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Cardiac TdP risk stratification modelling of anti-infective compounds including chloroquine and hydroxychloroquine.
Hydroxychloroquine (HCQ), the hydroxyl derivative of chloroquine (CQ), is widely used in the treatment of rheumatological conditions (systemic lupus erythematosus, rheumatoid arthritis) and is being studied for the treatment and prevention of COVID-19. Here, we investigate through mathematical modelling the safety profile of HCQ, CQ and other QT-prolonging anti-infective agents to determine their risk categories for Torsade de Pointes (TdP) arrhythmia. We performed safety modelling with uncertainty quantification using a risk classifier based on the qNet torsade metric score, a measure of the net charge carried by major currents during the action potential under inhibition of multiple ion channels by a compound. Modelling results for HCQ at a maximum free therapeutic plasma concentration (free Cmax) of approximately 1.2 µM (malaria dosing) indicated it is most likely to be in the high-intermediate-risk category for TdP, whereas CQ at a free Cmax of approximately 0.7 µM was predicted to most likely lie in the intermediate-risk category. Combining HCQ with the antibacterial moxifloxacin or the anti-malarial halofantrine (HAL) increased the degree of human ventricular action potential duration prolongation at some or all concentrations investigated, and was predicted to increase risk compared to HCQ alone. The combination of HCQ/HAL was predicted to be the riskiest for the free Cmax values investigated, whereas azithromycin administered individually was predicted to pose the lowest risk. Our simulation approach highlights that the torsadogenic potentials of HCQ, CQ and other QT-prolonging anti-infectives used in COVID-19 prevention and treatment increase with concentration and in combination with other QT-prolonging drugs.
The effects of trifluoperazine on brain edema, aquaporin-4 expression and metabolic markers during the acute phase of stroke using photothrombotic mouse model
© 2021 The Author(s) Stroke is the second leading cause of death and the third leading cause of disability globally. Edema is a hallmark of stroke resulting from dysregulation of water homeostasis in the central nervous system (CNS) and plays the major role in stroke-associated morbidity and mortality. The overlap between cellular and vasogenic edema makes treating this condition complicated, and to date, there is no pathogenically oriented drug treatment for edema. Water balance in the brain is tightly regulated, primarily by aquaporin 4 (AQP4) channels, which are mainly expressed in perivascular astrocytic end-feet. Targeting AQP4 could be a useful therapeutic approach for treating brain edema; however, there is no approved drug for stroke treatment that can directly block AQP4. In this study, we demonstrate that the FDA-approved drug trifluoperazine (TFP) effectively reduces cerebral edema during the early acute phase in post-stroke mice using a photothrombotic stroke model. This effect was combined with an inhibition of AQP4 expression at gene and protein levels. Importantly, TFP does not appear to induce any deleterious changes on brain electrolytes or metabolic markers, including total protein or lipid levels. Our results support a possible role for TFP in providing a beneficial extra-osmotic effect on brain energy metabolism, as indicated by the increase of glycogen levels. We propose that targeting AQP4-mediated brain edema using TFP is a viable therapeutic strategy during the early and acute phase of stroke that can be further investigated during later stages to help in developing novel CNS edema therapies.
On the 400th anniversary of the birth of Thomas Willis.
In 2021 we celebrate the 400th anniversary of the birth of one of the greatest neuroanatomists, Thomas Willis, the founder of clinical neurology. Willis' name is usually associated with 'the circle' and the word 'neurologia', but his work which comprised insightful descriptions of clinical cases and clear, well-illustrated and articulated scientific publications and case histories also formed the foundation of modern translational research and clinical medicine. In the 16th and 17th centuries classical medical training was based on academic, scholastic tradition, not empirical observations. Willis' work highlights the importance of first-hand clinical observations, and the personalized care of patients and their families. It also emphasizes the importance of interdisciplinary working and the significance of different viewpoints.
Exogenous ketosis in patients with type 2 diabetes: Safety, tolerability and effect on glycaemic control
Introduction: Ketogenic diets have shown to improve glycaemic control in patients with type 2 diabetes. This study investigated the safety, tolerability, and effects on glycaemic control in patients with type 2 diabetes of an exogenous ketone monoester (KE) capable of inducing fasting-like elevations in serum β-hydroxybutyrate (βHB) without the need for caloric or carbohydrate restriction. Methods: Twenty one participants (14 men and 7 women, aged 45 ± 11 years) with insulin-independent type 2 diabetes, and unchanged hypoglycaemic medication for the previous 6 months, were recruited for this non-randomised interventional study. Participants wore intermittent scanning glucose monitors (IS-GM) for a total of 6 weeks and were given 25 ml of KE 3 times daily for 4 weeks. Serum electrolytes, acid-base status, and βHB concentrations were measured weekly and cardiovascular risk markers were measured before and after the intervention. The primary endpoints were safety and tolerability, with the secondary endpoint being glycaemic control. Results: The 21 participants consumed a total of 1,588 drinks (39.7 litres) of KE over the course of the intervention. Adverse reactions were mild and infrequent, including mild nausea, headache, and gastric discomfort following fewer than 0.5% of the drinks. Serum electrolyte concentrations, acid-base status, and renal function remained normal throughout the study. Compared to baseline, exogenous ketosis induced a significant decrease in all glycaemic control markers, including fructosamine (335 ± 60 μmol/L to 290 ± 49 μmol/L, p
Insulin inhibits glucagon release by SGLT2-induced stimulation of somatostatin secretion.
Hypoglycaemia (low plasma glucose) is a serious and potentially fatal complication of insulin-treated diabetes. In healthy individuals, hypoglycaemia triggers glucagon secretion, which restores normal plasma glucose levels by stimulation of hepatic glucose production. This counterregulatory mechanism is impaired in diabetes. Here we show in mice that therapeutic concentrations of insulin inhibit glucagon secretion by an indirect (paracrine) mechanism mediated by stimulation of intra-islet somatostatin release. Insulin's capacity to inhibit glucagon secretion is lost following genetic ablation of insulin receptors in the somatostatin-secreting δ-cells, when insulin-induced somatostatin secretion is suppressed by dapagliflozin (an inhibitor of sodium-glucose co-tranporter-2; SGLT2) or when the action of secreted somatostatin is prevented by somatostatin receptor (SSTR) antagonists. Administration of these compounds in vivo antagonises insulin's hypoglycaemic effect. We extend these data to isolated human islets. We propose that SSTR or SGLT2 antagonists should be considered as adjuncts to insulin in diabetes therapy.
Reversible changes in pancreatic islet structure and function produced by elevated blood glucose.
Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.
A mutation causing increased KATP channel activity leads to reduced anxiety in mice.
Activating mutations in the Kir6.2 (KCNJ11) subunit of the ATP-sensitive potassium channel cause neonatal diabetes. Many patients also suffer from neurological complications. By using mice carrying a human Kir6.2 mutation (Val(59) to Met(59); nV59M mice) targeted to neurones, we show that these mutations also result in altered anxiety behaviour. The light/dark box, successive alleys and elevated plus maze tasks revealed that nV59M mice have reduced anxiety related responses. Additionally, nV59M mice displayed enhanced basal locomotor activity and exploratory behaviour, as assessed by the low anxiety open-field test. These findings, in combination with previously reported hyperactivity of nV59M mice, appear to correlate with the increased impulsivity and inattentiveness reported in iDEND/DEND patients.
Type 2 diabetes and congenital hyperinsulinism cause DNA double-strand breaks and p53 activity in β cells.
β cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with β cell replication and apoptosis. Here, we show that genetic activation of β cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues β cells from glucokinase-induced death, but only GLP-1 analog rescues β cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of β cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of β cell glucotoxicity and suggest pharmacological approaches to enhance β cell survival in diabetes.
ATP-sensitive potassium channels in health and disease
Since their discovery over 20 years ago, it has been recognized that adenosine triphosphate-sensitive potassium (K ) channels play a critical role in insulin secretion. When these channels are open, insulin secretion is inhibited, and when they are shut, secretion is initiated. Consequently drugs, mutations, or changes in beta-cell metabolism that open K channels decrease insulin secretion and may cause diabetes, whereas those manipulations that close K channels have the opposite effect, increasing insulin secretion and hypoglycemia. This chapter reviews our current knowledge of the pancreatic beta-cell K channel, and discusses new data on its structure, structure-function relationships, and role in disease. © 2008 Springer. ATP ATP ATP ATP
Na+ current properties in islet α- and β-cells reflect cell-specific Scn3a and Scn9a expression.
Mouse pancreatic β- and α-cells are equipped with voltage-gated Na(+) currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at -100 mV and -50 mV in β- and α-cells, respectively). Single-cell PCR analyses show that both α- and β-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: β-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na(+) current by 80%. In β-cells, knockout of Scn9a lowers the Na(+) current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a(-/-) islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na(+) channel α subunit in both α- and β-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in β-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.
Understanding molecular mechanisms for diabetes and obesity through mouse models
© 2014 S. Karger AG, Basel. Genome-wide association studies have identified several susceptibility genes for diabetes and obesity, whose exact function is often unknown. Mouse models provide a valuable tool for identifying their functional roles and how changes in their expression and/or function can lead to disease. Despite genetic and metabolic differences between mice and humans, the mouse is a good model organism as it breeds rapidly, its genome has been sequenced and it can be easily genetically manipulated. We illustrate the applicability of mouse models to the study of diabetes and/or obesity susceptibility genes using the fat mass- and obesity-associated (FTO) gene. When single-nucleotide polymorphisms in intron one of FTO were first found to be associated with obesity, little was known about the function of FTO. The use of mouse models has confirmed that FTO indeed plays a role in the regulation of energy homeostasis and provided much information about the various phenotypic characteristics it regulates. More detailed models promise much further information. This example underlines the importance of appropriate mouse models in providing insight into the function of other diabetes- and/or obesity-associated genes and the underlying mechanisms by which they cause disease.
The Nucleotide-Binding Sites of SUR1: A Mechanistic Model.
ATP-sensitive potassium (KATP) channels comprise four pore-forming Kir6.2 subunits and four modulatory sulfonylurea receptor (SUR) subunits. The latter belong to the ATP-binding cassette family of transporters. KATP channels are inhibited by ATP (or ADP) binding to Kir6.2 and activated by Mg-nucleotide interactions with SUR. This dual regulation enables the KATP channel to couple the metabolic state of a cell to its electrical excitability and is crucial for the KATP channel's role in regulating insulin secretion, cardiac and neuronal excitability, and vascular tone. Here, we review the regulation of the KATP channel by adenine nucleotides and present an equilibrium allosteric model for nucleotide activation and inhibition. The model can account for many experimental observations in the literature and provides testable predictions for future experiments.
Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels.
KATP channels act as key regulators of electrical excitability by coupling metabolic cues-mainly intracellular adenine nucleotide concentrations-to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.
Hyperglycaemia induces metabolic dysfunction and glycogen accumulation in pancreatic β-cells.
Insulin secretion from pancreatic β-cells is impaired in all forms of diabetes. The resultant hyperglycaemia has deleterious effects on many tissues, including β-cells. Here we show that chronic hyperglycaemia impairs glucose metabolism and alters expression of metabolic genes in pancreatic islets. In a mouse model of human neonatal diabetes, hyperglycaemia results in marked glycogen accumulation, and increased apoptosis in β-cells. Sulphonylurea therapy rapidly normalizes blood glucose levels, dissipates glycogen stores, increases autophagy and restores β-cell metabolism. Insulin therapy has the same effect but with slower kinetics. Similar changes are observed in mice expressing an activating glucokinase mutation, in in vitro models of hyperglycaemia, and in islets from type-2 diabetic patients. Altered β-cell metabolism may underlie both the progressive impairment of insulin secretion and reduced β-cell mass in diabetes.
Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time.
We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.