The German Fast Track Toward Reimbursement of Digital Health Applications (DiGA): Opportunities and Challenges for Manufacturers, Healthcare Providers, and People With Diabetes.

BACKGROUND: Digital health applications (DiGA) supporting the management of diabetes are among the most commonly available digital health technologies. However, transparent quality assurance of DiGA and clinical proof of a positive healthcare effect is often missing, which creates skepticism of some stakeholders regarding the usage and reimbursement of these applications. METHODS: This article reviews the recently established fast-track integration of DiGA in the German reimbursement market, with emphasis on the current impact for manufacturers, healthcare providers, and people with diabetes. The German DiGA fast track is contextualised with corresponding initiatives in Europe. RESULTS: The option of a provisional prescription and reimbursement of DiGA while proving a positive healthcare effect in parallel may expedite the adoption of DiGA in Germany and beyond. However, hurdles for a permanent prescription and reimbursement of DiGA are high and only one of 12 that have achieved this status specifically addresses people with diabetes. CONCLUSION: The DiGA fast track needs to be further enhanced to cope with remaining skepticism and contribute even more to a value-based diabetes care.

TIGER: technical variation elimination for metabolomics data using ensemble learning architecture.

Large metabolomics datasets inevitably contain unwanted technical variations which can obscure meaningful biological signals and affect how this information is applied to personalized healthcare. Many methods have been developed to handle unwanted variations. However, the underlying assumptions of many existing methods only hold for a few specific scenarios. Some tools remove technical variations with models trained on quality control (QC) samples which may not generalize well on subject samples. Additionally, almost none of the existing methods supports datasets with multiple types of QC samples, which greatly limits their performance and flexibility. To address these issues, a non-parametric method TIGER (Technical variation elImination with ensemble learninG architEctuRe) is developed in this study and released as an R package (https://CRAN.R-project.org/package=TIGERr). TIGER integrates the random forest algorithm into an adaptable ensemble learning architecture. Evaluation results show that TIGER outperforms four popular methods with respect to robustness and reliability on three human cohort datasets constructed with targeted or untargeted metabolomics data. Additionally, a case study aiming to identify age-associated metabolites is performed to illustrate how TIGER can be used for cross-kit adjustment in a longitudinal analysis with experimental data of three time-points generated by different analytical kits. A dynamic website is developed to help evaluate the performance of TIGER and examine the patterns revealed in our longitudinal analysis (https://han-siyu.github.io/TIGER_web/). Overall, TIGER is expected to be a powerful tool for metabolomics data analysis.

Validation of Candidate Phospholipid Biomarkers of Chronic Kidney Disease in Hyperglycemic Individuals and Their Organ-Specific Exploration in Leptin Receptor-Deficient db/db Mouse.

Biological exploration of early biomarkers for chronic kidney disease (CKD) in (pre)diabetic individuals is crucial for personalized management of diabetes. Here, we evaluated two candidate biomarkers of incident CKD (sphingomyelin (SM) C18:1 and phosphatidylcholine diacyl (PC aa) C38:0) concerning kidney function in hyperglycemic participants of the Cooperative Health Research in the Region of Augsburg (KORA) cohort, and in two biofluids and six organs of leptin receptor-deficient (db/db) mice and wild type controls. Higher serum concentrations of SM C18:1 and PC aa C38:0 in hyperglycemic individuals were found to be associated with lower estimated glomerular filtration rate (eGFR) and higher odds of CKD. In db/db mice, both metabolites had a significantly lower concentration in urine and adipose tissue, but higher in the lungs. Additionally, db/db mice had significantly higher SM C18:1 levels in plasma and liver, and PC aa C38:0 in adrenal glands. This cross-sectional human study confirms that SM C18:1 and PC aa C38:0 associate with kidney dysfunction in pre(diabetic) individuals, and the animal study suggests a potential implication of liver, lungs, adrenal glands, and visceral fat in their systemic regulation. Our results support further validation of the two phospholipids as early biomarkers of renal disease in patients with (pre)diabetes.

Integrated personalized diabetes management goes Europe: A multi-disciplinary approach to innovating type 2 diabetes care in Europe.

Type 2 diabetes mellitus represents a multi-dimensional challenge for European and global societies alike. Building on an iterative six-step disease management process that leverages feedback loops and utilizes commodity digital tools, the PDM-ProValue study program demonstrated that integrated personalized diabetes management, or iPDM, can improve the standard of care for persons living with diabetes in a sustainable way. The novel "iPDM Goes Europe" consortium strives to advance iPDM adoption by (1) implementing the concept in a value-based healthcare setting for the treatment of persons living with type 2 diabetes, (2) providing tools to assess the patient's physical and mental health status, and (3) exploring new avenues to take advantage of emerging big data resources.

Costs and its drivers for diabetes mellitus type 2 patients in France and Germany: a systematic review of economic studies.

BACKGROUND: Type 2 diabetes represents an increasingly critical challenge for health policy worldwide. It absorbs massive resources from both patients and national economies to sustain direct costs of the treatment of type 2 diabetes and its complications and indirect costs related to work loss and wages. More recently, there are innovations based on remote control and personalised programs that promise a more cost-effective diabetes management while reducing diabetes-related complications. In such a context, this work attempts to update cost analysis reviews on type 2 diabetes, focusing on France and Germany, in order to explore most significant cost drivers and cost-saving opportunities through innovations in diabetes care. Although both countries approach care delivery differently, France and Germany represent the primary European markets for diabetes technologies. METHODS: A systematic review of the literature listed in MEDLINE, Embase and EconLit has been carried out. It covered interventional, observational and modelling studies on expenditures for type 2 diabetes management in France or Germany published since 2012. Included articles were analysed for annual direct, associated and indirect costs of type 2 diabetes patients. An appraisal of study quality was performed. Results were summarised narratively. RESULTS: From 1260 records, the final sample was composed of 24 papers selected according to predefined inclusion/exclusion criteria. Both France and Germany revealed a predominant focus on direct costs. Comparability was limited due to different study populations and cost categories used. Indirect costs were only available in Germany. According to prior literature, reported cost drivers are hospitalisation, prescriptions, higher HbA1c and BMI, treatment with insulin and complications, all indicators of disease severity. The diversity of available data and included costs limits the results and may explain the differences found. CONCLUSIONS: Complication prevention and glycaemic control are widely recognized as the most effective ways to control diabetes treatment costs. The value propositions of self-based supports, such as hybrid closed-loop metabolic systems, already implemented in type 1 diabetes management, are the key points for further debates and policymaking, which should involve the perspectives of caregivers, patients and payers.

Machine Learning Approaches Reveal Metabolic Signatures of Incident Chronic Kidney Disease in Individuals With Prediabetes and Type 2 Diabetes.

Early and precise identification of individuals with prediabetes and type 2 diabetes (T2D) at risk for progressing to chronic kidney disease (CKD) is essential to prevent complications of diabetes. Here, we identify and evaluate prospective metabolite biomarkers and the best set of predictors of CKD in the longitudinal, population-based Cooperative Health Research in the Region of Augsburg (KORA) cohort by targeted metabolomics and machine learning approaches. Out of 125 targeted metabolites, sphingomyelin C18:1 and phosphatidylcholine diacyl C38:0 were identified as candidate metabolite biomarkers of incident CKD specifically in hyperglycemic individuals followed during 6.5 years. Sets of predictors for incident CKD developed from 125 metabolites and 14 clinical variables showed highly stable performances in all three machine learning approaches and outperformed the currently established clinical algorithm for CKD. The two metabolites in combination with five clinical variables were identified as the best set of predictors, and their predictive performance yielded a mean area value under the receiver operating characteristic curve of 0.857. The inclusion of metabolite variables in the clinical prediction of future CKD may thus improve the risk prediction in people with prediabetes and T2D. The metabolite link with hyperglycemia-related early kidney dysfunction warrants further investigation.

Effects of sequential treatment with lixisenatide, insulin glargine, or their combination on meal-related glycaemic excursions, insulin and glucagon secretion, and gastric emptying in patients with type 2 diabetes.

AIM: To examine the glucose-lowering mechanisms of the glucagon-like peptide-1 receptor agonist lixisenatide after two subsequent meals and in combination with basal insulin. MATERIALS AND METHODS: Twenty-eight metformin-treated patients with type 2 diabetes were randomly assigned to treatment sequences with either lixisenatide or insulin glargine alone for 4 weeks, and a combination of both treatments for 4 weeks. Metabolic examinations were performed before and after each treatment period following breakfast and a late lunch 8 hours later. RESULTS: Lixisenatide mainly reduced postprandial glycaemia, while insulin glargine mainly reduced fasting glucose after breakfast (P < 0.05). This was partially preserved after a late lunch (P < 0.05). After breakfast, lixisenatide reduced insulin secretion and glucagon levels significantly. These effects were lost after a late lunch. Insulin glargine did not significantly reduce glucagon or insulin secretion. Gastric emptying was slowed by lixisenatide, but not by insulin glargine after breakfast. After the late lunch, lixisenatide slightly accelerated gastric emptying. CONCLUSIONS: Lixisenatide decelerates gastric emptying after breakfast, thereby reducing glycaemic excursions, insulin secretion and glucagon levels. The glycaemic reduction persists until after a late lunch, despite accelerated gastric emptying. The combination with insulin glargine enhances the glucose-lowering effect because of complementary modes of action.

Artificial Pancreas Systems for People With Type 2 Diabetes: Conception and Design of the European CLOSE Project.

In the last 10 years tremendous progress has been made in the development of artificial pancreas (AP) systems for people with type 1 diabetes (T1D). The pan-European consortium CLOSE (Automated Glu cose Contro l at H ome for People with Chronic Disea se) is aiming to develop integrated AP solutions (APplus) tailored to the needs of people with type 2 diabetes (T2D). APplus comprises a product and service package complementing the AP system by obligatory training as well as home visits and telemedical consultations on demand. Outcome predictors and performance indicators shall help to identify people who could benefit most from AP usage and facilitate the measurement of AP impact in diabetes care. In a first step CLOSE will establish a scalable APplus model case working at the interface between patients, homecare service providers, and payers in France. CLOSE will then scale up APplus by pursuing geographic distribution, targeting additional audiences, and enhancing AP functionalities and interconnectedness. By being part of the European Institute of Innovation and Technology (EIT) Health public-private partnership, CLOSE is committed to the EIT "knowledge triangle" pursuing the integrated advancement of technology, education, and business creation. Putting stakeholders, education, and impact into the center of APplus advancement is considered key for achieving wide AP use in T2D care.

Impact of insulin glargine and lixisenatide on ß-cell function in patients with type 2 diabetes mellitus: A randomized open-label study.

It is known that ß-cell function can be enhanced by direct stimulation of insulin secretion or by induction of ß-cell rest, but whether both strategies can complement each other has not yet been examined. A total of 28 people with type 2 diabetes (glycated haemoglobin 7.8% ± 0.5%) were treated with either lixisenatide or titrated insulin glargine, followed by their combined administration, each over 4 weeks. First- and second-phase insulin secretion during an intravenous glucose challenge were calculated. First- and second-phase insulin secretion were not increased with glargine alone, but increased after addition of lixisenatide ( P < .001). Lixisenatide alone increased first- and second-phase insulin secretion ( P < .01). Addition of insulin glargine tended to further increase first-phase insulin secretion (P = .054), as well as insulin and C-peptide concentrations ( P < .05). Second-phase insulin secretion was not affected by the addition of glargine. The sequence of initiating lixisenatide or glargine had no effect on the final measures of glycaemia or insulin secretion. Thus, lixisenatide and, to a lesser extent, insulin glargine, increase glucose-stimulated insulin secretion in an additive manner.

Metabolic consequences of acute and chronic empagliflozin administration in treatment-naive and metformin pretreated patients with type 2 diabetes.

AIMS/HYPOTHESIS: Sodium glucose co-transporter 2 (SGLT2) inhibitors lower glycaemia by inducing glycosuria, but raise endogenous glucose production (EGP). Metformin lowers glycaemia mainly by suppressing EGP. We compared the effects of the SGLT2 inhibitor empagliflozin in treatment-naive (TN) and metformin pretreated (Met) patients with type 2 diabetes. METHODS: A total of 32 TN and 34 patients on a stable dose of metformin, two subgroups of a study that we previously reported, received a mixed meal with double-tracer glucose administration and indirect calorimetry at baseline, after a single 25 mg dose of empagliflozin, and after 4 weeks of treatment with empagliflozin 25 mg/day. RESULTS: At baseline, compared with the TN group, the Met group had higher fasting glycaemia (9.1 ± 1.7 vs 8.2 ± 1.3 mmol/l), lower fasting and postmeal insulin secretion, lower beta cell glucose sensitivity (37 [18] vs 58 [43] pmol min(-1) m(-2) [mmol/l](-1), median [interquartile range]) and insulin:glucagon ratio, and higher fasting EGP (15.9 [4.3] vs 12.1 [2.7] µmol kgFFM (-1) min(-1)). Change from baseline in fasting EGP after single dose and 4 weeks of treatment with empagliflozin was similar in the Met and TN groups (19.6 [4.2] and 19.0 [2.3] in Met vs 16.2 [3.6] and 15.5 [3.2] µmol kgFFM (-1) min(-1) in TN for acute and chronic dosing, respectively). Beta cell glucose sensitivity increased less in Met than TN patients, whereas substrate utilisation shifted from carbohydrate to fat more in Met than TN patients. CONCLUSIONS/INTERPRETATION: At baseline, Met patients with type 2 diabetes had more advanced disease than TN patients, featuring worse beta cell function and higher EGP. Empagliflozin induced similar glycosuria and metabolic and hormonal responses in Met and TN patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01248364; European Union Clinical Trials Register 2010-018708-99.

Upper gastrointestinal motility and symptoms in individuals with diabetes, prediabetes and normal glucose tolerance.

AIMS/HYPOTHESIS: Type 2 diabetes has been associated with upper gastrointestinal motility dysfunction, but the relationship with diabetes duration and glucose control is less well understood. Gastric emptying, oesophageal motility and gastrointestinal symptoms were examined in volunteers with diabetes, prediabetes (impaired fasting glucose [IFG] or impaired glucose tolerance [IGT]) and normal glucose tolerance (NGT). METHODS: The study included 41 patients with type 2 diabetes, 17 individuals with IFG/IGT and 31 individuals with NGT. A gastric emptying breath test and high-resolution oesophageal manometry were performed. Gastrointestinal symptoms were assessed using questionnaires. RESULTS: Gastric emptying was delayed in individuals with IFG/IGT (p < 0.05) but was normal in the diabetic group. Amongst the diabetic patients, gastric emptying rate was fastest in those with longer diabetes duration and the highest HbA1c levels (p < 0.001). Oesophageal motility variables were similar between the groups. However, the lower oesophagus resting pressure was reduced in patients with longer diabetes duration (p = 0.01). Abdominal pain/discomfort was more frequent amongst patients with diabetes (p = 0.04) but was unrelated to gastric emptying. Significant associations between various oesophageal motility variables and gastrointestinal symptoms were observed. CONCLUSIONS/INTERPRETATION: Gastric emptying and oesophageal motility are not generally altered in patients with type 2 diabetes. In more advanced disease stages, however, gastric emptying and oesophageal motility may be disturbed, probably as a consequence of autonomic neuropathy. Delayed gastric emptying in IFG/IGT individuals might be secondary to acute hyperglycaemia. Determination of gastric emptying and oesophageal manometry should be considered for the diagnostic workup of patients with diabetes and gastrointestinal symptoms.

Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment.

In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes.

Integrated metabolic spatial-temporal model for the prediction of ammonia detoxification during liver damage and regeneration.

UNLABELLED: The impairment of hepatic metabolism due to liver injury has high systemic relevance. However, it is difficult to calculate the impairment of metabolic capacity from a specific pattern of liver damage with conventional techniques. We established an integrated metabolic spatial-temporal model (IM) using hepatic ammonia detoxification as a paradigm. First, a metabolic model (MM) based on mass balancing and mouse liver perfusion data was established to describe ammonia detoxification and its zonation. Next, the MM was combined with a spatial-temporal model simulating liver tissue damage and regeneration after CCl4 intoxication. The resulting IM simulated and visualized whether, where, and to what extent liver damage compromised ammonia detoxification. It allowed us to enter the extent and spatial patterns of liver damage and then calculate the outflow concentrations of ammonia, glutamine, and urea in the hepatic vein. The model was validated through comparisons with (1) published data for isolated, perfused livers with and without CCl4 intoxication and (2) a set of in vivo experiments. Using the experimentally determined portal concentrations of ammonia, the model adequately predicted metabolite concentrations over time in the hepatic vein during toxin-induced liver damage and regeneration in rodents. Further simulations, especially in combination with a simplified model of blood circulation with three ammonia-detoxifying compartments, indicated a yet unidentified process of ammonia consumption during liver regeneration and revealed unexpected concomitant changes in amino acid metabolism in the liver and at extrahepatic sites. CONCLUSION: The IM of hepatic ammonia detoxification considerably improves our understanding of the metabolic impact of liver disease and highlights the importance of integrated modeling approaches on the way toward virtual organisms.

O-GlcNAcylation as a novel ammonia-induced posttranslational protein modification in cultured rat astrocytes.

Hepatic encephalopathy (HE) is a clinical manifestation of a low grade cerebral edema with a mutual interrelationship between osmotic- and oxidative stress. This leads to RNA oxidation and posttranslational protein modifications such as protein tyrosine nitration with pathophysiological relevance. Here, we report on O-GlcNAcylation as another ammonia-induced posttranslational protein modification in cultured rat astrocytes. NH4Cl induced O-GlcNAcylation of distinct proteins (25-250 kDa) in astrocytes in a dose- and time-dependent manner. Exposure of astrocytes to NH4Cl (5 mmol/l) for 48 h and 72 h significantly increased protein O-GlcNAcylation by about 2-fold and 4-fold, respectively. NH4Cl at a concentration of 1 mmol/l was sufficient to double protein O-GlcNAcylation in astrocytes after 72 h as compared to untreated controls. Ammonia-induced protein O-GlcNAcylation was sensitive towards glutamine-synthetase inhibition by methionine sulfoximine (MSO), but was not induced by hypoosmolarity (205 mosmol/l) or CH3NH3Cl (5 mmol/l). Increased protein O-GlcNAcylation in NH4Cl (5 mmol/l, 48 h)-treated astrocytes was fully reversible within 24 h after withdrawal of NH4Cl from culture medium. Amongst the proteins which are O-GlcNAcylated in response to ammonia, GAPDH was identified. It is concluded that ammonia induces reversible protein O-GlcNAcylation in astrocytes that depends on glutamine synthesis but not on astrocyte swelling per se or ammonia-induced pH-changes. In view of the complex involvement of O-GlcNAcylation in cell regulation, such as energy metabolism, apoptosis and circadian rhythmicity and in pathologies, such as neurodegenerative diseases, O-GlcNAcylation might contribute to the pathophysiology of hepatic encephalopathy.

Osmotic and oxidative/nitrosative stress in ammonia toxicity and hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric complication of acute or chronic liver failure. Currently, HE in cirrhotic patients is seen as a clinical manifestation of a low grade cerebral edema which exacerbates in response to a variety of precipitating factors after an ammonia-induced exhaustion of the volume-regulatory capacity of the astrocyte. Astrocyte swelling triggers a complex signaling cascade which relies on NMDA receptor activation, elevation of intracellular Ca(2+) concentration and prostanoid-driven glutamate exocytosis, which result in increased formation of reactive nitrogen and oxygen species (RNOS) through activation of NADPH oxidase and nitric oxide synthase. Since RNOS in turn promote astrocyte swelling, a self-amplifying signaling loop between osmotic- and oxidative stress ensues, which triggers a variety of downstream consequences. These include protein tyrosine nitration (PTN), oxidation of RNA, mobilization of zinc, alterations in intra- and intercellular signaling and multiple effects on gene transcription. Whereas PTN can affect the function of a variety of proteins, such as glutamine synthetase, oxidized RNA may affect local protein synthesis at synapses, thereby potentially interfering with protein synthesis-dependent memory formation. PTN and RNA oxidation are also found in post mortem human cerebral cortex of cirrhotic patients with HE but not in those without HE, thereby confirming a role for oxidative stress in the pathophysiology of HE. Evidence derived from animal experiments and human post mortem brain tissue also indicates an up-regulation of microglia activation markers in the absence of increased synthesis of pro-inflammatory cytokines. However, the role of activated microglia in the pathophysiology of HE needs to be worked out in more detail. Most recent observations made in whole genome micro-array analyses of post mortem human brain tissue point to a hitherto unrecognized activation of multiple anti-inflammatory signaling pathways.

Osmotic regulation of hepatic betaine metabolism.

Betaine critically contributes to the control of hepatocellular hydration and provides protection of the liver from different kinds of stress. To investigate how the hepatocellular hydration state affects gene expression of enzymes involved in the metabolism of betaine and related organic osmolytes, we used quantitative RT-PCR gene expression studies in rat hepatoma cells as well as metabolic and gene expression profiling in primary hepatocytes of both wild-type and 5,10-methylenetetrahydrofolate reductase (MTHFR)-deficient mice. Anisotonic incubation caused coordinated adaptive changes in the expression of various genes involved in betaine metabolism, in particular of betaine homocysteine methyltransferase, dimethylglycine dehydrogenase, and sarcosine dehydrogenase. The expression of betaine-degrading enzymes was downregulated by cell shrinking and strongly induced by an increase in cell volume under hypotonic conditions. Metabolite concentrations in the culture system changed accordingly. Expression changes were mediated through tyrosine kinases, cyclic nucleotide-dependent protein kinases, and JNK-dependent signaling. Assessment of hepatic gene expression using a customized microarray chip showed that hepatic betaine depletion in MTHFR(-/-) mice was associated with alterations that were comparable to those induced by cell swelling in hepatocytes. In conclusion, the adaptation of hepatocytes to changes in cell volume involves the coordinated regulation of betaine synthesis and degradation and concomitant changes in intracellular osmolyte concentrations. The existence of such a well-orchestrated response underlines the importance of cell volume homeostasis for liver function and of methylamine osmolytes such as betaine as hepatic osmolytes.

Ammonia increases nitric oxide, free Zn(2+), and metallothionein mRNA expression in cultured rat astrocytes.

Ammonia is a major player in the pathogenesis of hepatic encephalopathy (HE) and affects astrocyte function by triggering a self-amplifying cycle between osmotic and oxidative stress. We recently demonstrated that hypoosmotic astrocyte swelling rapidly stimulates nitric oxide (NO) production and increases intracellular free Zn(2+) concentration ([Zn(2+)](i)). Here we report effects of ammonia on [Zn(2+)](i) homeostasis and NO synthesis. In cultured rat astrocytes, NH(4)Cl (5 mm) increased within 6 h both cytosolic and mitochondrial [Zn(2+)]. The [Zn(2+)](i) increase was transient and was mimicked by the nonmetabolizable CH(3)NH(3)Cl, and it was dependent on NO formation, as evidenced by the sensitivity toward the nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine. The NH(4)Cl-induced NO formation was sensitive to the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester and increases in both NO and [Zn(2+)](i) were blocked by the N-methyl-d-aspartate receptor antagonist MK-801. The NH(4)Cl-triggered increase in [Zn(2+)](i) was followed by a Zn(2+)-dependent nuclear appearance of the metal response element-binding transcription factor and metallothionein messenger RNA (mRNA) induction. Metallothionein mRNA was also increased in vivo in rat cerebral cortex 6 h after an NH(4)Ac challenge. NH(4)Cl increased peripheral-type benzodiazepine receptor (PBR) protein expression, whereas PBR mRNA levels were decreased in a Zn(2+)-independent manner. The Zn(2+)-dependent upregulation of metallothionein following ammonia intoxication may reflect a cytoprotective response, whereas the increase in PBR expression may augment HE development.

RNA oxidation and zinc in hepatic encephalopathy and hyperammonemia.

Hepatic encephalopathy is a neuropsychiatric manifestation of acute and chronic liver failure. Ammonia plays a key role in the pathogenesis of hepatic encephalopathy by inducing astrocyte swelling and/or sensitizing astrocytes to swelling by a heterogeneous panel of precipitating factors and conditions. Whereas astrocyte swelling in acute liver failure contributes to a clinically overt brain edema, a low grade glial edema without clinically overt brain edema is observed in hepatic encephalopathy in liver cirrhosis. Astrocyte swelling produces reactive oxygen and nitrogen oxide species (ROS/RNOS), which again increase astrocyte swelling, thereby creating a self-amplifying signaling loop. Astroglial swelling and ROS/RNOS increase protein tyrosine nitration and may account for neurotoxic effects of ammonia and other precipitants of hepatic encephalopathy. Recently, RNA oxidation and an increase of free intracellular zinc ([Zn(2+)](i)) were identified as further consequences of astrocyte swelling and ROS/RNOS production. An elevation of [Zn(2+)](i) mediates mRNA expression of metallothionein and the peripheral benzodiazepine receptor (PBR) induced by hypoosmotic astrocyte swelling. Further, Zn(2+) mediates RNA oxidation in ammonia-treated astrocytes. In the brain of hyperammonemic rats oxidized RNA localizes in part to perivascular astrocyte processes and to postsynaptic dendritic spines. RNA oxidation may impair postsynaptic protein synthesis, which is critically involved in learning and memory consolidation. RNA oxidation offers a novel explanation for multiple disturbances of neurotransmitter systems and gene expression and the cognitive deficits observed in hepatic encephalopathy.

Hypoosmotic swelling affects zinc homeostasis in cultured rat astrocytes.

Astrocyte swelling is observed in different types of brain injury including hepatic encephalopathy (HE). This study investigates the role of astrocyte swelling on Zn2+ homeostasis in hypoosmotically treated astrocytes by using the Zn2+ indicators Newport-Green, Zinquin, and RhodZin-3. Hypoosmolarity (205 mosmol/L) led to a persistent increase of the intracellular "free" Zn2+ concentration [Zn2+](i) within 15 min, which was reversible after reinstitution of normoosmolarity (305 mosmol/L). The hypoosmotic [Zn2+](i) increase was abolished in the presence of the Zn2+ chelator TPEN, the NMDA receptor antagonists MK-801 and AP5, the antioxidant epigallocatechin gallate, and the nitric oxide synthase inhibitors L-NMMA and TRIM. Hypoosmolarity triggered nuclear accumulation of the metal response element-binding transcription factor MTF-1 and the specificity protein Sp1 and expression of the mRNAs encoding metallothionein and the Sp1-regulated peripheral-type benzodiazepine receptor (PBR). These effects were abolished by the Zn2+ chelator TPEN. The data suggest that astrocyte swelling affects gene expression by modulation of [Zn2+](i). Whereas Zn2+-dependent upregulation of metallothionein may help to counteract excessive astrocyte swelling and production of reactive oxygen and nitrogen oxide species, stimulation of PBR expression may augment HE development.

Ammonia induces RNA oxidation in cultured astrocytes and brain in vivo.

UNLABELLED: Oxidative stress plays a major role in cerebral ammonia toxicity and the pathogenesis of hepatic encephalopathy (HE). As shown in this study, ammonia induces a rapid RNA oxidation in cultured rat astrocytes, vital mouse brain slices, and rat brain in vivo. Ammonia-induced RNA oxidation in cultured astrocytes is reversible and sensitive to MK-801, 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, apocynin, epigallocatechin gallate, and polyphenon 60, suggesting the involvement of N-methyl-D-aspartic acid (NMDA) receptor activation, Ca(2+), nicotinamide adenine dinucleotide phosphate, and reduced form (NADPH) oxidase-dependent oxidative stress. Also, hypo-osmolarity, tumor necrosis factor alpha (TNF-alpha), and diazepam increase RNA oxidation in cultured astrocytes, suggesting that the action of different HE-precipitating factors converges at the level of RNA oxidation. Among the oxidized RNA species, 18S-rRNA and the messenger RNA (mRNA) coding for the glutamate/aspartate transporter (GLAST) were identified. Cerebral RNA oxidation in acutely ammonia-loaded rats in vivo is reversible and predominates in neuronal soma and perivascular astrocyte processes. In neuronal dendrites, oxidized RNA colocalizes with the RNA-binding splicing protein neurooncological ventral antigen (NOVA)-2 within putative RNA transport granules, which are also found in close vicinity to postsynaptic spines. This indicates that oxidized RNA species may participate in postsynaptic protein synthesis, which is a biochemical substrate for learning and memory consolidation. Neuronal and astroglial RNA oxidation increases also in vital mouse brain slices treated with ammonia and TNF-alpha, respectively. CONCLUSION: Cerebral RNA oxidation is identified as a not yet recognized consequence of acute ammonia intoxication. RNA oxidation may affect gene expression and local protein synthesis and thereby provide another link between reactive oxygen species (ROS)/reactive nitrogen oxide species (RNOS) production and ammonia toxicity.