Biomedical subjects

N Moustaïd

Publications and source records attributed to N Moustaïd.

9 recordsLinked to original sources

PubMed1997

Angiotensinogen gene expression in adipose tissue: analysis of obese models and hormonal and nutritional control.

Synthesis of angiotensin II (ANG II) has recently been described in adipose cells and has been linked to regulation of adiposity. Angiotensinogen (AGT), the substrate from which ANG II is formed, was previously shown to be elevated in adipose tissue of obese (ob/ob and db/db) mice and regulated by nutritional manipulation. It is unknown, however, whether overexpression of adipose AGT can be extended to other models of obesity and whether hormonal and/or nutritional factors directly regulate AGT expression in adipocytes. We investigated these possibilities by analyzing AGT mRNA levels in adipose tissue of obese Zucker rats, viable yellow (Avy) mice, and humans and by treating 3T3-L1 adipocytes with insulin, glucose, and a beta-adrenergic agonist. We demonstrate that AGT mRNA is decreased by approximately 50 and 80%, respectively, in adipose tissue of obese vs. lean Zucker rats and Avy mice. We also report that AGT is expressed at variable levels in human adipose tissue. Finally, we show that AGT mRNA is upregulated by insulin and downregulated by beta-adrenergic stimulation in adipocytes.

3T3 Cells↗

PubMed1996

Insulin increases lipogenic enzyme activity in human adipocytes in primary culture.

Studies with human adipose tissue have demonstrated the presence of key enzymes of fat synthesis. However, long-term regulation of these enzymes has not been reported. To address this issue, we used human adipocytes in primary culture. Human adipose tissue was obtained from abdominal fat of patients undergoing abdominal surgery. Adipocytes were isolated by collagenase digestion and cultured in media supplemented with 1% fetal bovine serum. To evaluate metabolic activity of cultured cells, we assessed the following during the culture: DNA pattern, cell size, glucose consumption and activities for two lipogenic enzymes, fatty acid synthase (FAS) and glycerol-3-phosphate dehydrogenase (GPDH). Analysis of DNA pattern showed that human adipocytes cultured under the above condition did not undergo cell apoptosis. In addition, no significant change in the cell size occurred during 22 d of culture. Glucose consumption by cultured cells was also constant during the culture and was 60% greater in the presence of 10 nmol/L of insulin. Treatment of cultured human adipocytes with insulin for 3-22 d increased GPDH and FAS activity by 60% and 2.8-fold, respectively, compared to cells cultured without insulin. Furthermore, the increase in FAS activity due to insulin treatment was dose dependent and maximal at 10 nmol/L. Our studies show for the first time that human adipocytes can be maintained viable and metabolically active for 2-3 wk in culture. Interestingly, cultured cells remain responsive to insulin. Therefore, this system will allow further characterization of long-term regulation of lipogenesis in human adipocytes and will be useful for developing pharmacological treatments of obesity.

Adipocytes↗

PubMed1996

Adipose tissue stearoyl-CoA desaturase mRNA is increased by obesity and decreased by polyunsaturated fatty acids.

Stearoyl-CoA desaturase (SCD) is a key regulatory enzyme in the synthesis of unsaturated fatty acids. Although regulation of hepatic SCD by obesity and polyunsaturated fatty acids (PUFA) has been well investigated, no studies have addressed whether similar regulation occurs in adipose tissue. We addressed these questions by feeding control (12% corn oil) and high-PUFA (48% corn oil) diets to lean and obese Zucker rats and analyzing SCD mRNA levels in adipose tissue and liver. We report that SCD mRNA content was dramatically elevated in adipose tissue of obese vs. lean rats on both diets and was significantly decreased by PUFA in both genotypes. Interestingly, we demonstrate that SCD expression was directly downregulated in a dose dependent manner by PUFA in 3T3-L1 adipocytes. We conclude that 1) obese Zucker rats overexpress the SCD gene in both liver and adipose tissue and 2) PUFA directly suppress SCD expression in adipocytes. Further studies will elucidate the mechanisms responsible for obesity- and PUFA-mediated regulation of SCD in adipose cells.

Adipocytes↗

PubMed1994

Localization of sequences for the basal and insulin-like growth factor-I inducible activity of the fatty acid synthase promoter in 3T3-L1 fibroblasts.

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -19 of the FAS gene were compared. Deletions of the promoter sequences from -136 to -19 resulted in a step-wise decrease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the -136 to -19 region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 fibroblasts transiently transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Identification of an insulin response element in the fatty acid synthase promoter.

We have previously reported that insulin increases fatty acid synthase (FAS) gene transcription, and that sequences responsible for positive regulation are located within the first 332 base pairs of the FAS promoter. To define minimal sequences required for insulin regulation within this region, chimeric constructs containing serial 5' deletions starting at -318 and extending through position +67 of the rat FAS gene ligated to the luciferase reporter gene were transfected into 3T3-L1 adipocytes. Insulin treatment at 10 nM increased luciferase activity 2-3-fold in 3T3-L1 adipocytes transfected with constructs containing progressive deletions from -318 to -67. This stimulation of the FAS promoter activity by insulin was dose-dependent. However, no effect of insulin was observed when fusion constructs containing FAS promoter sequences spanning from -25 or from -19 to +67 were transfected into adipocytes. These results suggest that the insulin response sequences of the FAS gene may be located in the region from -67 to -25. DNase I footprinting using liver nuclear extracts revealed a protected region spanning -71 and -50 in addition to a region near the putative TATA box. Gel mobility shift assays using the sequence from -71 to -50 as a probe revealed nuclear factor(s) from mouse liver and 3T3-L1 adipocytes that specifically complexed with this sequence. Mutational analysis of this region showed that sequences between -68 and -60 are essential for recognition and interaction with a trans-acting factor(s). Moreover, when three tandem repeats of the sequences spanning -68 to -52 were linked to the SV40 promoter and used for transfection, luciferase activity increased 3.6-fold in response to insulin treatment. Thus, we have identified novel cis-acting DNA sequences responsible for insulin regulation of the FAS gene, which interact with nuclear protein(s) from liver and adipocytes and which are found to share limited homology to insulin response sequences present in other genes.

3T3 Cells↗

PubMed1993

Regulation of fatty acid synthase gene transcription. Sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3-L1 preadipocytes are present in the 332 bp promoter.

We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5'-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5' flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 +/- 0.2-, 2.6 +/- 0.1-, 2.0 +/- 0.2- and 1.7 +/- 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 +/- 0.3- and 3.5 +/- 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 +/- 0.8-, 3.0 +/- 0.7- and 2.3 +/- 0.5-fold respectively in serum-free media, and by 2.6 +/- 0.4-, 3.3 +/- 0.9-, 3.1 +/- 0.4- and 2.9 +/- 0.6-fold respectively in the presence of 0.5% serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.

3T3 Cells↗

PubMed1991

Transcriptional regulation of p90 with sequence homology to Escherichia coli glycerol-3-phosphate acyltransferase.

We have previously isolated cDNA clones for several mRNAs that are dramatically increased in livers of fasted mice refed a high carbohydrate diet. We report here the sequence and regulation of one such mRNA; the 6.8-kilobase mRNA has an open reading frame of 2481 nucleotides, and the coded protein contains 827 amino acid residues (Mr of 90,000) with a 30% identity and an additional 42% similarity in an approximately 300-amino acid stretch to Escherichia coli glycerol-3-phosphate acyltransferase. The p90 mRNA is highly expressed in liver and in adipose tissue. When previously fasted mice were refed a high carbohydrate, fat-free diet, the liver mRNA level for p90 was increased about 20-fold at 8 h. Administration of dibutyryl cAMP at the time of refeeding prevented the increase in the p90 mRNA by 70%. In addition, there was no increase in the p90 mRNA level when previously starved streptozotocin-diabetic mice were refed. In diabetic animals, the p90 mRNA level increased by 2-fold 1 h after insulin injection and reached a maximum of 19-fold after 6 h. The increase in transcription rate of the p90 gene preceded that of steady state mRNA level caused by fasting/refeeding, and cAMP abolished the increase in transcription. Transcription of the p90 gene was not detectable in either fasted or refed streptozotocin-diabetic mice, but increased 4-fold 30 min after insulin administration and further increased up to 8-fold at 2 h. On-going protein synthesis was necessary for this increase.

Amino Acid Sequence↗

PubMed1991

Regulation of expression of the fatty acid synthase gene in 3T3-L1 cells by differentiation and triiodothyronine.

We have previously reported that fatty acid synthase mRNA levels increase 10-15-fold during the differentiation of 3T3-L1 cells to adipocytes, correlating well with the increase in the relative rate of synthesis for this enzyme. Here we show by transcription run-on assays that fatty acid synthase is highly transcribed in both preadipocytes and adipocytes. Furthermore, the transcription rate of the fatty acid synthase gene increased only 1.5-fold during the adipose conversion, whereas the apparent mRNA half-life increased from 2.5 h in preadipocytes to approximately 20 h in adipocytes. These results indicate that the increase in mRNA level during adipose conversion is not only due to the transcriptional activation of this gene but reflects the post-transcriptional stabilization of the message in adipocytes compared to preadipocytes. As thyroid hormone has been reported to increase lipogenic enzyme activities including fatty acid synthase in adipose tissue and in differentiating adipocytes in vitro, we used fully differentiated 3T3-L1 adipocytes to study T3 regulation of mammalian fatty acid synthase expression. We measured the effect of T3 on the relative rate of protein synthesis, mRNA content, and transcription rate of this gene. When mature adipocytes were treated with 10 nM T3, the relative rate of synthesis of fatty acid synthase increased 1.9-fold at 6 h, and it reached a maximum of 2.9-fold at 12 h. In addition, Northern blot analysis showed that T3 increased the steady-state mRNA level for fatty acid synthase by 2.4-fold at 12 h and 4.5-fold at 24 h. Furthermore, run-on transcription analysis with isolated nuclei from cells treated with T3 showed that the transcription rate of the fatty acid synthase gene increased 4.1-fold after 6 h of T3 treatment and remained at the stimulated level for 24 h. These results demonstrate that the increase in transcription of the fatty acid synthase gene preceded that of the steady-state mRNA level, indicating that T3 regulates expression of fatty acid synthase primarily by modulating the transcription rate of the gene. In conclusion, while the differentiation-dependent increase in fatty acid synthase is mediated by both transcriptional and posttranscriptional processes, T3 regulation is primarily at the transcriptional level.

3T3 Cells↗

PubMed1990

[Decrease of gene expression of glycerophosphate dehydrogenase by dexamethasone in differentiated 3T3-F442A cells: antagonism with insulin and antiglucocorticoid RU38486].

Preadipocyte subclones derived from mouse 3T3 cells differentiate into adipocytes; this differentiation is characterized by an increased activity of numerous enzymes required for triglyceride synthesis and/or mobilization. Among these enzymes, the role of glycerophosphate dehydrogenase in the differentiation process has been previously reported. In the present work, we studied the hormonal regulation of glycerophosphate dehydrogenase gene expression (G3PDH) in differentiated 3T3-F442A adipocytes. Dexamethasone (DEX) elicited a 50% decrease in both mRNA content and specific activity of G3PDH. This effect was due to a posttranscriptional event since DEX shortened the half life of the mRNA, whereas it did not modify the transcription rate of this gene. The DEX effect is specific to G3PDH, since the expression of another adipose-specific gene, namely adipsin, is not modified by DEX treatment. Insulin counteracts the inhibitory effect of DEX, mainly by stabilizing the mRNA encoding for G3PDH. The antiglucocorticoid RU38486 is able to reverse DEX inhibition. Latter phenomenon suggests that DEX action on G3PDH gene expression could be mediated by glucocorticoid receptors.

Animals↗