Transketolase Activity but not Thiamine Membrane Transport Change in Response to Hyperglycaemia and Kidney Dysfunction

2017 ◽  
Vol 126 (04) ◽  
pp. 255-262 ◽  
Author(s):  
Katarína Chalásová ◽  
Lukáš Pácal ◽  
Anna Pleskačová ◽  
Lucia Knopfová ◽  
Jitka Řehořová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kidney Disease ◽  
Pentose Phosphate ◽  
Protective Mechanism ◽  
Protein Levels ◽  
Human Experiments ◽  
Key Enzyme ◽  
Vitro Analysis

Abstract Aim Pentose phosphate pathway (PPP) with key enzyme transketolase (TKT), represents a potentially ‘protective’ mechanism in hyperglycaemia. Diabetic kidney disease (DKD), a common complication of both type 1 and type 2 diabetes associated with significant morbidity and mortality, represents the most common cause of chronic kidney disease (CKD). We hypothesized that protective PPP action in diabetes and eventually even more severely in concomitant DKD might be compromised by limited intracellular availability of an active TKT cofactor thiamine diphosphate (TDP). Methods Effect of hyperglycaemia on gene expression and protein levels of key PPP loci was studied in vitro using human cell lines relevant to diabetes (HUVEC and HRGEC) and (together with measurement of TKT activity, plasma thiamine and erythrocyte TDP concentration) in vivo in diabetic vs. non-diabetic subjects with comparable renal function (n=83 in total). Results Hyperglycaemia significantly decreased protein levels of RFC-1, THTR1, THTR2 and TKT (P<0.05) in vitro. Analysis of blood samples from CKD patients with and without diabetes and from controls did not reveal any difference in gene expression and protein levels of thiamine transporters while TKT activity and TDP in erythrocytes gradually increased with decreasing kidney function being highest in patients with CKD3-4 of both diabetic and non-diabetic aetiology. Hyperglycaemia and uremic serum mimicking CKD in diabetes did not affect TKT activity in vitro (P<0.05). Conclusion Both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while TKT activity in parallel with intracellular TDP was increased in CKD patients with or without diabetes. Therefore, lack of adaptive increase of thiamine transmembrane transport allowing further increase of TKT activity might contribute to compromised PPP function in diabetes and CKD and to the development of glycotoxic injury.

Multifactorial Regulation of Cardiac Gene Expression: an In Vivo and In Vitro Analysis

1995 ◽  
Vol 752 (1 Cardiac Growt) ◽  
pp. 370-386 ◽  
Author(s):  
J. L. SAMUEL ◽  
I. DUBUS ◽  
F. FARHADIAN ◽  
F. MAROTTE ◽  
P. OLIVIERO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Vitro Analysis ◽  
In Vitro Analysis

255 THE ESTROGENIC EVALUATION OF PARABENS USING RAT UTERINE CALBINDIN-D9k

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
T. T. B. Vo ◽  
E. B. Jeung
Keyword(s):  
Gene Expression ◽  
Adverse Effects ◽  
Treated Group ◽  
Environmental Chemicals ◽  
Female Rats ◽  
Protein Levels ◽  
Calbindin D9k ◽  
The Difference

In the current study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutyl-paraben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17?-ethinylestradiol (EE, 1 mg/kg of body weight (BW) per day) or parabens (62.5, 250, and 1000 mg/kg of BW per day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the greatest dose of isopropyl-, butyl- and isobutyl-paraben. In addition, these parabens induced uterine CaBP-9k mRNA and protein levels, whereas co-treatment of parabens and fulvestrant (Faslodex, formerly known as ICI 182, 780), a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERα and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER? mRNA and protein levels, whereas co-treatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER? expression, suggesting that CaBP-9k might not express via ER? pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER regulating gene, at both transcriptional and translational levels was indicated in the greatest dose of isopropyl- and butyl-paraben. These parabens induced PR gene expression that was completely blocked by fulvestrant. This result indicates that CaBP-9k expression might involve PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which might be mediated by a PR and/or ER? signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

scholarly journals Gene expression of GLUT3 glucose transporter regulated by glucose in vivo in mouse brain and in vitro in neuronal cell cultures from rat embryos

1994 ◽  
Vol 300 (1) ◽  
pp. 125-131 ◽  
Author(s):  
S Nagamatsu ◽  
H Sawa ◽  
N Inoue ◽  
Y Nakamichi ◽  
H Takeshima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Mouse Brain ◽  
Glucose Transporter ◽  
Neuronal Cultures ◽  
Protective Mechanism ◽  
Rat Embryos ◽  
Glut1 Mrna

This study was designed to determine whether glucose regulates the gene expression of glucose transporter GLUT3 in neurons. We examined the regulation of GLUT3 mRNA by glucose in vivo in mouse brain and in vitro by using neuronal cultures from rat embryos. Hypoglycaemia (< 30 mg/dl), produced by 72 h of starvation, increased GLUT3 mRNA in mouse brain by 2-fold. Hybridization studies in situ demonstrated that hypoglycaemia-induced increases in GLUT3 mRNA expression were observed selectively in brain regions including the hippocampus, dentate gyrus, cerebral cortex and piriform cortex, but not the cerebellum. Primary neuronal cultures from rat embryos deprived of glucose for 48 h also showed an increase (4-fold over control) in GLUT3 mRNA, indicating that glucose can directly regulate expression of GLUT3 mRNA. In contrast with hypoglycaemia, hyperglycaemia produced by streptozotocin did not alter the expression of GLUT3 mRNA. We also confirmed previous findings that hypoglycaemia increases GLUT1 mRNA expression in brain. The increase in GLUT1 expression was probably limited to the blood-brain barrier in vivo, since GLUT1 mRNA could not be detected in neurons of the mouse cerebrum. Thus we conclude that up-regulation of neuronal GLUT3 in response to glucose starvation represents a protective mechanism against energy depletion in neurons.

scholarly journals Investigation of exposure to lifestyle and environmental factors on cumulus-oocyte complex function

2021 ◽  
Author(s):  
◽  
Kelly Anne Campen
Keyword(s):  
Gene Expression ◽  
Oocyte Quality ◽  
Protein Levels ◽  
Time Interaction ◽  
Cumulus Oocyte Complex ◽  
In Vitro Exposure ◽  
In Vitro Effects ◽  
In Vivo Effects

<p>Pathways involved in bi-directional communication within the cumulus-oocyte complex (COC) include gap junction (GJ) communication, oocyte growth factor production, and glucose metabolism and are essential for oocyte health. Perturbation of these pathways may result in reduced oocyte quality due to altered COC function. Using rats as a model, in vitro effects of exposure to bisphenol A (BPA), caffeine, nicotine, ethanol, methylenedioxymeth- amphetamine (MDMA), or Δ⁹-tetrahydrocannabinol (THC) on COC function were investigated. Furthermore, MDMA was administered to rats to compare in vitro with in vivo effects.  The transfer of a fluorescent dye (calcein) from cumulus cells (CC) to the oocyte was used as a measure of GJ communication. Expression of CC-derived (Atr, Cx43, Cycs, Gfpt1, Pfkp) and oocyte-derived (Atr, Bmp15, Cx37, Gdf9) genes were measured using multiplex TaqMan quantitative PCR. Levels of CX43 and GDF9 proteins were quantified using Western blotting.  Optimisation of the GJ bioassay included the addition of phosphodiesterase inhibitors (rolipram and dipyridamole), and a 1 hour post-calcein incubation period to allow dye transfer. Quantification of gene expression in calcein-treated CC and oocytes was validated, enabling direct comparisons between GJ communication and gene expression.  To determine the in vitro effects, COC were incubated with test factors at high physiological concentrations over 25 hours. GJ communication decreased over time in control COC. This reduction was attenuated after exposure to BPA and nicotine, and partially by caffeine. Furthermore, exposure to ethanol maintained oocyte meiotic arrest, whereas MDMA and THC promoted meiotic resumption.  Oocyte-derived gene expression was mostly unaffected by in vitro exposure to the lifestyle and environmental factors, although a treatment x time interaction for Cx37 levels following nicotine exposure was observed. Of the CC-derived genes, Cx43 was the most sensitive where BPA, MDMA, and THC increased, and caffeine and ethanol decreased, expression. In CC, exposure to MDMA and THC increased Gfpt1 levels and exposure to MDMA resulted in a treatment x time interaction in Cycs and Pfkp expression.  In COC, caffeine increased CX43 protein levels after 1 hour. Nicotine initially reduced, but with time increased CX43 levels. Furthermore, CX43 levels decreased and increased after 25 hour exposures to ethanol and MDMA, respectively. GDF9 protein levels in COC exhibited wide within-treatment variation. Overall, BPA and caffeine reduced GDF9 levels after 1 hour whereas GDF9 levels were increased following exposure to BPA, caffeine, MDMA, and THC for 25 hours.  To determine in vivo effects, female rats were administered saline or 5 mg/kg/day MDMA for 3 days. COC from MDMA-treated rats had higher levels of CX43 protein but gene expression and meiotic reactivation were unaffected.  In conclusion, COC function was altered by in vitro exposure to BPA, caffeine, ethanol, nicotine, MDMA, and THC. Furthermore, in vivo exposure to MDMA elicits similar, albeit reduced, effects on COC function. A role for CC in protecting the oocyte from harmful contaminants is proposed. Perturbation of the bi-directional communication pathway is likely to influence oocyte quality due to alterations in nutrient availability and timing of follicular events, although these may not be associated with negative outcomes. This study provides evidence that exposure to lifestyle factors and environmental contaminants affect COC function.</p>

scholarly journals Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway

2009 ◽  
Vol 296 (6) ◽  
pp. C1321-C1328 ◽  
Author(s):  
R. P. Rhoads ◽  
R. M. Johnson ◽  
C. R. Rathbone ◽  
X. Liu ◽  
C. Temm-Grove ◽  
...  
Keyword(s):  
Gene Expression ◽  
Satellite Cell ◽  
Hypoxia Inducible Factor ◽  
Collagen Gel ◽  
Culture Conditions ◽  
Cell Physiology ◽  
Vegf Gene ◽  
Protein Levels

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1α activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

Transcriptional regulation of pulmonary elastin gene expression in elastase-induced injury

2003 ◽  
Vol 285 (2) ◽  
pp. L354-L362 ◽  
Author(s):  
Celeste B. Rich ◽  
Isabel Carreras ◽  
Edgar C. Lucey ◽  
Julie A. Jaworski ◽  
Jo Ann Buczek-Thomas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Injury ◽  
Gene Promoter ◽  
Activity Levels ◽  
Intratracheal Administration ◽  
Protein Levels ◽  
Elastin Gene ◽  
Pulmonary Fibroblast

Previously we have shown that treatment of confluent, pulmonary fibroblast cultures with elastase results in upregulation of elastin mRNA and protein levels. In the present study we focused on determining the level at which elastin expression is upregulated after elastase exposure. We examined as models for this investigation elastin gene expression in primary pulmonary fibroblast cells during the transition from subconfluent to confluent cultures and in confluent, matrix-laden cultures treated briefly with elastase. In addition, we extended our studies to mice that were given an intratracheal dose of elastase; the effects on lung elastin mRNA and elastin promoter activity levels were measured and compared with results from in vitro cell models. The results demonstrate that upregulation of elastin gene expression during the transition of subconfluent to confluent cultures and after elastase injury is associated with an increase in the level of transcription both in vitro and in vivo. Furthermore, intratracheal administration of elastase to transgenic mice illustrates that the increased levels of elastin mRNA are accompanied by increased activity of the elastin gene promoter in cells spatially positioned near major sites of tissue injury.

In Vivo and In Vitro Analysis of Hyperoxia-Induced Gene Expression in Mouse Lung and Mouse Transformed Clara Cells

2006 ◽  
Vol 923 (1) ◽  
pp. 346-347 ◽  
Author(s):  
BIHONG ZHAO ◽  
PATRICIA L. RAMSAY ◽  
MOON S. PARK ◽  
STEPHEN E. WELTY ◽  
FRANCESCO J. DEMAYO
Keyword(s):  
Gene Expression ◽  
Mouse Lung ◽  
Clara Cells ◽  
Vitro Analysis ◽  
In Vitro Analysis

scholarly journals miR-29a and miR-30c negatively regulate DNMT 3a in cardiac ischemic tissues: implications for cardiac remodelling

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Carolina Gambacciani ◽  
Claudia Kusmic ◽  
Elena Chiavacci ◽  
Francesco Meghini ◽  
Milena Rizzo ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Ischemia Reperfusion ◽  
Chromatin Accessibility ◽  
Response To Therapy ◽  
Vitro Assay ◽  
Protein Levels ◽  
Transcriptional Reprogramming

AbstractRecent evidences indicate that epigenetic changes play an important role in the transcriptional reprogramming of gene expression that characterizes cardiac hypertrophy and failure and may dictate response to therapy. Several data demonstrate that microRNAs (miRNAs) play critical roles both in normal cardiac function and under pathological conditions. Here we assessed, in in vivo rat models of myocardial infarction (MI) and ischemia-reperfusion (IR), the relationship between two miRNAs (miR-29a and miR-30c) and de novo methyltransferase (DNMT3a) which, altering the chromatin accessibility for transcription factors, deeply impacts gene expression. We showed that the levels of members of miR-29 and miR- 30 families were down regulated in ischemic tissues whilst the protein levels of DNMT3a were increased, such a relation was not present in healthy tissues. Furthermore, by an in vitro assay, we demonstrated that both miRNAs are able to down regulate DNMT3a by directly interacting with DNMT3a 3’UTR and that miR-29a or miR-30c overexpression in the cardiac HL1 cell line causes decrease of DNMT3a enzyme both at the mRNA and protein levels. Our data, besides confirming the down regulation of the miR-29a and miR-30c in infarcted tissues, envisage a cross-talk between microRNAs and chromatin modifying enzymes suggesting a new mechanism that might generate the alterations of DNA methylation often observed in myocardial pathophysiology.

scholarly journals Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

2008 ◽  
Vol 74 (7) ◽  
pp. 2161-2170 ◽  
Author(s):  
Joseph Horzempa ◽  
Deanna M. Tarwacki ◽  
Paul E. Carlson ◽  
Cory M. Robinson ◽  
Gerard J. Nau
Keyword(s):  
Gene Expression ◽  
Francisella Tularensis ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Genomic Sequence ◽  
Protein A ◽  
Hypothetical Protein ◽  
Protein Levels

ABSTRACT Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

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