Browsing by Subject "hyperglycemia"
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Item Impact Of Culture Conditions on Primary Astrocyte Phenotype(2019-05) Prah, Jude K.; Yang, Shaohua; Singh, Meharvan; Forster, Michael J.; Yan, Liang-Jun; Fudala, RafalAlthough previously thought to be passive support cells in the central nervous system (CNS), recent findings introduced critical contributions of astrocytes to numerous CNS functions like energy metabolism, ion and water homeostasis, blood brain barrier formation and neurotransmission. Their dysfunction has been implicated in the initiation and progression of specific CNS pathologies with astrocyte now given serious attention as cellular target for neuroprotection and treatment of neurological disorders. In spite of the aforementioned advances, our understanding of the mechanisms and pathways regulating astrocytic function, dysfunction and astrogliosis is still rudimentary. This is as a result of the complex interwoven nature of different cells in the CNS. Because of the complexities of the brain structure and function in vivo, methods of in vitro primary culture that overcome the influence of complex brain environment provide critical tools for understanding brain cell function at the cellular and molecular levels. The current primary astrocytes cultures are mostly maintained in serum-containing hyperglycemic medium which is non-physiological and produces astrocyte with a reactive, morphological and functional phenotype different from in vivo quiescent astrocytes. The first study presented in the dissertation delineates a serum free astrocyte culture condition that maintains primary astrocytes in a quiescent state. Results showed that primary astrocytes isolated from the cerebral cortex of postnatal day 1 C57BL6 mice and cultured in an astrocyte base medium supplemented with fibroblast growth factor (FGF2) and epidermal growth factor (EGF) (ABM- FGF2-EGF) have higher process bearing morphologies similar to in vivo astrocytes and different from the flat polygonal fibroblast like morphologies exhibited by astrocytes cultured under the traditional FBS condition developed by McCarthy and de Vellis (1980) (MD-10% FBS). Additionally astrocytes cultured in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP and vimentin, increased glutamine synthase and glutamate transporter mRNA levels compared to astrocytes in the MD-10% FBS condition. These findings strongly indicates that astrocytes cultured in ABM-FGF2-EGF medium compared to the usual FBS medium promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. This media provides a novel method for studying astrocytes function in vitro under physiological and pathological condition. Hyperglycemia could increase neuronal glucose level which leads to neuronal damage in a phenomenal referred to as glucose neurotoxicity. On the other hand the impact of hyperglycemia on astrocytes has been less explored although astrocytes are critical for glucose uptake and metabolism and many primary astrocyte cultures are maintained in high glucose conditions. In the second part of this dissertation we investigated the impact of hyperglycemia on astrocyte phenotype and function. Our studies demonstrated that hyperglycemic levels (25 mM) induce cell cycle arrest, ROS production, cytokine expression and inhibited astrocyte proliferation. High glucose enhanced glycolysis and increased metabolic potentials of astrocyte. In addition high glucose activated AMP-activated kinase (AMPK) signaling pathways and induces reactive astrocyte phenotype. In conclusion both studies presented a unique perspective of how culture conditions influence astrocyte phenotype and experimental outcome. Our study also provided a mechanism which may underline the role of astrocytes in hyperglycemia induced neurological complications.Item The Effects of Elevated Glucose Upon Na+/K+-ATPase in Bovine Retinal Pigment Epithelial Cells(1994-12-01) Crider, Julie Y.; Thomas Yorio; John Lane; Edward OrrCrider, Julie Y., The Effects of Elevated Glucose Upon Na+/K+-ATPase in Bovine Retinal Pigment Epithelial Cells. Doctor of Philosophy (Biomedical Sciences, Pharmacology), December, 1994, 154 pp., 14 tables, 31 illustrations, bibliography, 288 titles. Bovine retinal pigment epithelial (RPE) cells were cultured under 1, 4.5 and 10 g/l glucose conditions in order to characterize the effects of hyperglycemia upon Na+/K+-ATPase. Functional activity of Na+/K+-ATPase was measured as ouabain-sensitive Rb+ uptake. 3H ouabain was used to assess binding characteristics of Na+/K+-ATPase. The major contributors to rubidium (mRb+) uptake activity were the ouabain-sensitive Na+/K+-ATpase and a bumetanide-sensitive NA+/K+/Cl- cotransporter. Dose response curves for ouabain and bumetanide produced IC50 values for 86Rb+ uptake of 60-100 nM and 120 nM, respectively. At elevated glucose concentrations, the aldose reductase inhibitor (ARI) AL-1576 stimulated 86Rb+ uptake upon chronic treatment. A sensitive new nonradioactive Rb+ uptake assay was developed which utilized suppressed conductivity detection and provided several advantages over the radioactive method. The average ouabain IC50 value was confirmed to be 100nM and was not significantly affected by elevated glucose concentrations. The bumetanide sensitive component was responsible for approximately 30% of Rb+ uptake at all glucose concentrations. Potassium efflux out of the cells was observed that was sensitive to the Na+/K+/Cl- cotransport inhibitor bumetanide. Elevated glucose appeared to increase Rb+ transport through potassium channels was also reduced Rb+ uptake indicating a decrease in Na+/K+-ATPase activity. Bovine RPE cells exposed to both high glucose and AL-1576 for one month showed mild stimulation of Rb+ uptake compared to the activity in high glucose alone. Ouabain and strophanthidin inhibition of 3H ouabain binding, in bovine RPE cells, appeared to be unaffected by hyperglycemia. The average IC50 values for these compounds were 5.02 x 10^-8 M, respectively. The results of this study indicate that Na+/K+-ATPase activity in bovine RPE is decreased by hyperglycemic state, and can be stimulated by treatment with an aldose reductase inhibitor administered from the onset of the hyperglycemic insult.Item The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response(Elsevier B.V., 2022-12-15) Chamseddine, Douja; Mahmud, Siraje A.; Westfall, Aundrea K.; Castoe, Todd A.; Berg, Rance E.; Pellegrino, Mark W.Organisms use several strategies to mitigate mitochondrial stress, including the activation of the mitochondrial unfolded protein response (UPR(mt)). The UPR(mt) in Caenorhabditis elegans, regulated by the transcription factor ATFS-1, expands on this recovery program by inducing an antimicrobial response against pathogens that target mitochondrial function. Here, we show that the mammalian ortholog of ATFS-1, ATF5, protects the host during infection with enteric pathogens but, unexpectedly, by maintaining the integrity of the intestinal barrier. Intriguingly, ATF5 supports intestinal barrier function by promoting a satiety response that prevents obesity and associated hyperglycemia. This consequently averts dysregulated glucose metabolism that is detrimental to barrier function. Mechanistically, we show that intestinal ATF5 stimulates the satiety response by transcriptionally regulating the gastrointestinal peptide hormone cholecystokinin, which promotes the secretion of the hormone leptin. We propose that ATF5 protects the host from enteric pathogens by promoting intestinal barrier function through a satiety-response-mediated metabolic control mechanism.