scholarly journals Regulation of GLUT4 gene expression by SREBP-1c in adipocytes

2006 ◽  
Vol 399 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Seung-Soon Im ◽  
Sool-Ki Kwon ◽  
Seung-Youn Kang ◽  
Tae-Hyun Kim ◽  
Ha-Il Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Glucose Transporter ◽  
Regulatory Element ◽  
Diabetic Rats ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Response Element ◽  
Glut4 Gene Expression

Expression of the GLUT4 (glucose transporter type 4 isoform) gene in adipocytes is subject to hormonal or metabolic control. In the present study, we have characterized an adipose tissue transcription factor that is influenced by fasting/refeeding regimens and insulin. Northern blotting showed that refeeding increased GLUT4 mRNA levels for 24 h in adipose tissue. Consistent with an increased GLUT4 gene expression, the mRNA levels of SREBP (sterol-regulatory-element-binding protein)-1c in adipose tissue were also increased by refeeding. In streptozotocin-induced diabetic rats, insulin treatment increased the mRNA levels of GLUT4 in adipose tissue. Serial deletion, luciferase reporter assays and electrophoretic mobility-shift assay studies indicated that the putative sterol response element is located in the region between bases −109 and −100 of the human GLUT4 promoter. Transduction of the SREBP-1c dominant negative form to differentiated 3T3-L1 adipocytes caused a reduction in the mRNA levels of GLUT4, suggesting that SREBP-1c mediates the transcription of GLUT4. In vivo chromatin immunoprecipitation revealed that refeeding increased the binding of SREBP-1 to the putative sterol-response element in the GLUT4. Furthermore, treating streptozotocin-induced diabetic rats with insulin restored SREBP-1 binding. In addition, we have identified an Sp1 binding site adjacent to the functional sterol-response element in the GLUT4 promoter. The Sp1 site appears to play an additive role in SREBP-1c mediated GLUT4 gene upregulation. These results suggest that upregulation of GLUT4 gene transcription might be directly mediated by SREBP-1c in adipose tissue.

scholarly journals Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

1993 ◽  
Vol 13 (1) ◽  
pp. 257-265 ◽  
Author(s):  
H Pöpperl ◽  
M S Featherstone
Keyword(s):  
Retinoic Acid ◽  
Hox Genes ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Response Element ◽  
Retinoic Acid Response Element ◽  
Ec Cells

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.

scholarly journals Insulin and insulin-like growth factor 1 antagonize the stimulation of ob gene expression by dexamethasone in cultured rat adipose tissue

1997 ◽  
Vol 324 (2) ◽  
pp. 605-610 ◽  
Author(s):  
Bénédicte A. REUL ◽  
Lumbe N. ONGEMBA ◽  
Anne-Marie Marie ◽  
Jean-Claude HENQUIN ◽  
Sonia M. BRICHARD
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Growth Factor ◽  
Fatty Acid Synthase ◽  
Glucose Transporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Ob Gene ◽  
Rat Adipose Tissue

The ob gene, specifically expressed in fat cells, encodes leptin, a hormone that induces satiety and increases energy expenditure. In this study, we investigated the interactions between glucocorticoids and insulin on ob gene expression in cultured explants of rat adipose tissue. Only low levels of ob mRNA were detected when adipose tissue from fasted rats was cultured for 12–24 h in minimal essential medium. However, the addition of dexamethasone to the medium increased ob gene expression in a concentration-dependent manner (EC50 10 nM). With 1 μM dexamethasone, ob mRNA levels were similar to those in fresh fat pads from fed rats, reaching a maximum after 12 h. The effect of dexamethasone was blocked by actinomycin D, which indicates an action on transcription. This effect was increased when a minimum amount of fuel (glucose or a mixture of lactate and pyruvate) was supplied in the medium. Unlike dexamethasone, insulin, even when combined with high glucose concentrations, did not induce ob expression, although it strongly increased the accumulation of mRNA species for fatty acid synthase (FAS), the insulin-sensitive glucose transporter GLUT4 and the γ isoform of peroxisome proliferator-activated receptor (PPARγ). Unexpectedly, insulin dose-dependently inhibited dexamethasone-induced ob mRNA accumulation. This effect was observed at low concentrations of insulin (IC50 1 nM) and was delayed in onset, beginning after 6–9 h of culture. It was mimicked by insulin-like growth factor 1 (IGF-1) (100 nM). The inhibition by insulin was only detectable when fuels were present and/or when a critical level of ob expression was reached. As this inhibitory effect was reversed by cycloheximide, this suggests that it required ongoing protein synthesis. In conclusion, unlike dexamethasone, insulin had no direct stimulatory effect on ob gene expression. On the other hand, insulin (and IGF-1) even inhibited the dexamethasone-induced accumulation of ob mRNA. The underlying mechanism involved ongoing synthesis of an inhibitory protein by insulin, which is in keeping with its delayed effect. Moreover, the expression of genes for FAS, GLUT4 and PPARγ may be inversely related to that of ob.

scholarly journals The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element

2000 ◽  
Vol 278 (6) ◽  
pp. H1769-H1774 ◽  
Author(s):  
Michael R. Morissette ◽  
Valerie P. Sah ◽  
Christopher C. Glembotski ◽  
Joan Heller Brown
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Common Element ◽  
Luciferase Reporter ◽  
Serum Response Element ◽  
Transcriptional Responses ◽  
Response Element ◽  
Serum Response

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c- fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene

1993 ◽  
Vol 13 (1) ◽  
pp. 257-265 ◽  
Author(s):  
H Pöpperl ◽  
M S Featherstone
Keyword(s):  
Retinoic Acid ◽  
Hox Genes ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Response Element ◽  
Retinoic Acid Response Element ◽  
Ec Cells

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.

Metallothionein gene expression and secretion in white adipose tissue

2000 ◽  
Vol 279 (6) ◽  
pp. R2329-R2335 ◽  
Author(s):  
Paul Trayhurn ◽  
Jacqueline S. Duncan ◽  
Anne M. Wood ◽  
John H. Beattie
Keyword(s):  
Gene Expression ◽  
Molecular Weight ◽  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Stromal Vascular Fraction ◽  
Low Molecular Weight ◽  
Secretory Product ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Adrenoceptor Agonist

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese ( ob/ ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a β3-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.

scholarly journals Central Leptin Regulates Total Ceramide Content and Sterol Regulatory Element Binding Protein-1C Proteolytic Maturation in Rat White Adipose Tissue

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Elena Bonzón-Kulichenko ◽  
Dominik Schwudke ◽  
Nilda Gallardo ◽  
Eduardo Moltó ◽  
Teresa Fernández-Agulló ◽  
...  
Keyword(s):  
Nervous System ◽  
Adipose Tissue ◽  
Insulin Sensitivity ◽  
White Adipose Tissue ◽  
Binding Protein ◽  
Regulatory Element ◽  
Mrna Levels ◽  
Element Binding Protein ◽  
Ceramide Content ◽  
Sterol Regulatory Element

Obesity and type 2 diabetes are associated with insulin and leptin resistance, and increased ceramide contents in target tissues. Because the adipose tissue has become a central focus in these diseases, and leptin-induced increases in insulin sensitivity may be related to effects of leptin on lipid metabolism, we investigated herein whether central leptin was able to regulate total ceramide levels and the expression of enzymes involved in ceramide metabolism in rat white adipose tissue (WAT). After 7 d central leptin treatment, the total content of ceramides was analyzed by quantitative shotgun lipidomics mass spectrometry. The effects of leptin on the expression of several enzymes of the sphingolipid metabolism, sterol regulatory element binding protein (SREBP)-1c, and insulin-induced gene 1 (INSIG-1) in this tissue were studied. Total ceramide levels were also determined after surgical WAT denervation. Central leptin infusion significantly decreased both total ceramide content and the long-chain fatty acid ceramide species in WAT. Concomitant with these results, leptin decreased the mRNA levels of enzymes involved in de novo ceramide synthesis (SPT-1, LASS2, LASS4) and ceramide production from sphingomyelin (SMPD-1/2). The mRNA levels of enzymes of ceramide degradation (Asah1/2) and utilization (sphingomyelin synthase, ceramide kinase, glycosyl-ceramide synthase, GM3 synthase) were also down-regulated. Ceramide-lowering effects of central leptin were prevented by local autonomic nervous system denervation of WAT. Finally, central leptin treatment markedly increased INSIG-1 mRNA expression and impaired SREBP-1c activation in epididymal WAT. These observations indicate that in vivo central leptin, acting through the autonomic nervous system, regulates total ceramide levels and SREBP-1c proteolytic maturation in WAT, probably contributing to improve the overall insulin sensitivity. Central leptin decreases total ceramide levels and prevents sterol regulatory element binding protein (SREBP-1C) proteolytic maturation in white adipose tissue, and probably, in this way, contributes to improve the overall insulin sensitivity.

scholarly journals Microarray analysis reveals novel gene expression changes associated with erectile dysfunction in diabetic rats

2005 ◽  
Vol 23 (2) ◽  
pp. 192-205 ◽  
Author(s):  
Chris J. Sullivan ◽  
Thomas H. Teal ◽  
Ian P. Luttrell ◽  
Khoa B. Tran ◽  
Mette A. Peters ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Erectile Dysfunction ◽  
Splice Variants ◽  
Full Range ◽  
Diabetic Rats ◽  
Differentially Expressed ◽  
Diabetic Rat ◽  
Literature Mining ◽  
Mrna Levels

To investigate the full range of molecular changes associated with erectile dysfunction (ED) in Type 1 diabetes, we examined alterations in penile gene expression in streptozotocin-induced diabetic rats and littermate controls. With the use of Affymetrix GeneChip arrays and statistical filtering, 529 genes/transcripts were considered to be differentially expressed in the diabetic rat cavernosum compared with control. Gene Ontology (GO) classification indicated that there was a decrease in numerous extracellular matrix genes (e.g., collagen and elastin related) and an increase in oxidative stress-associated genes in the diabetic rat cavernosum. In addition, PubMatrix literature mining identified differentially expressed genes previously shown to mediate vascular dysfunction [e.g., ceruloplasmin ( Cp), lipoprotein lipase, and Cd36] as well as genes involved in the modulation of the smooth muscle phenotype (e.g., Kruppel-like factor 5 and chemokine C-X3-C motif ligand 1). Real-time PCR was used to confirm changes in expression for 23 relevant genes. Further validation of Cp expression in the diabetic rat cavernosum demonstrated increased mRNA levels of the secreted and anchored splice variants of Cp. CP protein levels showed a 1.9-fold increase in tissues from diabetic rats versus controls. Immunohistochemistry demonstrated localization of CP protein in cavernosal sinusoids of control and diabetic animals, including endothelial and smooth muscle layers. Overall, this study broadens the scope of candidate genes and pathways that may be relevant to the pathophysiology of diabetes-induced ED as well as highlights the potential complexity of this disorder.

scholarly journals Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter

2005 ◽  
Vol 34 (1) ◽  
pp. 177-197 ◽  
Author(s):  
A Cote-Vélez ◽  
L Pérez-Martínez ◽  
M Y Díaz-Gallardo ◽  
C Pérez-Monter ◽  
A Carreón-Rodríguez ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Camp Response Element ◽  
Glucocorticoid Response Element ◽  
Response Element ◽  
Nuclear Extracts ◽  
Glucocorticoid Response ◽  
Hypothalamic Cells

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity

2002 ◽  
Vol 282 (1) ◽  
pp. E46-E51 ◽  
Author(s):  
Frédérique Diraison ◽  
Eric Dusserre ◽  
Hubert Vidal ◽  
Monique Sothier ◽  
Michel Beylot
Keyword(s):  
Adipose Tissue ◽  
Fatty Acid Synthase ◽  
Regulatory Element ◽  
Energy Restriction ◽  
Mrna Levels ◽  
Hepatic Lipogenesis ◽  
Deuterated Water ◽  
Human Obesity ◽  
Fas Mrna ◽  
Obese Subjects

To determine whether increased lipogenesis contributes to human obesity, we measured (postabsorptive state), in lean and obese subjects, lipid synthesis (deuterated water method) and the mRNA concentration (RT-competitive PCR) in subcutaneous adipose tissue of fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1c. Before energy restriction, obese subjects had an increased contribution of hepatic lipogenesis to the circulating triglyceride pool (14.5 ± 1.3 vs. 7.5 ± 1.9%, P < 0.01) without enhancement of cholesterol synthesis. This increased hepatic lipogenesis represented an excess of 2–5 g/day of triglycerides, which would represent 0.7–1.8 kg on a yearly basis. The lipogenic capacity of adipose tissue appeared, on the contrary, decreased with lower FAS mRNA levels ( P < 0.01) and a trend for decreased SREBP-1c mRNA ( P = 0.06). Energy restriction in obese patients decreased plama insulin ( P < 0.05) and leptin ( P < 0.05) and normalized hepatic lipogenesis. FAS mRNA levels were unchanged, whereas SREBP-1c increased. In conclusion, subjects with established obesity have an increased hepatic lipogenesis that could contribute to their excessive fat mass but no evidence for an increased lipogenic capacity of adipose tissue.

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