Browsing by Author "Kempson, Stephen A."

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    Betaine chemistry, roles, and potential use in liver disease
    (Elsevier, 2016-06) Day, Christopher R.; Kempson, Stephen A.; Department of Cellular & Integrative Physiology, IU School of Medicine
    Background Betaine is the trimethyl derivative of glycine and is normally present in human plasma due to dietary intake and endogenous synthesis in liver and kidney. Betaine is utilized in the kidney primarily as an osmoprotectant, whereas in the liver its primary role is in metabolism as a methyl group donor. In both organs, a specific betaine transporter mediates cellular uptake of betaine from plasma. The abundance of both betaine and the betaine transporter in liver greatly exceeds that of other organs. Scope of review The remarkable contributions of betaine to normal human and animal health are summarized together with a discussion of the mechanisms and potential beneficial effects of dietary betaine supplements on liver disease. Major conclusions A significant amount of data from animal models of liver disease indicates that administration of betaine can halt and even reverse progression of the disruption of liver function. Betaine is well-tolerated, inexpensive, effective over a wide range of doses, and is already used in livestock feeding practices. General significance The accumulated data indicate that carefully controlled additional investigations in humans are merited. The focus should be on the long-term use of betaine in large patient populations with liver diseases characterized by development of fatty liver, especially non-alcoholic fatty liver disease and alcoholic liver disease.
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    Mechanisms of hexosamine-induced cholesterol accumulation and therapeutic actions of chromium
    (2014-01-03) Penque, Brent A.; Elmendorf, Jeffrey S.; Atkinson, Simon; Considine, Robert V.; Evans-Molina, Carmella; Kempson, Stephen A.
    Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.
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    Noninvasive monitoringn of CCl4 induced acute and chronic liver damage in rat by single quantum and triple quantum filtered 23Na magnetic resonance imaging
    (2008) Gao, Yong; Bansal, Navin; Babsky, Andriy M.; Kempson, Stephen A.; Basile, David P.
    In present study, single quantum (SQ) and triple quantum filtered (TQF) 23Na magnetic resonance imaging (MRI) was used to monitor the severity and progression of CCl4 induced acute and chronic liver damage in rat model. SQ 23Na MRI was proposed to measure the 23Na signal intensity (SI) of total tissue sodium ions, and TQF 23Na MRI was proposed to measure the SI of intracellular sodium ions. In addition, shift reagent aided 23Na and 31P magnetic resonance spectroscopy (MRS) was used to measure in vivo intracellular sodium concentration ([Na+i]), total tissue sodium concentration ([Na+t]) and relative extracellular space (rECS) of liver in the same model. In acute high dose CCl4 intoxication, 24 hours after single dose of CCl4 in 5ml per kg body weight of mixture of CCl4 and oil in 1:1 ratio, SQ 23Na SI increased by 83% and TQF 23Na SI increased by 174% compared to the baseline level. According to SR-aided 23Na and 31P MRS, [Na+i] increased by 188% and [Na+t] increased by 43%. In addition, there was significant decrease in cellular energetic level, represented by ATP/Pi ratio. Histology examination showed pronounced inflammatory response in centrilobular regions, with neutrophiles infiltration, fatty accumulation and swollen hepatocytes. In chronic 8-week experiment, chronic damage was induced by biweekly administration of CCl4 in a dosage of 0.5 ml per kg body weight. From week 1 to week 6, SQ 23Na SI remained relatively constant, and then increased by 15% from week 6 to week 8. TQF 23Na SI progressively increased from week 1 to week 8, totally by 56%. Both SQ and TQF 23Na SI showed significant difference between treated group and control at every week. SR-aided 23Na and 31P MRS experiment showed that, at the end of 8-week CCl4 intoxication, both [Na+t] and rECS were higher than control, by 49% and 47% respectively; however, there was no significant difference for [Na+i] between two groups. Histology examination showed excessive deposition of extracellular matrix. In conclusion, SQ and TQF 23Na MRI appears valuable in the functional assessment of liver in noninvasive approach, and could be a promising diagnostic modality for liver diseases in clinical area.
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    A Nutrient Network Regulating Cellular Cholesterol and Glucose Metabolism
    (2014) Pattar, Guruprasad R.; Elmendorf, Jeffrey S.; Considine, Robert V.; Deeg, Mark A.; Herring, B. Paul; Kempson, Stephen A.
    Insulin resistance, a hallmark of type 2 diabetes (T2D), is associated with accompanying derangements such as hyperinsulinemia that promote the progression of insulin resistance, yet a mechanism(s) is imperfectly understood. Data have demonstrated that hyperinsulinemia promotes insulin resistance as evidenced by diminished ability of insulin to mobilize glucose transporter GLUT4 to the plasma membrane (PM). We found that loss of PM phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure contributes to hyperinsulinemia-induced insulin resistance. We tested if increased glucose flux through hexosamine biosynthesis pathway (HBP) causes dysregulation of PM components necessary for GLUT4 translocation. Increased HBP activity was detected in 3T3-L1 adipocytes cultured in hyperinsulinemia (5 nM Ins; 12 h) and also 2 mM glucosamine (GlcN), a distal HBP activator, inducing losses of PM PIP2 and F-actin. In accordance with HBP flux directly weakening PIP2/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored F-actin and insulin responsiveness. Furthermore, less invasive challenges with glucose led to PIP2/F-actin dysregulation. New findings support a negative correlation between PM cholesterol accrual, PIP2/F-actin structure and GLUT4 regulation. These data stemmed from parallel study aimed at understanding the antidiabetic mechanism of the nutrient chromium (Cr3+). We found that chromium picolinate (CrPic) enhanced insulin-stimulated GLUT4 trafficking via reduction in PM cholesterol. In line with glucose/cholesterol toxicity findings, we demonstrated that therapeutic effects of CrPic occurred solely in adipocytes with increased HBP activity and a concomitant elevation in PM cholesterol. Mechanistically, data are consistent with a role of AMP-activated protein kinase (AMPK) in CrPic action. These data show that CrPic increases AMPK activity and perhaps suppresses cholesterol synthesis via distal phosphorylation and inactivation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), a rate-limiting enzyme in cholesterol synthesis. Continued study of the consequence of increased HBP activity revealed alterations in cholesterogenic transcription factors – Sp1, SREBP-1, and NFY – with Sp1 showing a significant increase in O-linked glycosylation. Consistent with Sp1 modification eliciting maximal transcriptional activation of SREBP-1, Hmgr mRNA was significantly enhanced. In conclusion, these data are consistent with a central role of PM cholesterol in glucose transport and suggest perturbations in this lipid have a contributory role in developing insulin resistance.
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    Role of N-glycosylation in renal betaine transport
    (Portland Press, 2015-08) Schweikhard, Eva S.; Burckhardt, Birgitta C.; Joos, Friedericke; Fenollar-Ferrer, Cristina; Forrest, Lucy R.; Kempson, Stephen A.; Ziegler, Christine; Department of Cellular & Integrative Physiology, IU School of Medicine
    The osmolyte and folding chaperone betaine is transported by the renal Na+-coupled GABA (γ-aminobutyric acid) symporter BGT-1 (betaine/GABA transporter 1), a member of the SLC6 (solute carrier 6) family. Under hypertonic conditions, the transcription, translation and plasma membrane (PM) insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves PM depletion of BGT-1. The molecular mechanism of the regulated PM insertion of BGT-1 during changes in osmotic stress is unknown. In the present study, we reveal a link between regulated PM insertion and N-glycosylation. Based on homology modelling, we identified two sites (Asn171 and Asn183) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion and transport by immunogold-labelling, electron microscopy (EM), mutagenesis and two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radiolabelled substrate into MDCK (Madin–Darby canine kidney) and HEK293 (human embryonic kidney) cells. Trafficking and PM insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at Asn171 and Asn183 contributed equally to protein activity and substrate affinity. Substitution of Asn171 and Asn183 by aspartate individually caused no loss of BGT-1 activity, whereas the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells PM insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.