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A Vidal-Puig

Publications and source records attributed to A Vidal-Puig.

36 records · Page 2Linked to original sources
PubMed1998

Mice expressing human but not murine beta3-adrenergic receptors under the control of human gene regulatory elements.

Beta-adrenergic receptors (ARs) are expressed predominantly in adipose tissue, and beta3-selective agonists are effective anti-obesity drugs in rodents. Rodent and human beta3-ARs differ with respect to expression in white versus brown adipocytes as well as their ability to be stimulated by beta3-AR-selective agonists. Humans express beta3-AR mRNA abundantly in brown but not white adipocytes, while rodents express beta3-AR mRNA abundantly in both sites. To determine the basis for this difference, we have transgenically introduced 74 kilobases (kb) of human beta3-AR genomic sequence into gene knockout mice lacking beta3-ARs. Importantly, human beta3-AR mRNA was expressed only in brown adipose tissue (BAT) of transgenic mice, with little or no expression being detected in white adipose tissue (WAT), liver, stomach, small intestine, skeletal muscle, and heart. This pattern of expression differed from that observed in mice bearing a murine beta3-AR genomic transgene in which beta3-AR mRNA was expressed in both WAT and BAT, but not in other sites. Furthermore, we have transgenically introduced smaller human constructs containing -14.5 and -0.6 kb of upstream sequence into beta3-AR gene knockout mice. Both -14.5 and -0.6 kb constructs were expressed in BAT but not WAT. Thus, human but not murine cis-regulatory elements direct beta3-AR gene expression preferentially to brown adipocytes. Identification of responsible cis-regulatory element(s) and relevant trans-acting factor(s) should provide insight into mechanisms controlling human beta3-AR gene expression. In addition, the beta3-AR agonist, CGP-12177, stimulated oxygen consumption in mice expressing human but not murine beta3-ARs by 91% compared with only 49% in control beta3-AR gene knockout mice, demonstrating that the human beta3-AR can functionally couple with energy expenditure. These "humanized" mice should assist us in the development of drugs that may become effective anti-obesity agents in humans.

Adipose Tissue↗
PubMed1997

The human uncoupling protein-3 gene. Genomic structure, chromosomal localization, and genetic basis for short and long form transcripts.

Uncoupling protein-3 (UCP3) is a recently identified candidate mediator of adaptive thermogenesis in humans. Unlike UCP1 and UCP2, UCP3 is expressed preferentially and at high levels in human skeletal muscle and exists as short and long form transcripts, UCP3S and UCP3L. UCP3S is predicted to encode a protein which lacks the last 37 C-terminal residues of UCP3L. In the present study, we have defined the intron-exon structure for the human UCP3 gene and determined that UCP3S is generated when a cleavage and polyadenylation signal (AATAAA) located in the last intron prematurely terminates message elongation. In addition we have mapped UCP3 to the distal segment of human chromosome 11q13 (between framework markers D11S916 and D11S911), adjacent to UCP2. Of note, UCP2 and UCP3 in both mice and humans colocalize in P1 and BAC genomic clones indicating that these two UCPs are located within 75-150 kilobases of each other and most likely resulted from a gene duplication event. Previous studies have noted that mouse UCP2 maps to a region of chromosome 7 which is coincident with three independently mapped quantitative trait loci for obesity. Our study shows that UCP3 is also coincident with these quantitative trait loci raising the possibility that abnormalities in UCP3 are responsible for obesity in these models.

Animals↗
PubMed1997

The organization, promoter analysis, and expression of the human PPARgamma gene.

PPARgamma is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARgamma cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARgamma1 and PPARgamma2, were detected in man. The relative expression of human PPARgamma was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARgamma1 and gamma2 mRNA. In all tissues analyzed, PPARgamma2 was much less abundant than PPARgamma1. Adipose tissue and large intestine have the highest levels of PPARgamma mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARgamma is barely detectable in muscle. This high level expression of PPARgamma in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARgammas, the human PPARgammas are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARgamma gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARgamma1 and PPARgamma2 mRNAs, which differ at their 5'-ends. PPARgamma1 is encoded by eight exons, and PPARgamma2 is encoded by seven exons. The 5'-untranslated sequence of PPARgamma1 is comprised of exons A1 and A2, whereas that of PPARgamma2 plus the additional PPARgamma2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARgamma1 and gamma2. Knowledge of the gene structure will allow screening for PPARgamma mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.

3T3 Cells↗
PubMed1997

UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue.

Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. UCP1 is expressed exclusively in brown adipocytes while UCP2 is expressed widely. We now report the molecular cloning of a third uncoupling protein homologue, designated UCP3. At the amino acid level, hUCP3 is 71% identical to hUCP2 and 57% identical to hUCP1. UCP3 is distinguished from UCP1 and UCP2 by its abundant and preferential expression in skeletal muscle in humans, and brown adipose tissue and skeletal muscle in rodents. Since skeletal muscle and brown adipose tissue are believed to be important sites for regulated energy expenditure in humans and rodents, respectively, UCP3 may be an important mediator of adaptive thermogenesis. Since UCP3 is minimally expressed in human heart and other critical organs, it is a promising target for anti-obesity drug development aimed at increasing thermogenesis.

Adipose Tissue, Brown↗
PubMed1996

Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.

The orphan nuclear receptor, peroxisome proliferator-activated receptor (PPAR) gamma, is implicated in mediating expression of fat-specific genes and in activating the program of adipocyte differentiation. The potential for regulation of PPAR gamma gene expression in vivo is unknown. We cloned a partial mouse PPAR gamma cDNA and developed an RNase protection assay that permits simultaneous quantitation of mRNAs for both gamma l and gamma 2 isoforms encoded by the PPAR gamma gene. Probes for detection of adipocyte P2, the obese gene product, leptin, and 18S mRNAs were also employed. Both gamma l and gamma 2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma 1 expression was also detected at lower levels in liver, spleen, and heart; whereas, gamma l and gamma 2 mRNA were expressed at low levels in skeletal muscle. Adipose tissue levels of gamma l and gamma 2 were not altered in two murine models of obesity (gold thioglucose and ob/ob), but were modestly increased in mice with toxigene-induced brown fat ablation uncoupling protein diphtheria toxin A mice. Fasting (12-48 h) was associated with an 80% fall in PPAR gamma 2 and a 50% fall in PPAR gamma mRNA levels in adipose tissue. Western blot analysis demonstrated a marked effect of fasting to reduce PPAR gamma protein levels in adipose tissue. Similar effects of fasting on PPAR gamma mRNAs were noted in all three models of obesity. Insulin-deficient (streptozotocin) diabetes suppressed adipose tissue gamma l and gamma 2 expression by 75% in normal mice with partial restoration during insulin treatment. Levels of adipose tissue PPAR gamma 2 mRNA were increased by 50% in normal mice exposed to a high fat diet. In obese uncoupling protein diphtheria toxin A mice, high fat feeding resulted in de novo induction of PPAR gamma 2 expression in liver. We conclude (a) PPAR gamma 2 mRNA expression is most abundant in adipocytes in normal mice, but lower level expression is seen in skeletal muscle; (b) expression of adipose tissue gamma1 or gamma2 mRNAs is increased in only one of the three models of obesity; (c) PPAR gamma 1 and gamma 2 expression is downregulated by fasting and insulin-deficient diabetes; and (d) exposure of mice to a high fat diet increases adipose tissue expression of PPAR gamma (in normal mice) and induces PPAR gamma 2 mRNA expression in liver (in obese mice). These findings demonstrate in vivo modulation of PPAR gamma mRNA levels over a fourfold range and provide an additional level of regulation for the control of adipocyte development and function.

Adipocytes↗
PubMed1996

Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus.

Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed.

Adolescent↗
PubMed1996

Candidate genes for insulin resistance.

Insulin resistance confers increased susceptibility to NIDDM, atherosclerotic cardiovascular disease, ovarian hyperandrogenism, and possibly hypertension. Insulin resistance is largely inherited, in rare cases as a monogenic disorder or more commonly as a complex trait. The search for insulin resistance genes relies mainly on two complementary approaches: 1) positional cloning using random DNA markers present throughout the genome; and 2) the analysis of specific candidate genes. This report briefly summarizes the candidate gene approach to insulin resistance. Progress related to the analysis of genes encoding molecules that participate in insulin action is reviewed. In addition, the spectrum of potential genetic defects that might contribute to insulin resistance, both at the level of the target cell and secondarily (e.g., obesity genes), is discussed.

Animals↗
PubMed1995

Genetic variability in the TNF-alpha promoter is not associated with type II diabetes mellitus (NIDDM).

Recent evidence suggests that expression of tumor necrosis factor-alpha (TNF-alpha) by adipocytes is a molecular mediator of insulin resistance in obesity. We have therefore tested the hypothesis that variations within the regulatory region of the TNF-alpha gene, which might cause increased adipocyte or generalized TNF-alpha synthesis, are associated with NIDDM, a state in which insulin resistance is routinely observed. Neither the previously known variants within the TNF-alpha promoter at position -308 and -238, nor two newly identified polymorphisms at position -376 and -163, were found at a significantly higher frequency in Caucasian NIDDM patients compared to non-diabetic controls. No genetic variants were found in Pima Indians. These data make it unlikely that mutations within regulatory elements of the TNF-alpha gene are associated with an increase in the prevalence of NIDDM.

Adult↗
PubMed1995

Analysis of the hexokinase II gene in subjects with insulin resistance and NIDDM and detection of a Gln142-->His substitution.

Hexokinase II (HKII) is the predominant hexokinase isozyme expressed in insulin-responsive tissues. Since defects involving glucose transport and/or its phosphorylation to glucose-6-phosphate are present in muscle of insulin-resistant humans, HKII should be viewed as a candidate gene for inherited insulin resistance and susceptibility to non-insulin-dependent diabetes mellitus (NIDDM). To investigate the prevalence of potential mutations in the gene encoding HKII, we used the polymerase chain reaction (PCR) to amplify each of the 18 exons of the HKII gene from genomic DNA derived from 59 subjects: 25 insulin-resistant probands with clinical features of the type A syndrome and 34 NIDDM subjects enrolled in the United Kingdom Prospective Study of Therapies of NIDDM (UKPDS) who represented the highest percentile of fasting hyperinsulinemia in the UKPDS population of 5,098 subjects. PCR products corresponding to individual HKII exons derived from each subject were screened for the presence of nucleotide variation using a sensitive nonradioactive single-strand conformation polymorphism (SSCP) protocol. Variant SSCP patterns indicative of genetic variation were detected only in PCR amplimers containing exons 4-7, 10, 15, and 17. Direct sequencing of amplified DNA from individuals affected with variant SSCP patterns revealed the presence of the following silent polymorphisms: Asp251 (GAT/C) in exon 7 and Asn692 (AAT/C) in exon 15. SSCP variants detected in PCR products containing exons 5, 10, and 17 were due to single base substitutions in flanking intronic sequences. A polymorphic GGA repeat was identified within intron 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Hyperinsulinemia in polycystic ovary syndrome: relationship to clinical and hormonal factors.

We analyzed the association between hyperandrogenism and hyperinsulinemia, and their relationship to body mass index, in a large series of patients with polycystic ovary syndrome (PCOS). A characteristic hormonal profile was sought in women with marked hyperinsulinemia. The patient group consisted of 73 women with PCOS, ranging in age from 16 to 29 years. The control group consisted of 34 healthy women with no evidence of hyperandrogenism, aged 19-30 years. None of the patients or control women had a body mass index above 27 kg/m2. Follicle-stimulating hormone, luteinizing hormone, prolactin, testosterone, estradiol, androstenedione, dehydroepiandrosterone sulfate, sex hormone binding globulin, 17-hydroxyprogesterone, and free cortisol were determined by radioimmunoassay. The free testosterone index was calculated. The oral glucose tolerance test was used to analyze basal insulinemia, maximum insulin peak, and the insulinemia/glycemia index. In the group with PCOS body mass index was greater, free testosterone index was higher, and levels of dehydroepiandrosterone sulfate, testosterone, 17-hydroxyprogesterone (P < 0.001) and androstenedione (P < 0.05) were higher than in the control group. Of the insulin parameters, basal insulinemia, maximum insulin peak, and insulinemia/glycemia index were higher in the patient group (P < 0.001). In patients with marked insulinemia, free testosterone index was more markedly elevated, and gonadotrophin levels were normal. Our data confirm that a characteristic pattern of hyperinsulinemia is associated with PCOS. We found no causal relationship between hyperinsulinemia and androgen levels. A characteristic hormonal pattern was found in patients with marked hyperinsulinemia.

Adolescent↗
PubMed1994

Reduction of endogenous, ovarian and adrenal androgens with ketoconazole does not alter insulin response in the polycystic ovary syndrome.

Several different strategies were used to investigate the relationship between hyperandrogenism and hyperinsulinemia associated with polycystic ovary syndrome. Ketoconazole was given orally (400 mg/day) for 9 months to evaluate the effect of reduction in ovarian and adrenal androgens on insulin response (oral glucose tolerance test) in 35 women with polycystic ovary syndrome. Androgenic steroids (testosterone, androstenedione, dehydroepiandrosterone sulphate, and free testosterone index) decreased (p < 0.01), but basal insulinemia, maximum peak insulin, and insulin/glucose ratio showed no significant changes. One month after treatment was stopped, free testosterone index, and serum concentrations of androstenedione and testosterone, increased (p < 0.05), but no alterations were noted in insulin parameters. Body mass index was stable throughout the ten-month study period. Our findings suggest that endogenous androgens, no matter whether they are of ovarian or adrenal origin, do not play a major role in the modulation of hyperinsulinemia in patients with polycystic ovary syndrome.

Adolescent↗
PubMed1994

Comparative sensitivity of alternative single-strand conformation polymorphism (SSCP) methods.

Single-strand conformation polymorphism (SSCP) analysis is a genetic screening technique that allows for the rapid detection of single nucleotide substitutions in fragments of PCR-amplified genomic DNA or cDNA. Several alternative protocols are now being commonly used to resolve differences in electrophoretic mobility of single-stranded DNA fragments. The aim of this study was to directly compare the sensitivity of three popular SSCP methods using a panel of 19 known human mutations/polymorphisms present in genomic DNA samples. Using a single electrophoresis protocol, 95% of the mutations were detected using small-format PhastGel and the PhastSystem. Large-format gels (5% polyacrylamide and Hydrolink-MDE) were tested both with and without the addition of 10% glycerol. The sensitivity for polyacrylamide and Hydrolink-MDE gels without glycerol was 89% and 79%, respectively, and 68% or 63%, respectively, for glycerol-containing gels. However, all mutations were detected with either polyacrylamide or Hydrolink-MDE when both glycerol and non-glycerol gels were examined. We conclude that comparable very high detection efficiency can be achieved using the PhastSystem or by using a combination of two large-gel conditions with either polyacrylamide or Hydrolink-MDE.

Humans↗
PubMed1993

[Lipid profile of hyperandrogenic women undergoing treatment with ketoconazole and its relationship with the evolution of the hormone pattern].

BACKGROUND: Previous studies have demonstrated changes in the lipidic profile following treatment with ketoconazole. However, the possible influence in patients with hyperandrogenism treated with this drug and its possible relation with the evolution of the hormonal pattern is unknown. RESULTS: Thirty-seven women with no tumoral hyperandrogenism treated with ketoconazole for 9 months (400 mg/day) were studied during treatment and one month after suppression of the medication. The study included the evaluation of total cholesterol, HDL cholesterol, triglycerides, LDL cholesterol and hormonal determinations of estradiol, androstenedione, testosterone, dehydroepiandrosterone, sulphate and sex hormone binding globulin. RESULTS: A rapid and significant decrease of LDL cholesterol and total cholesterol was observed (p < 0.01) which was reversible upon discontinuation of treatment. The HDL cholesterol levels and triglycerides did not modify. The final hormonal profile was more favorable with a significant reduction of testosterone and dehydroepiandrosterone sulphate (p < 0.01) and an increase in estradiol (p < 0.01). No statistical correlation was observed between the changes of sexual steroids and the changes in the lipidic pattern. CONCLUSIONS: In patients with non tumoral hyperandrogenism treated with ketoconazole the modifications of the steroid hormone profile do not appear to be related with changes in lipidic metabolism.

Adolescent↗
PubMed1993

[Adrenal androgens: traditionally forgotten hormones. Today, of current importance].

Androgens have been some traditionally forgotten hormones because, apparently, they do not play a main role in any pathology. However, the conviction that hormones circulating at such a high plasmatic concentration must have a biologic function, have promote an active research in this field, resulting in a better understanding of the regulation mechanisms of the synthesis of these hormones, as well as demonstrating its potential relationship with the etiopathogenicity of such important pathologies as obesity, arteriosclerosis or diabetes. In the present review, we approach the specific regulation mechanisms of synthesis of adrenal androgens, as well as their implications in severe physiological and pathological conditions.

Adrenal Glands↗
PubMed1992

Dehydroepiandrosterone sulfate and other possible influencing factors that modulate sex hormone-binding globulin levels in the hirsute patient.

This study was designed to investigate the most important factors affecting serum concentrations of sex hormone-binding globulin (SHBG) in women with hirsutism. We compared endocrine profiles based on biochemical measurements of LH, FSH, oestradiol, testosterone (T), prolactin, 17-hydroxy-progesterone, dehydroepiandrosterone sulphate (DHEAS), SHBG, cortisol and insulin in the follicular phase in 32 healthy women and 52 patients. The study group was subdivided according to SHBG levels into Group A (low level) and Group B (high level). Significant differences between Groups A and B were found in DHEAS and T levels, but not in body mass index or insulinaemia. There was a relationship between DHEAS and SHBG levels (r = 0.51) and between T and SHBG (r = 0.31). We conclude that DHEAS may be a significant modulator of SHBG in the female hirsute patient, an observation seldom mentioned in previous reports.

Adult↗