Excessive fructose consumption is closely linked to the pathogenesis of metabolic disease. Carbohydrate response element-binding protein (ChREBP) is a transcription factor essential for fructose tolerance in mice. However, the functional significance of liver ChREBP in fructose metabolism remains unclear. Here, we show that liver ChREBP protects mice against fructose-induced hepatotoxicity by regulating liver glycogen metabolism and ATP homeostasis. Liver-specific ablation of ChREBP did not compromise fructose tolerance, but rather caused severe transaminitis and hepatomegaly with massive glycogen overload in mice fed a high-fructose diet, while no obvious inflammation, cell death, or fibrosis was detected in the liver. In addition, liver ATP contents were significantly decreased by ChREBP deficiency in the fed state, which was rendered more pronounced by fructose feeding. Mechanistically, liver contents of glucose-6-phosphate (G6P), an allosteric activator of glycogen synthase, were markedly increased in the absence of liver ChREBP, while fasting-induced glycogen breakdown was not compromised. Furthermore, hepatic overexpression of LPK, a ChREBP target gene in glycolysis, could effectively rescue glycogen overload and ATP reduction, as well as mitigate fructose-induced hepatotoxicity in ChREBP-deficient mice. Taken together, our findings establish a critical role of liver ChREBP in coping with hepatic fructose stress and protecting from hepatotoxicity by regulating LPK.
Glycogenolysis, in brain parenchyma an astrocyte-specific process, has changed from being envisaged as an emergency procedure to playing central roles during brain response to whisker stimulation, memory formation, astrocytic K(+) uptake and stimulated release of ATP. It is activated by several transmitters and by even very small increases in extracellular K(+) concentration, and to be critically dependent upon an increase in free cytosolic Ca(2+) concentration ([Ca(2+)]i), whereas cAMP plays only a facilitatory role together with increased [Ca(2+)]i. Detailed knowledge about the signaling pathways eliciting glycogenolysis is therefore of interest and was investigated in the present study in well differentiated cultures of mouse astrocytes. The β-adrenergic agonist isoproterenol stimulated glycogenolysis by a β1-adrenergic effect, which initiated a pathway in which cAMP/protein kinase A activated a Gi/Gs shift, leading to Ca(2+)-activated glycogenolysis. Inhibition of this pathway downstream of cAMP but upstream of the Gi/Gs shift abolished the glycogenolysis. However, inhibitors operating downstream of the Ca(2+)-sensitive step, but preventing transactivation-mediated epidermal growth factor (EGF) receptor stimulation, a later step in the activated pathway, also caused inhibition of glycogenolysis. For this reason the effect of EGF was investigated and it was found to be glycogenolytic. Large increases in extracellular K(+) activated glycogenolysis by a nifedipine-inhibited L-channel opening allowing influx of Ca(2+), known to be glycogenolysis-dependent. Small increases (addition of 5 mM KCl) caused a smaller effect by a similarly glycogenolysis-reliant opening of an IP3 receptor-dependent ouabain signaling pathway. The same pathway could be activated by GABA (also in brain slices) due to its depolarizing effect in astrocytes.
Here we present the data indicating that chronic treatment with fluoxetine regulates Cav-1/PTEN/PI3K/AKT/GSK-3β signalling pathway and glycogen content in primary cultures of astrocytes with bi-phasic concentration dependence. At lower concentrations fluoxetine down-regulates gene expression of Cav-1, decreases membrane content of PTEN, increases activity of PI3K/AKT, and elevates GSK-3β phosphorylation thus suppressing its activity. At higher concentrations fluoxetine acts in an inverse fashion. As expected, fluoxetine at lower concentrations increased while at higher concentrations decreased glycogen content in astrocytes. Our findings indicate that bi-phasic regulation of glycogen content via Cav-1/PTEN/AKT/GSK-3β pathway by fluoxetine may be responsible for both therapeutic and side effects of the drug.
This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations.
The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral non-sensory region. Here we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear non-sensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane, a flattened spiral prominence, and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the non-sensory epithelium covering the outer border Claudius cells, outer sulcus root cells and spiral prominence epithelial cells. Transcriptome analysis shows ZBTB20 regulates genes coding for tectorial membrane proteins in the greater epithelial ridge, and those preferentially expressed in root cells and spiral prominence epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral non-sensory domain.
Clinical evidence suggest astrocytic abnormality in major depression (MD) while treatment with anti-psychotic drugs affects astroglial functions. Astroglial cells are involved in pH homeostasis of the brain by transporting protons (through sodium-proton transporter 1, NHE1, glutamate transporters EAAT1/2 and proton-lactate co-transporter MCT1) and bicarbonate (through the sodium-bicarbonate co-transporter NBC or the chloride-bicarbonate exchanger AE). Here we show that chronic treatment with fluoxetine increases astroglial pHi by stimulating NHE1-mediated proton extrusion. At a clinically relevant concentration of 1 μM, fluoxetine significantly increased astroglial pHi from 7.05 to 7.34 after 3 weeks and from 7.18 to 7.58 after 4 weeks of drug treatment. Stimulation of NHE1 is a result of transporter phosphorylation mediated by several intracellular signaling cascades that include MAPK/ERK1/2, PI3K/AKT and ribosomal S6 kinase (RSK). Fluoxetine stimulated phosphorylation of ERK1/2, AKT and RSK in a concentration dependent manner. Positive crosstalk exists between two signal pathways, MAPK/ERK1/2 and PI3K/AKT activated by fluoxetine since ERK1/2 phosphrylation could be abolished by inhibitors of PI3K, LY294002 and AKT, triciribine, and AKT phosphorylation by inhibitor of MAPK, U0126. As a result, RSK phosphorylation was not only inhibited by U0126 but also by inhibitor of LY294002. The NHE1 phoshorylation resulted in stimulation of NHE1 activity as revealed by the NH4Cl-prepulse technique; the increase of NHE1 activity was dependent on fluoxetine concentration, and could be inhibited by both U0126 and LY294002. Our findings suggest that regulation of astrocytic pHi and brain pH may be one of the mechanisms underlying fluoxetine action.
In this work, we utilized adsorbed BSA and multiwalled carbon nanoparticles (BSA/MWCNTs) as a stationary phase in open tubular (OT) capillary for separation of chiral drugs. (3-Aminopropyl)triethoxysilane was used to assist fabrication of BSA/MWCNTscoated OT column by covalent bonding. Incorporation of MWCNTs nanomaterials into a polymer matrix could increase the phase ratio and take advantage of the easy preparation of an open tubular CEC column. SEM was carried out to characterize the BSA/MWCNTs OT columns. The electrochromatographic performance of the OT columns was evaluated by separation of ketoprofen, ibuprofen, uniconazole, and hesperidin. The effects of MWCNTs concentration, background solution pH and concentration, and applied voltage on separation were investigated. Chiral separations of ketoprofen, ibuprofen, uniconazole, and hesperidin were achieved using the BSA/MWCNTs-coated OT column with resolutions of 24.20, 12.81, 1.50, and 1.85, respectively. Their optimas were found in the 30 mM phosphate buffers at pH 5.0, 6.5, 7.0, and 6.5, respectively. In addition, the columns demonstrated good repeatability and stability with the run-to-run, day-today , and batch-to-batch RSDs of migration times less than 3.5%.
Learning from the design concept of antibody–drug conjugates (ADCs), we attempted to construct siRNA conjugated polymer brush by attaching a multiple of siRNA to the units of poly(amino acids) [poly(lysine) derivatives] through an intracellular cleavable disulfide bond. Note that the disulfide linkage is stable at extracellular milieu yet subjected to cleavage into free thiol residues at the intracellular reducing compartments. Consequently, ready release of arrays of active siRNA was achieved selectively in the intracellular compartments. Furthermore, tumor-targeted cyclic Asp-Gly-Arg (RGD) was conjugated to the aforementioned polymer brush in view that the RGD receptors (αVβ3 and αVβ5 integrins) were overexpressed over a wide spectrum of cancerous cells. Our subsequent results have achieved potent gene silencing in cultured cancerous cells from our proposed siRNA delivery construct. To our best knowledge, our proposed conjugate should be the first example of using an ADC platform in successful intracellular transportation of larger macromolecular biological payloads rather than small molecular chemotherapeutic drugs. Hence, the proposed strategy may serve as a promising avenue for targeted delivery of macromolecular pharmaceutical payloads.
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