing and gene expression, we generated an algorithm that allowed discrimination between the group of patients who developed a treatment-emergent depression and those who did not. The current model of drug-induced depression identified A-I RNA editing biomarkers as useful tools for the identification of individuals at risk of developing depression in an objective, quantifiable biological blood test.

Effects of high-fat diet feeding on Znt8-null mice: differences between ß-cell and global knockout of Znt8.

Genomewide association studies have linked a polymorphism in the zinc transporter 8 (Znt8) gene to higher risk of developing type 2 diabetes. Znt8 is highly expressed in pancreatic ß-cells where it is involved in the regulation of zinc transport into granules. However, Znt8 is also expressed in other tissues including α-cells, where its function is as yet unknown. Previous work demonstrated that mice lacking Znt8 globally were more susceptible to diet-induced obesity (Lemaire et al., Proc Natl Acad Sci USA 106: 14872-14877, 2009; Nicolson et al., Diabetes 58: 2070-2083, 2009). Therefore, the main goal of this study was to examine the physiological impact of ß-cell-specific Znt8 deficiency in mice during high-fat high-calorie (HFHC) diet feeding. For these studies, we used ß-cell-specific Znt8 knockout (Ins2Cre:Znt8loxP/loxP) and whole body Znt8 knockout (Cre-:Znt8(-/-)) mice placed on a HFHC diet for 16 wk. Ins2Cre:Znt8loxP/loxP mice on HFHC diet had similar body weights throughout the study but displayed impaired insulin biosynthesis and secretion and were glucose intolerant compared with littermate control Ins2Cre mice. In contrast, Cre-:Znt8(-/-) mice became remarkably obese, hyperglycemic, hyperinsulinemic, insulin resistant, and glucose intolerant compared with littermate control Cre- mice. These data show that ß-cell Znt8 alone does not considerably aggravate weight gain and glucose intolerance during metabolic stress imposed by an HFHC diet. However, global loss of Znt8 is involved in exacerbating diet-induced obesity and resulting insulin resistance, and this may be due to the loss of Znt8 activity in a tissue other than the ß-cell. Thus, our data suggest that Znt8 contributes to the risk of developing type 2 diabetes through ß-cell- and non-ß-cell-specific effects.

Regulation of glucagon secretion by zinc: lessons from the ß cell-specific Znt8 knockout mouse model.

In type-2 diabetes, hyperglucagonaemia aggravates elevated blood glucose levels. Relative to our knowledge of the ß-cell and insulin secretion, there remains a limited understanding of glucagon secretion in α-cells. Regulation of glucagon may be dependent on a combination of factors, which include direct glucose sensing by the α-cell, innervations from the autonomic nervous system and potential 'paracrine' actions by hormones and factors that are released by adjacent endocrine cells within the islets. The list of potential 'paracrine' regulators within the islet includes insulin, somatostatin, γ-aminobutyric acid, glutamate and zinc. Zinc crystallises with insulin in ß-cells and is co-secreted with insulin. In the scientific literature, the effect of exogeneous zinc on glucagon secretion has been debated. Here, we confirm that an increase in exogeneous zinc does inhibit glucagon secretion. To determine if there are physiological effects of zinc on glucagon secretion we used a ß-cell-specific ZnT8 knockout (Znt8BKO) mouse model. Znt8BKO mice, despite showing lower granular zinc content in ß-cells, showed no changes in fasted plasma glucagon levels and glucose regulated glucagon secretion. These findings suggest that zinc secreted from ß-cell does not regulate glucagon secretion.

Beta cell-specific Znt8 deletion in mice causes marked defects in insulin processing, crystallisation and secretion.

AIMS/HYPOTHESIS: Zinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues. METHODS: We generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis. RESULTS: Znt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis. CONCLUSIONS/INTERPRETATION: This is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.

Zinc, a regulator of islet function and glucose homeostasis.

It is well known that zinc is required in pancreatic beta-cells in the process of insulin biosynthesis and the maturation of insulin secretory granules. In fact, the zinc level in pancreatic islets is amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. High concentrations of zinc can also be toxic because of enhanced oxidative damage. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genomewide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we explore the importance of both zinc and ZnT8 to islet biology and whole body glucose homeostasis.

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

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules," which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Resveratrol enhances UVA-induced DNA damage in HaCaT human keratinocytes.

Resveratrol, a polyphenolic phytoalexin, is a very effective antioxidant that also exhibits strong antiproliferative and anti-inflammatory properties. Recent studies have provided support for the use of resveratrol in human cancer chemoprevention, in combination with either chemotherapeutic drugs or cytotoxic factors for a most efficient treatment of drug refractory tumor cells. Resveratrol is also widely used in topical preparations, as a chemoprotective compound against development of several cutaneous disorders, including skin cancer. Nevertheless, the combined effect of resveratrol and UVA irradiation on cellular toxicity and DNA damage has never been assessed. The aim of this work was to investigate the effect of resveratrol on cell fate in immortalized human keratinocytes HaCaT cells. The results indicated that resveratrol potentiates the production of significant amounts of 8-oxo-7,8-dihydro-2'-deoxyguanosine in UVA-irradiated genomic DNA. Moreover, the combination of resveratrol with UVA significantly enhances the induction of DNA strand breaks and cell death in HaCaT keratinocytes. The conclusion is a potential hazardous effect of topical application of resveratrol, particularly on regions exposed to sunlight.

Zinc homeostasis-regulating proteins: new drug targets for triggering cell fate.

Zinc is an essential trace element for life. Zinc is not only an important nutrient, cofactor of numerous enzymes and transcription factors, but also it acts as an intracellular mediator, similarly to calcium. The recent discovery of its intracellular molecular pathways opens the door to new fields of drug design. Zinc homeostasis results from a coordinated regulation by different proteins involved in uptake excretion and intracellular storage/trafficking of zinc. These proteins are membranous transporters, belonging to the ZIP and ZnT families, and metallothioneins. Their principal function is to provide zinc to new synthesized proteins, important for several functions such as gene expression, immunity, reproduction or protection against free radicals damage. Zinc intracellular concentration is correlated to cell fate, ie proliferation, differentiation or apoptosis, and modifications of zinc homeostasis are observed in several pathologies affecting humans at any stage of life. Two zinc-related diseases, acrodermatitis enteropathica and the lethal milk syndrome, have been recently related to mutations in zinc transporters, SLC39A4 and ZnT-4, respectively. Zinc acts as an inhibitor of apoptosis, while its depletion induces programmed cell death in many cell lines. However, excess zinc can also be cytotoxic, and zinc transporters as well as metallothioneins serve as zinc detoxificating systems. Several zinc channels, controlling the intracellular zinc movements and the free form of the metal, maintain the intracellular zinc homeostasis, and thus the balance between life and cell death. Apart from these general activities, zinc has particular biological roles in some specialized cells. It acts as a paracrine regulator in pancreatic cell, neuron or neutrophil activity by a mechanism of vesicles-mediated metal excretion and uptake. A well knowledge on zinc transporters will be useful to develop new molecular targets to act on these zinc-regulated biological functions.

[Role of intracellular zinc in programmed cell death]. / Rôle du zinc intracellulaire dans la mort cellulaire programmée.

Apoptosis is a type of cell death involved in several biological events during tissue development, remodelling or involution. It could be induced by several extracellular or intracellular stimuli with an important role for metals like zinc or calcium. Cellular zinc is described as an inhibitor of apoptosis, while its depletion induces death in many cell lines. Using different chemical tools like specific zinc-chelators or ionophores, it is possible to study and understand the mechanisms of programmed cell death induction. The decrease in intracellular zinc concentration induces a characteristic apoptosis with apoptotic bodies formation and nuclear DNA condensation and fragmentation. This zinc depletion activates the caspases-3, -8 and -9, responsible for the proteolysis of several target proteins like poly(ADP-ribose) polymerase or transcription factors. Zinc addition in cell culture medium prevents the apparition of morphological and biochemical signs induced by intracellular zinc chelation, but also by other apoptosis inducers like etoposide or tumour necrosis factor alpha (TNF alpha). However, excess of zinc can also be cytotoxic. The balance between life and cell death is maintained by several zinc channels, controlling the intracellular zinc movements and the free amount of the metal.

Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression.

To analyze the effects of high concentrations of zinc ions on oxidative stress protection, we developed an original model of zinc-resistant HeLa cells (HZR), by using a 200 microM zinc sulfate-supplemented medium. Resistant cells specifically accumulate high zinc levels in intracellular vesicles. These resistant cells also exhibit high expression of metallothioneins (MT), mainly located in the cytoplasm. Exposure of HZR to Zn-depleted medium for 3 or 7 d decreases the intracellular zinc content, but only slightly reduces MT levels of resistant cells. No changes of the intracellular redox status were detected, but zinc resistance enhanced H2O2-mediated cytotoxicity. Conversely, zinc-depleted resistant cells were protected against H2O2-induced cell death. Basal- and oxidant-induced DNA damage was increased in zinc resistant cells. Moreover, measurement of DNA damage on zinc-depleted resistant cells suggests that cytoplasmic metal-free MT ensures an efficient protection against oxidative DNA damage, while Zn-MT does not. This newly developed Zn-resistant HeLa model demonstrates that high intracellular concentrations of zinc enhance oxidative DNA damage and subsequent cell death. Effective protection against oxidative damage is provided by metallothionein under nonsaturating zinc conditions. Thus, induction of MT by zinc may mediate the main cellular protective effect of zinc against oxidative injury.

Role of cellular zinc in programmed cell death: temporal relationship between zinc depletion, activation of caspases, and cleavage of Sp family transcription factors.

Zinc is a potent inhibitor of apoptosis, whereas zinc depletion induces apoptosis in many cell lines. To investigate the mechanisms of zinc depletion-induced apoptosis, HeLa cells were treated with the membrane permeable metal ion chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). TPEN decreased the intracellular level of zinc and induced apoptosis with a characteristic cellular pattern, i.e. cell shrinkage and formation of apoptotic bodies, with DNA fragmentation and formation of a typical DNA ladder pattern. Following TPEN treatment, caspases-3, -8, and -9 were activated and caspase target proteins, poly(ADP-ribose) polymerase, and Sp transcription factors were cleaved. These effects were inhibited by adding zinc to the medium. To assess the role of zinc in the activation of the caspase cascade, we compared zinc inhibition during tumor necrosis factor alpha/cycloheximide- and etoposide-induced apoptosis with that induced by TPEN. Zinc addition partially inhibited caspase-3 activation, but not caspase-8 and -9 cleavage in HeLa cells treated with tumor necrosis factor alpha or etoposide. These results suggest that caspase-3 is rapidly and directly activated by zinc chelation, without a requirement for an upstream event. Caspase-3 activation is therefore the main event leading to apoptosis after intracellular zinc chelation. Finally, we conclude that cellular zinc inhibits apoptosis by maintaining caspase-3 inactive.