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Biomedical subjects

J Grober

Publications and source records attributed to J Grober.

17 recordsLinked to original sources
PubMed2006

Cholesterol dependent downregulation of mouse and human apical sodium dependent bile acid transporter (ASBT) gene expression: molecular mechanism and physiological consequences.

BACKGROUND AND AIMS: Faecal bile acid elimination greatly contributes to cholesterol homeostasis. Synthesised from cholesterol in the liver, bile acids are actively reclaimed in the ileum by the apical sodium dependent bile acid transporter (ASBT). Although the expression level of ASBT affects body cholesterol balance, the impact of cholesterol on ASBT gene expression remains unclear. In this study, the effect of cholesterol on ASBT expression and ileal bile acid uptake was explored in vivo and in vitro. METHODS: ASBT gene expression was assessed by real time quantitative polymerase chain reaction and northern or western blotting, or both, in mice subjected to a 2% cholesterol diet for two weeks, in mouse ileal explants, or in human enterocyte-like Caco-2 cells cultured in sterol enriched or depleted media. Bile acid uptake was determined by measuring [3H]-taurocholic acid influx into in situ isolated ileal loops from mice or into differentiated Caco-2 cells. Molecular analysis of mouse and human ASBT promoters was undertaken with reporter assays, site directed mutagenesis, and electrophoretic mobility shift assays. RESULTS: In mice, cholesterol enriched diet triggered a downregulation of ASBT expression (mRNA and protein), a fall in ileal bile acid uptake, and a rise in the faecal excretion of bile acids. This effect was direct as it was reproduced ex vivo using mouse ileal explants and in vitro in differentiated Caco-2 cells. CONCLUSIONS: This regulation, which involves an original partnership between SREBP-2 and HNF-1alpha transcription factors, affects ileal bile acid recycling and thus might participate in the maintenance of body cholesterol homeostasis.

Animals↗
PubMed2005

The gene encoding the human ileal bile acid-binding protein (I-BABP) is regulated by peroxisome proliferator-activated receptors.

Peroxisome proliferator-activator receptors (PPAR) are involved in cholesterol homeostasis through the regulation of bile acids synthesis, composition, and reclamation. As ileal bile acid-binding protein (I-BABP) is thought to play a crucial role in the enterohepatic circulation of bile acids, we investigated whether I-BABP gene expression could also be affected by PPAR. Indeed, treatment with the PPARalpha-PPARbeta/delta agonist bezafibrate led to the up-regulation of I-BABP mRNA levels in the human intestine-derived Caco-2 cells. Cotransfections of the reporter-linked human I-BABP promoter (hI-BABP-2769/+44) together with PPAR and RXR expression vectors demonstrated that the fibrate-mediated induction of the I-BABP gene is dependent on PPARalpha or PPARbeta/delta. Using progressive 5' deletions of the hI-BABP promoter and sequence analysis, we identified a putative PPAR-binding site located at the position -198 and -186 upstream of the transcription initiation site. Electrophoretic mobility shift assays showed that the PPAR/RXR heterodimer can specifically bind to this PPRE-like motif. The deletion of the PPRE within the hI-BABP promoter abolished the PPAR-mediated transactivation in transient transfection assays. The regulation of the I-BABP promoter by PPAR appears species-specific, as the mouse I-BABP promoter, which lacks a conserved PPRE, was not responsive to exogenous PPAR expression in the presence of bezafibrate. Our findings show that the I-BABP gene may be a novel target for PPAR in humans and further emphasize the role for PPAR in the control of bile acid homeostasis.

Animals↗
PubMed1999

Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer.

Intestinal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acids (BAs) with a high affinity. In the small intestine, its expression is restricted to the ileum where it is involved in the enterohepatic circulation of BAs. Using the human enterocyte-like Caco-2 cell line, we have recently shown that BAs increased I-BABP gene expression. To determine whether this regulation occurs in vivo, the effect of BA depletion or supplementation was studied in mice. A dramatic drop in I-BABP mRNA levels was observed in mice treated with the BA-binding resin cholestyramine, whereas an increase was found in animals fed with taurocholic acid. BAs are physiological ligands for the nuclear farnesoid X receptor (FXR). Both FXR and I-BABP are co-expressed along the small intestine and in Caco-2 cells. To determine the role of FXR in the regulation of I-BABP expression, the promoter of the human I-BABP gene was cloned. In Caco-2 cells, cotransfection of FXR and RXRalpha is required to obtain the full transactivation of the I-BABP promoter by BAs. Deletion and mutation analyses demonstrate that the FXR/RXRalpha heterodimer activates transcription through an inverted repeat bile acid responsive element located in position -160/-148 of the human I-BABP promoter. In conclusion, we show that FXR is a physiological BA sensor that is likely to play an essential role in BA homeostasis through the regulation of genes involved in their enterohepatic circulation.

Animals↗
PubMed1999

Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins.

The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL.

Animals↗
PubMed1998

Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells.

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. The activity of HSL is thought to be primarily regulated by reversible phosphorylation. However, the regulation of HSL activity by pre-translational mechanisms has been poorly studied. The present studies were undertaken to explore the relationship between the levels of HSL protein and mRNA expressions and the lipolytic capacity. The study was performed in human abdominal subcutaneous adipocytes with identical sizes but having either a high (HL) or low (LL) lipolytic capacity (n = 16). Basal and maximal lipolysis induced by catecholamines, an adenylyl cyclase activator forskolin, and a cyclic AMP analogue dibutyryl cAMP were 50% lower in LL- in comparison with HL-fat cells (P < 0.05 or better). No differences in drug sensitivity were found. HSL activity and quantity were about 50% lower in LL- compared with HL-fat cells (P < 0.05). Moreover, the mRNA ratio between HSL and gamma-actin was 35% lower in LL- compared with HL-fat cells (P < 0.05). There was a strong linear correlation between the protein and enzymatic HSL measurements (r2 = 0.91). In addition, the maximum lipolytic capacity was significantly correlated with HSL activity (r2 = 0.75) and HSL protein amount (r2 = 0.64). It is concluded that hormone-sensitive lipase (HSL) expression, measured either as total HSL protein by Western blot analysis or as total amount of activatable HSL enzyme, is a major determinant of the maximum lipolytic capacity of human fat cells. In addition, HSL protein expression is at least, in part, determined by HSL mRNA expression.

Adipocytes↗
PubMed1997

Characterization of the promoter of human adipocyte hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5'-UTR is mutually exclusive. The short and long 5'-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

Adipocytes↗
PubMed1997

Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.

Alternative Splicing↗
PubMed1996

Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.

3T3 Cells↗
PubMed1996

Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase.

By catalyzing the rate-limiting step in adipose tissue lipolysis, hormone-sensitive lipase (HSL) is an important regulator of energy homeostasis. The role and importance of HSL in tissues other than adipose are poorly understood. We report here the cloning and expression of a testicular isoform, designated HSLtes. Due to an addition of amino acids at the NH2-termini, rat and human HSLtes consist of 1068 and 1076 amino acids, respectively, compared to the 768 and 775 amino acids, respectively, of the adipocyte isoform (HSLadi). A novel exon of 1.2 kb, encoding the human testis-specific amino acids, was isolated and mapped to the HSL gene, 16 kb upstream of the exons encoding HSLadi. The transcribed mRNA of 3.9 kb was specifically expressed in testis. No significant similarity with other known proteins was found for the testis-specific sequence. The amino acid composition differs from the HSLadi sequence, with a notable hydrophilic character and a high content of prolines and glutamines. COS cells, transfected by the 3.9-kb human testis cDNA, expressed a protein of the expected molecular mass (M(r) approximately 120,000) that exhibited catalytic activity similar to that of HSLadi. Immunocytochemistry localized HSL to elongating spermatids and spermatozoa; HSL was not detected in interstitial cells.

Amino Acid Sequence↗
PubMed1995

Expression of hormone-sensitive lipase in the human colon adenocarcinoma cell line HT29.

Hormone-sensitive lipase expression was studied in the human colon adenocarcinoma cell line, HT29. Diacylglycerol lipase and cholesterol esterase [corrected] activities in HT29 cells were inhibited by known inhibitors of hormone-sensitive lipase (diethyl-p-nitrophenyl phosphate, NaF and HgCl2) to the same extent as in human adipocytes. A polyclonal antiserum directed against rat hormone-sensitive lipase inhibited 89% of HT29 cell lipase activity. HT29 hormone-sensitive lipase was the same size as the adipocyte enzyme as was its mRNA. Complete homology between mRNA sequences in HT29 and adipocyte was demonstrated using ribonuclease protection assay. These data are consistent with the expression of a protein closely related, if not identical, to the enzyme expressed in human adipose tissue. HT29 is the first human cell line where hormone-sensitive lipase expression has been shown.

Adenocarcinoma↗
PubMed1995

The hormone-sensitive lipase (LIPE) gene located on chromosome 19q13.1-->13.2 is not duplicated on 19p13.3.

The existence of a DNA polymorphism at the hormone-sensitive lipase locus could be of great interest for genetic analysis of obesity and related disorders since hormone-sensitive lipase is the rate-limiting enzyme of adipose tissue lipolysis and therefore plays a key role in energy metabolism. The polymorphic dinucleotide repeat D19S120 was identified within a human genomic clone selected with a rat hormone-sensitive lipase cDNA. This marker was subsequently localized to the short arm of chromosome 19 (p13.3) whereas human hormone-sensitive lipase (LIPE) had been mapped to the long arm of chromosome 19 (q13.1-->13.2). A duplication of the hormone-sensitive lipase gene or the presence of a pseudogene could explain the discrepancy. Cosmids from the two regions were analyzed in Southern blot experiments. A human adipose tissue hormone-sensitive lipase full-length cDNA probe hybridized only to cosmids from the 19q13.1-->13.2 region whereas the D19S120 amplicon probe hybridized only to cosmids from the p13.3 region. These data show that the occurrence of gene duplication or the presence of a pseudogene on the short arm of chromosome 19 is very unlikely and that D19S120 is unrelated to the hormone-sensitive lipase gene.

Adipose Tissue↗
PubMedDate not supplied

[BWS-mobility in patients with an impingement syndrome compared to healthy subjects--an inclinometric study].

AIM: The aim of this study was to analyse the relation between the mobility of the thoracic spine and an impingement syndrome of the shoulder. METHOD: In a prospective study, 50 patients with an impingement syndrome and 50 healthy test subjects were examined for the mobility of their thoracic spines. All patients and test subjects were examined according to a standardized protocol. The experiments were carried out in the biomechanical laboratory of our clinic with the Plurimetercompass and the Inclinometer of Rippstein. RESULTS: In 23 patients a tendinosis calcarea was diagnosed radiologically, 27 patients suffered from a plain impingement without calcification, hence both groups were analyzed separately. The mobility of the thoracic spine in the sagittal and frontal planes and in rotation was significantly different between the three groups. The highest mobility was found in the healthy test subjects, the lowest in patients with a plain impingement. No differences were found concerning the initial posture of the thoracic spine. CONCLUSION: There is a relation between mobility of the thoracic spine and an impingement syndrome. This should be respected in diagnosis and therapy.

Adult↗