Myo-inositol Uptake Research Articles

Myo‐inositol transport and metabolism participate in salt tolerance of halophyte ice plant seedlings

Myo-inositol and its metabolic derivatives such as pinitol, galactinol, and raffinose affect growth and development and are also involved in stress adaptation. Previous studies have identified myo-inositol transporters (INTs) as transporters of Na+ from root to shoot in the halophyte ice plant (Mesembryanthemum crystallinum). We found that the supply of myo-inositol could alleviate the dehydration effects of salt-stressed ice plant seedlings by decreasing the Na/K ratio in roots and increasing the Na/K ratio in shoots. Analyses of the uptake of exogenous myo-inositol revealed that ice plant seedlings contained intrinsic high-affinity transporters and inducible low-affinity uptake systems. The presence of Na+ facilitated both high- and low-affinity myo-inositol uptake. Six INT genes were identified from the ice plant transcriptome and named McINT1a, 1b, 2, 4a, 4b, and 4c, according to the classification of the Arabidopsis INT family. In seedlings treated with myo-inositol, salt, or myo-inositol plus salt, the expression patterns of all McINT members differed in shoot and root, which indicates organ-specific regulation of McINTs by salt and myo-inositol. The expression of McINT2, 4a, 4b, and 4c was induced by salt stress in shoot and root, but that of McINT1a and 1b was salt-induced only in shoot. The expression of pinitol biosynthesis gene IMT1 was induced by salt and myo-inositol, and their combination had a synergistic effect on the accumulation of pinitol. Supply of myo-inositol to salt-treated seedlings alleviated the detrimental effects by maintaining a low root Na/K ratio and providing precursors for the synthesis of compatible solute to maintain the osmotic balance.

Maternal Inositol Status and Neural Tube Defects: A Role for the Human Yolk Sac in Embryonic Inositol Delivery?

ABSTRACTSupplementation with myo-inositol during the periconceptional period of pregnancy may ameliorate the recurrence risk of having a fetus affected by a neural tube defect (NTD; e.g., spina bifida). This could be of particular importance in providing a means for preventing NTDs that are unresponsive to folic acid. This review highlights the characteristics of inositol and describes the role of myo-inositol in the prevention of NTDs in rodent studies and the evidence for its efficacy in reducing NTD risk in human pregnancy. The possible reduction in NTD risk by maternal myo-inositol implies functional and developmentally important maternal–embryonic inositol interrelationships and also suggests that embryonic uptake of myo-inositol is crucial for embryonic development. The establishment of active myo-inositol cellular uptake mechanisms in the embryonic stages of human pregnancy, when the neural tube is closing, is likely to be an important determinant of normal development. We draw attention to the generation of materno-fetal inositol concentration gradients and relationships, and outline a transport pathway by which myo-inositol may be delivered to the early developing human embryo. These considerations provide novel insights into the mechanisms that may underpin inositol's ability to confer embryonic developmental benefit.

Myo‐Inositol Improves Ischemic Stroke Outcome After both Nicotine Containing Electronic Cigarette and Tobacco Smoke Exposure

Ischemic stroke occurs in 87% of all strokes and is the leading cause of disability in the United States. Smoking has been shown to increase brain edema after ischemic stroke and worsen long‐term clinical outcomes. Electronic nicotine delivery systems or e‐cigarettes (e‐Cigs) are gaining popularity over traditional tobacco smoke cigarettes, and have been reported influence stroke outcome. Previous work from our lab reported nicotine and e‐Cig exposure decreased glucose utilization in the brain in ischemic stroke models, further exacerbating the effects of ischemic injury. Myo‐inositol(MI), a major organic osmolyte that promotes cell membrane stability and various cellular functions, has been reported to reduce infarcted area and edema ratio in a diabetic ischemic mouse model. We also sought to understand the role of MI protection with regard to additional stroke comorbidities, such as e‐Cig or tobacco smoke exposure. In this study, we investigated the neuroprotective role of MI transporters during stroke and post‐stroke outcomes, especially related to the role of glial cells and post‐stroke reorganization. We hypothesize MI plays a role in increased recovery of motor function, after stroke, in animal groups exposed to tobacco smoke or e‐Cig vape. Primary astrocytes and primary neurons were used for in vitro studies and a middle cerebral artery occlusion (MCAO) mouse model for in vivo stroke studies. Immunocytochemistry (ICC) studies showed astrocytes express sodium dependent myo‐inositol transporter 1 (SMIT1) and sodium‐dependent glucose transporter 6 (SGLT6). Our data illustrated expression of both transporters increased in a time‐dependent manner during oxygen‐glucose deprivation (OGD), in tobacco smoke extract (TSE), and e‐Cig extract (ECE) treated cells. Further, this was confirmed by an increase of MI uptake in TSE treated astrocytes. Altered cell mitochondrial respiration levels were observed in astrocytes and neurons incubated for 24hr with TSE and ECE followed by OGD exposure using Seahorse XF Cell Mito Stress. Animal studies demonstrated post‐stroke administration of MI, reduced infarction size and edema ratios in tobacco smoke compared to non‐tobacco smoke animals. An improvement of motor function was also seen in selected behavioral tests. E‐cig exposed animals had a slight increase in infarction size compared to non‐e‐Cig animals. This preliminary data suggest myo‐inositol may have a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome in both tobacco smokers and e‐Cig users. Further investigation will include assessment of sex differences with regard to the neuroprotective mechanisms of MI in e‐Cig and tobacco smoke exposed animals.Support or Funding Information1R01DA0497375 and R01DA029121 to TJA and LC

Role of Myo‐inositol in Ischemic Stroke Outcome in a Preclinical Tobacco Smoke Exposed Mouse Model

Tobacco smoking is a preventable risk factor for ischemic stroke in both males and females and contributes up to 14% of all stroke deaths. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome, such as seen in tobacco smokers. Myo‐inositol (MI) is a major organic “non‐perturbing” osmolyte that promotes cell membrane stability and also a precursor for phosphoinositide signaling pathway. Our previous work reported that MI attenuated brain infarct and edema ratio in an ischemic stroke model coupled to preclinical diabetes and suggests that MI and MI transporters provide neuroprotection during/following stroke both in non‐diabetic and diabetic conditions through protection from brain edema. Since our lab has shown that tobacco smoke exposure worsens stroke edema similarly to diabetic conditions, the aim of the present study was to assess the neuroprotective effects of MI associated with ischemic mice exposed to tobacco smoke. In this study, we also investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of astrocytes cells, which are the most abundant glial cells in the brain and play an important role in post‐stroke reorganization, and indirectly protecting neuronal metabolic support. Primary mouse astrocytes were incubated with tobacco smoke extract (TSE) overnight prior for in vitro studies. Toxicity of TSE was measured using MTT assay after incubating astrocytes with TSE 6hr and 24hr. Therapeutic efficacy of MI was evaluated in a transient‐middle cerebral artery occlusion (tMCAO) mouse model after smoking 3R4F research cigarettes for 10 days. Uptake studies showed an increase of MI uptake increased with longer incubation of TSE in normoxic conditions. Additionally, it was also observed that uptake of MI increased during Oxygen Glucose Deprivation(OGD) conditions with prior exposure of TSE. These results are in support with our previous reports, where we found an increased expression of MI transports, Sodium Myo‐inositol Transporter 1 (SMIT1) and Sodium Dependent Glucose Transporter 6 (SGLT6) during OGD exposure. Preliminary animal studies using middle cerebral artery occlusion mouse model for in vivo stroke studies, demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in tobacco smoke exposed animals compared to non‐tobacco smoked ischemic animals and also improved motor function in behavioral tests. Taken together with previous work, this study provides an insight into how MI may play a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome, especially in tobacco smoke populations. Further studies are required to understand the neuroprotective mechanisms of MI in ischemic stroke injury in reducing brain edema.Support or Funding InformationR01NS076012 and R01DA029121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tamoxifen inhibits the biosynthesis of inositolphosphorylceramide in Leishmania

Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo-[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C18:0)alkyl -2-O-(C18:1)acylglycerol-3-HPO4-inositol and sn-1-O-(C18:0)acyl-2-O-(C18:1)acylglycerol-3-HPO4-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC50 value of 8.48 μM (95% CI 7.68–9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites.

Potential role of myo-inositol to improve ischemic stroke outcome in diabetic mouse

Brain edema is one of the critical factors causing hightened disability and mortality in stroke patients, which is exaggerated further in diabetic patients. Organic osmolytes could play a critical role in the maintenance of cytotoxic edema. The present study was aimed to assess the role of myo-inositol, an organic osmolyte, on stroke outcome in diabetic and non-diabetic animals. In situ brain perfusion and acute brain slice methods were used to assess transport of myo-inositol across the blood-brain barrier and uptake by brain cells using non-diabetic (C57BL/6) and diabetic (streptozotocin-induced) mice, respectively. In vitro studies were conducted to assess the role of myo-inositol during and after ischemia utilizing oxygen glucose deprivation (OGD) and reperfusion. Further, the expression of transporters, such as SGLT6, SMIT1 and AQP4 were measured using immunofluorescence. Therapeutic efficacy of myo-inositol was evaluated in a transient middle cerebral artery occlusion (tMCAO) mouse model using non-diabetic (C57BL/6) and diabetic (db/db) mice. Myo-inositol release from and uptake in astrocytes and altered expression of myo-inositol transporters at different OGD timepoints revealed the role of myo-inositol and myo-inositol transporters during ischemia reperfusion. Further, hyperglycemic conditions reduced myo-inositol uptake in astrocytes. Interestingly, in in-vivo tMCAO, infarct and edema ratios following 24 h reperfusion decreased in myo-inositol treated mice. These results were supported by improvement in behavioral outcomes in open-field test, corner test and neurological score in both non-diabetic and db/db animals. Our data suggest that myo-inositol and myo-inositol transporters may provide neuroprotection during/following stroke both in non-diabetic and diabetic conditions.

Osmolyte taurine induction in UVA exposed human retinal pigment epithelial cells

Aim: To explore the osmolytes expression in ultraviolet (UVA) stressed human retinal pigment epithelial cells.Methods: Osmolyte transporters and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) were determined by real time polymerase chain reaction (PCR). Osmolyte uptake was measured by radioimmunoassay. VEGF concentrations were determined by immunoassay and enzyme-linked immunosorbent assay (ELISA). Osmolyte taurine transporter (TAUT) were silenced by siRNA technology.Results: Hypertonicity accelerated osmolyte betaine uptake, myoinositol uptake, and taurine uptake, compared to normotonic stress. UVA irradiation also accelerated osmolyte transporters expression and osmolytes uptake. Especially, osmolyte taurine remarkably inhibited VEGF release induced by UVA irradiation. VEGF in the UVA stressed retinal pigment epithelial cell supernatant was accumulated slow after taurine preincubation. VEGF expression increased significantly in UVA-stressed cells after TAUT silencing. Moreover, taurine reduced the VEGF level in human ocular aqueous humor.Conclusion: The inhibition of VEGF by osmolyte taurine plays the crucial role in retina adaption to UVA irradiation.

Role of Myo‐inositol in Ischemic Stroke Outcome in a Type 2 Diabetic Mouse Model

Stroke is the leading cause of disability in the United States; ischemic stroke is the common form that occurs in 87% of all strokes. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome. Myo‐inositol (MI), a major organic osmolyte that promotes cell membrane stability and various cellular functions, has been reported to be detected at lower brain levels in stroke patients. Furthermore, glucose can compete with MI transport which could lead to further lowering of brain levels of MI in diabetic patients which is compounded with higher brain edema reported in diabetic stroke patients. Therefore, understanding the relationship between MI and diabetic stroke injury is vital. In this study, we investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of glial cells and post‐stroke reorganization. Our hypothesis is that MI plays a putative role in recovery of motor function after stroke. Restoration of activity in the primary motor cortex has been associated with better motor function in stroke patients. Primary astrocytes were used for in vitro studies and middle cerebral artery occlusion (MCAO) mouse model for in vivo stroke studies. Immunocytochemistry (ICC) studies showed that astrocytes express sodium dependent myo‐inositol transporter 1 (SMIT1) and sodium‐dependent glucose transporter 6 (SGLT6) transporters and level of expression changed in a time‐dependent manner during oxygen‐glucose deprivation (OGD). Further, this was confirmed by time‐dependent uptake of MI during normoxic and OGD conditions. Preliminary animal studies demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in a db/db mouse model of type 2 diabetes compared to nondiabetic ischemic animals and also improved motor function in behavioral tests. This preliminary data suggests that myo‐inositol may have a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome. More studies are required to understand the neuroprotective mechanisms of MI in stroke injury.Support or Funding InformationRO1 NS070612This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Taurine reduces blue light-induced retinal neuronal cell apoptosis in vitro

Background: The massive uptake of organic compatible osmolytes is a self-protective response to multiple stressors.Objective: This study aimed to determine the protective effects of the osmolyte taurine against blue light-induced apoptosis in retinal neuronal cells in vitro.Methods: Real-time PCR was used to measure osmolyte transport. Radioimmunoassays were performed to measure osmolyte uptake. Cell Counting Kit-8 assays were conducted to measure cellular viability. Flow cytometry analysis was used to measure apoptosis.Results: Compared with normotonic stress, hypertonic stress-induced uptake of osmolytes, including betaine, myoinositol, and taurine, into the retinal neuronal cells. Blue light increased osmolyte transporter mRNA expression together with osmolyte uptake. Furthermore, taurine significantly suppressed blue light-induced retinal neuronal cell apoptosis.Conclusion: The compatible osmolyte taurine may have an important role in cell resistance to blue light and cell survival.

Potential utility of aldose reductase-deficient Schwann cells IKARS1 for the study of axonal degeneration and regeneration.

Potential utility of aldose reductase-deficient Schwann cells IKARS1 for the study of axonal degeneration and regeneration.

Ameliorative effect of myo-inositol on red blood cell alterations in polycystic ovary syndrome: in vitro study

Polycystic ovary syndrome (PCOS)is a gynecological endocrine disorder which is associated with systemic inflammatory status inducing red blood cells (RBC) membrane alterations related to insulin resistance and testosterone levels which could be greatly improved by myo-inositol (MYO) uptake. In this study we aim to evaluate the effect of MYO in reducing oxidative-related alterations through in vitro study on PCOS RBC. Blood samples from two groups of volunteers, control group (CG, n = 12) and PCOS patient group (PG, n = 12), were analyzed for band 3 tyrosine phosphorylation (Tyr-P), high molecular weight aggregate (HMWA), IgG in RBC membranes, and glutathione (GSH) in cytosol, following O/N incubation in the presence or absence of MYO. PCOS RBC underwent oxidative stress as indicated by higher band 3 Tyr-P and HMWA and increased membrane bound autologous IgG. Twenty four hours (but not shorter time) MYO incubation, significantly improved both Tyr-P level and HMWA formation and concomitant membrane IgG binding. However, no relevant modification of GSH content was detected. PCOS RBC membranes are characterized by increased oxidized level and enhanced sensitivity to oxidative injuries leading to potential premature RBC removal. MYO treatment is effective in reducing oxidative related abnormalities in PCOS patients probably restoring the inositol phospholipid pools of the membranes.

KCNQ-SMIT complex formation facilitates ion channel-solute transporter cross talk.

Voltage-gated potassium channels formed by KCNQ2 and KCNQ3 are essential for normal neuronal excitability. KCNQ2/3 channel activity is augmented in vivo by phosphatidylinositol 4,5-bisphosphate (PIP2), which is generated from myo-inositol, an osmolyte transported into cells by sodium-dependent myo-inositol transporters (SMITs). Here, we discovered that KCNQ2/3 channels isoform-specifically colocalize with SMIT1 and SMIT2 at sciatic nerve nodes of Ranvier and in axon initial segments, and form channel-transporter complexes in vitro and in vivo KCNQ2/3 coexpression protected SMIT1 activity from the otherwise inhibitory effects of cellular depolarization imposed by elevating extracellular [K+], and KCNQ2 was required for potentiation of SMIT activity by myo-inositol preincubation. Cytoskeletal disruption, which speeds PIP2 dispersion, attenuated potentiation of KCNQ2/3 currents by SMIT1-mediated myo-inositol uptake, suggesting close channel-transporter juxtaposition ensures KCNQ2/3 exposure to locally high myo-inositol-derived PIP2 concentrations. Thus, KCNQ2/3-SMIT1/2 coassembly permits cross talk via physical interaction, and may also be required for optimal, reciprocal indirect regulation via membrane potential and PIP2, especially within the specialized architecture of axons.-Neverisky, D. L., Abbott, G. W. KCNQ-SMIT complex formation facilitates ion channel-solute transporter cross talk.

Abstract 231: Uncovering the Mechanism and Function of Newly Discovered KCNQ1-Transporter Complexes

The KCNQ1 voltage-gated potassium channel is essential for human ventricular repolarization, permitting potassium efflux from excited cardiomyocytes to end each action potential and repolarize the heart. In cardiomyocytes, KCNQ1 is modulated by interaction with beta-subunits from the KCNE gene family, each of which significantly alters KCNQ1 channel function. KCNQ1 mutations are the most common identified genetic basis for Long QT syndrome (LQTS) and are also associated with lone atrial fibrillation (AF). The sodium-dependent myo-inositol transporter 1 (SMIT1) mediates cellular uptake of myo-inositol, an essential osmolyte that also represents an important substrate for phosphatidylinositol signaling pathways that regulate a plethora of ion channels including those essential for human cardiac function. We recently discovered that KCNQ1 can form heteromeric, co-regulatory complexes with Na+-coupled solute transporters including SMIT1, SMIT2 and glucose transporter SGLT1. These findings represent the first reported example of formation of an ion channel-solute transporter complex. Having discovered KCNQ1-SMIT1 complexes in mouse choroid plexus epithelium, we are currently investigating whether these types of complexes occur in the heart, how their function is altered by the various cardiac-expressed KCNE regulatory subunits or by arrhythmia-associated mutations, and which parts of KCNQ1 coordinate complex formation. Here, we present evidence of KCNQ1-SMIT1 co-assembly in pig heart based on co-immunoprecipitation experiments. Using KCNQ1-KCNQ4 chimeras we also begin to define which specific regions of KCNQ1 are required for complex formation with SMIT1. Finally, we present data showing the effects of SMIT1 on complexes formed by KCNQ1 and KCNE1, 2 and 3. KCNQ1-transporter complexes provide a potential hub for electrochemical crosstalk in normal cardiac function and in arrhythmogenesis.

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ΔramA, ΔgntR1 ΔgntR2, and ΔiolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the ΔiolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5'-KGWCHTRACA-3') which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.

Myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

The periplasmic binding protein (PBP) IbpA mediates the uptake of myo-inositol by the IatP-IatA ATP-binding cassette transmembrane transporter. We report a crystal structure of Caulobacter crescentus IbpA bound to myo-inositol at 1.45 Å resolution. This constitutes the first structure of a PBP bound to inositol. IbpA adopts a type I PBP fold consisting of two α-β lobes that surround a central hinge. A pocket positioned between the lobes contains the myo-inositol ligand, which binds with submicromolar affinity (0.76 ± 0.08 μM). IbpA is homologous to ribose-binding proteins and binds D-ribose with low affinity (50.8 ± 3.4 μM). On the basis of IbpA and ribose-binding protein structures, we have designed variants of IbpA with inverted binding specificity for myo-inositol and D-ribose. Five mutations in the ligand-binding pocket are sufficient to increase the affinity of IbpA for D-ribose by 10-fold while completely abolishing binding to myo-inositol. Replacement of ibpA with these mutant alleles unable to bind myo-inositol abolishes C. crescentus growth in medium containing myo-inositol as the sole carbon source. Neither deletion of ibpA nor replacement of ibpA with the high-affinity ribose binding allele affected C. crescentus growth on D-ribose as a carbon source, providing evidence that the IatP-IatA transporter is specific for myo-inositol. This study outlines the evolutionary relationship between ribose- and inositol-binding proteins and provides insight into the molecular basis upon which these two related, but functionally distinct, classes of periplasmic proteins specifically bind carbohydrate ligands.

New Therapies for Diabetes Management

New Therapies for Diabetes Management

Chronic Treatment with Anti-bipolar Drugs Causes Intracellular Alkalinization in Astrocytes, Altering Their Functions

Bipolar disorder I and II are affective disorders with mood changes between depressive and manic (bipolar I) or hypomanic (bipolar II) periods. Current therapy of these conditions is chronic treatment with one or more of the anti-bipolar drugs, Li(+) ('lithium'), carbamazepine and valproic acid. The pathophysiology of bipolar disorder is multifactorial and far from clear. Recent data on the dependence of normal brain function on neuronal-astrocytic interactions raise the possibility of astrocytic involvement. We will discuss our previously published and new results on effects of chronic treatment of primary cultures of normal mouse astrocytes with any of three conventional anti-bipolar drugs. The focus will be on several drug-induced events in relation to therapeutic effects of the drugs, such as myo-inositol uptake, intracellular pH and alkalinization, drug-induced modulation of glutamatergic activity in astrocytes and release of astrocytic 'gliotransmitters'. Finally, we will discuss the importance of phospholipase A2 (PLA(2)) and arachidonic acid cascade in drug-treated astrocytes, partly based on Dr. Barneda Cuirana's published thesis. All three drugs cause gradual intracellular alkalinization through different mechanisms. Alkalinization inhibit myo-inositol uptake, resulting in reduced inositolphosphate/phospholipid signaling. Accordingly, transmitter-induced increase in free intracellular Ca(2+) ([Ca(2+)](i)) becomes inhibited, aborting release of astrocytic 'gliotransmitters'. The reduction of "gliotransmitter" effects on neurons may have therapeutic effects in mania. Alkalinization also up-regulates expression of cPLA(2), an enzyme releasing arachidonic acid, and triggered arachidonic acid cascade and production, but perhaps not release, of prostaglandins. Whenever tested, identical effects were observed in freshly isolated astrocytes, but not neurons, from carbamazepine-treated healthy animals.

Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH

myo-Inositol is important for cell signaling both in cytoplasm and in intracellular organelles. It is required in the plasma membrane and cytoplasm for maintained synthesis of the second messengers, inositoltrisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP2), and in organelles as precursor for synthesis of complex signaling phospholipids and inositolphosphates from IP3 and PIP2. myo-Inositol must be taken up into the cell where its is used, because neither neurons nor astrocytes synthesize it. It is also an osmolyte, taken up in response to surrounding hyperosmolarity and released during hypo-osmolarity. There are three myo-inositol transporters, the Na+-dependent SMIT1 and SMIT2, and HMIT, which co-transports myo-inositol with H+. Their relative expressions in astrocytes and neurons are unknown. Uptake kinetics for myo-inositol in astrocytes has repeatedly been determined, but always on the assumption of only one component, leaving kinetics for the individual transporters unknown. This paper demonstrates that astrocytes obtained directly from the brain express SMIT1 and HMIT, but little SMIT2, and that all three transporters are expressed in neurons. Cultured mouse astrocytes show a high-affinity/low-capacity myo-inositol uptake (Vmax: 60.0±3.0pmol/min per mg protein; Km: 16.7±2.6μM), mediated by SMIT1 and perhaps partly by SMIT2. It was determined in cells pre-treated with HMIT-siRNA and confirmed by specific inhibition of SMIT. However at physiologically relevant myo-inositol concentrations most uptake is by a lower-affinity/higher-capacity uptake, mediated by HMIT (Vmax: 358±60pmol/min per mg protein; Km: 143±36μM) and determined by subtraction of SMIT-mediated from total uptake. At high myo-inositol concentrations, its uptake is inhibited by incubation in medium with increased pH, and increased during intracellular acidification with NH4Cl. This is in agreement with literature data for HMIT alone. At low concentration, where SMIT1/2 activity gains importance, myo-inositol uptake is reduced by ammonia-induced intracellular acidification, consistent with the transporter’s pH sensitivity reported in the literature.

Myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.

Empagliflozin, a novel selective sodium glucose cotransporter‐2 (SGLT‐2) inhibitor: characterisation and comparison with other SGLT‐2 inhibitors

Empagliflozin is a selective sodium glucose cotransporter-2 (SGLT-2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT-2 inhibitors. [(14)C]-alpha-methyl glucopyranoside (AMG) uptake experiments were performed with stable cell lines over-expressing human (h) SGLT-1, 2 and 4. Two new cell lines over-expressing hSGLT-5 and hSGLT-6 were established and [(14)C]-mannose and [(14)C]-myo-inositol uptake assays developed. Binding kinetics were analysed using a radioligand binding assay with [(3)H]-labelled empagliflozin and HEK293-hSGLT-2 cell membranes. Acute in vivo assessment of pharmacokinetics was performed with normoglycaemic beagle dogs and Zucker diabetic fatty (ZDF) rats. Empagliflozin has an IC(50) of 3.1 nM for hSGLT-2. Its binding to SGLT-2 is competitive with glucose (half-life approximately 1 h). Compared with other SGLT-2 inhibitors, empagliflozin has a high degree of selectivity over SGLT-1, 4, 5 and 6. Species differences in SGLT-1 selectivity were identified. Empagliflozin pharmacokinetics in ZDF rats were characterised by moderate total plasma clearance (CL) and bioavailability (BA), while in beagle dogs CL was low and BA was high. Empagliflozin is a potent and competitive SGLT-2 inhibitor with an excellent selectivity profile and the highest selectivity window of the tested SGLT-2 inhibitors over hSGLT-1. Empagliflozin represents an innovative therapeutic approach to treat diabetes.