Catherine Mounier

Hormonal and nutritional regulation of SCD1 gene expression.

Stearoyl-CoA Desaturase 1 (SCD1) is the rate limiting enzyme catalyzing the biosynthesis of monounsaturated fatty acids preferentially from palmitoyl-CoA and stearoyl-CoA forming respectively palmitoleyl-CoA and oleyl-CoA. These monounsaturated fatty acids are the key components of triglycerides and membrane phospholipids. Studying the regulation of SCD1 is of particular interest since alterations in phospholipids composition have been implicated in a variety of diseases including cancers, diabetes and cardiovascular disorders. Furthermore, oleic acid, the main product of SCD1 reaction, is the predominant fatty acid of human adipose tissue triacylglycerols, associating SCD1 with the development of obesity and the metabolic syndrome. In light of the key role of SCD1 in general metabolism, it is not surprising to observe a very tight and complex regulation of SCD1 gene expression in response to various parameters including hormonal and nutrient factors. In this review we analyze the anatomy and index the transcription factors that have been characterized to bind the SCD1 promoter. Then we present the current knowledge on how hormones regulate SCD1 expression with a particular interest on the role of insulin and leptin. We also describe how nutrients especially polyunsaturated fatty acids and carbohydrates modulate SCD1 gene expression.

Epidermal Growth Factor-induced Phosphatidylinositol 3-Kinase Activation and DNA Synthesis IDENTIFICATION OF Grb2-ASSOCIATED BINDER 2 AS THE MAJOR MEDIATOR IN RAT HEPATOCYTES

In previous work we showed that the phosphatidylinositol 3-kinase (PI3-kinase), not the mitogen-activated protein kinase, pathway is necessary and sufficient to account for insulin- and epidermal growth factor (EGF)-induced DNA synthesis in rat hepatocytes. Here, using a dominant-negative p85, we confirmed the key role of EGF-induced PI3-kinase activation and sought to identify the mechanism by which this is effected. Our results show that EGF activates PI3-kinase with a time course similar to that of the association of p85 with three principal phosphotyrosine proteins (i.e. PY180, PY105, and PY52). We demonstrated that each formed a distinct p85-associated complex. PY180 and PY52 each constituted about 10% of EGF-activated PI3-kinase, whereas PY105 was responsible for 80%. PY105 associated with Grb2 and SHP-2, and although it behaved like Gab1, none of the latter was detected in rat liver. We therefore cloned a cDNA from rat liver, which was found to be 95% homologous to the mouse Grb2-associated binder 2 (Gab2) cDNA sequence. Using a specific Gab2 antibody, we demonstrated its expression in and association with p85, SHP-2, and Grb2 upon EGF treatment of rat hepatocytes. Gab2 accounted for most if not all of the PY105 species, since immunoprecipitation of Gab2 with specific antibodies demonstrated parallel immunodepletion of Gab2 and PY105 from the residual supernatants. We also found that the PI3-kinase activity associated with Gab2 was totally abolished by dominant negative p85. Thus, Gab2 appears to be the principal EGF-induced PY protein recruiting and activating PI3-kinase and mitogenesis.

Epidermal Growth Factor and Insulin-Induced Deoxyribonucleic Acid Synthesis in Primary Rat Hepatocytes Is Phosphatidylinositol 3-Kinase Dependent and Dissociated from Protooncogene Induction*

The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhap...

Hepatic regulation of fatty acid synthase by insulin and T3: evidence for T3 genomic and nongenomic actions

Fatty acid synthase (FAS) is a key enzyme of hepatic lipogenesis responsible for the synthesis of long-chain saturated fatty acids. This enzyme is mainly regulated at the transcriptional level by nutrients and hormones. In particular, glucose, insulin, and T(3) increase FAS activity, whereas glucagon and saturated and polyunsaturated fatty acids decrease it. In the present study we show that, in liver, T(3) and insulin were able to activate FAS enzymatic activity, mRNA expression, and gene transcription. We localized the T(3) response element (TRE) that mediates the T(3) genomic effect, on the FAS promoter between -741 and -696 bp that mediates the T(3) genomic effect. We show that both T(3) and insulin regulate FAS transcription via this sequence. The TRE binds a TR/RXR heterodimer even in the absence of hormone, and this binding is increased in response to T(3) and/or insulin treatment. The use of H7, a serine/threonine kinase inhibitor, reveals that a phosphorylation mechanism is implicated in the transcriptional regulation of FAS in response to both hormones. Specifically, we show that T(3) is able to modulate FAS transcription via a nongenomic action targeting the TRE through the activation of a PI 3-kinase-ERK1/2-MAPK-dependent pathway. Insulin also targets the TRE sequence, probably via the activation of two parallel pathways: Ras/ERK1/2 MAPK and PI 3-kinase/Akt. Finally, our data suggest that the nongenomic actions of T(3) and insulin are probably common to several TREs, as we observed similar effects on a classical DR4 consensus sequence.

Key role of the ERK1/2 MAPK pathway in the transcriptional regulation of the Stearoyl-CoA Desaturase (SCD1) gene expression in response to leptin.

Stearoyl-CoA Desaturase-1 (SCD1) is the rate limiting enzyme catalyzing the synthesis of monounsaturated fatty acids. Variation of SCD1 activity and the ratio of saturated to unsaturated fatty acids have been implicated in a variety of diseases including obesity, type II diabetes and cancers. In liver, many factors regulate SCD1 expression including dietary and hormonal factors such as insulin and leptin. We previously showed in hepatic cells that insulin acts through the PI3K and mTOR pathways to upregulate SCD1 expression. In the present study, using HepG2 cells, we characterized the signaling pathway mediating the leptin inhibitory response on SCD1 gene expression. We showed that leptin inhibits SCD1 at the transcriptional level. Inhibition of the ERK1/2 MAPK pathway with the PD98059 reverses the effect of leptin on SCD1 expression. Our data also demonstrated that the effect of leptin is entirely independent of the effect of insulin. Using the pharmaceutical inhibitors Ag490 and SL0101, we showed that the inhibitory effect of leptin is also mediated by the Janus Kinase 2 (Jak2) and p90RSK. EMSA and transfection experiments suggest a key role for the Sp1 transcription factor, which in turn may compete for the binding of other transcription factors such as AP-1, leading to the inhibition of SCD1 transcription. Taken together, our observations showed that, independently of insulin action, leptin exerts an inhibitory effect on SCD1 transcription via a signaling pathway implicating Jak2, ERK1/2, and p90RSK which probably targets the downstream transcription factor Sp1 on the SCD1 promoter.

Specific inhibition by hGRB10ζ of insulin-induced glycogen synthase activation: evidence for a novel signaling pathway☆

Grb10 is a member of a family of adapter proteins that binds to tyrosine-phosphorylated receptors including the insulin receptor kinase (IRK). In this study recombinant adenovirus was used to over-express hGrb10zeta, a new Grb10 isoform, in primary rat hepatocytes and the consequences for insulin signaling were evaluated. Over-expression of hGrb10zeta resulted in 50% inhibition of insulin-stimulated IRK autophosphorylation and activation. Analysis of downstream events showed that hGrb10zeta over-expression specifically inhibits insulin-stimulated glycogen synthase (GS) activity and glycogen synthesis without affecting insulin-induced IRS1/2 phosphorylation, PI3-kinase activation, insulin like growth factor binding protein-1 (IGFBP-1) mRNA expression, and ERK1/2 MAP kinase activity. The classical pathway from PI3-kinase through Akt-PKB/GSK-3 leading to GS activation by insulin was also not affected by hGrb10zeta over-expression. These results indicate that hGrb10zeta inhibits a novel and presently unidentified insulin signaling pathway leading to GS activation in liver.

Lipogenesis in cancer progression (Review)

In normal tissues, energy-providing lipids come principally from circulating lipids. However, in growing tumors, energy supply is mainly provided by lipids coming from de novo synthesis. It is not surprising to see elevated expression of several lipogenic genes in tumors from different origins. The role of lipogenic genes in the establishment of the primary tumor has been clearly established. A large number of studies demonstrate a role of fatty acid synthase in the activation of cell cycle and inhibition of apoptosis in tumor cells. Other lipogenic genes such as the acetyl CoA carboxylase (ACC) and the stearoyl CoA desaturase 1 (SCD1) are highly expressed in primary tumors and also appear to play a role in their development. However, the role of lipogenesis in the metastatic process is less clear. In the present review, we aim to present the most recent evidences for the key role of lipogenic enzymes in the metastatic process and in epithelial to mesenchymal transition.

Expression of Calbindin-D28k (CaBP28k) in Trophoblasts from Human Term Placenta

Abstract Calbindin-D28k (CaBP28k) belongs to a large class of eucaryotic proteins that bind calcium (Ca2+) to a specific helix-loop-helix structure. To date, this protein was mainly linked to brain, kidneys, and pancreas. Here, we demonstrate for the first time the existence of CaBP8k in the human placental trophoblasts of the human term placenta. Placental Ca2+ transfer from maternal to fetus is crucial for fetal development, although the biochemical mechanisms responsible for this process are largely unknown. In the current study, we have investigated the 45Ca2+ uptake by human trophoblast cells in correlation with the expression CaBP28k. The expression of CaBP28k was determined by Northern blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), immunochemistry, and Western blot analysis. Indeed, Northern blot analysis revealed the presence of a CaBP28k transcript in syncytiotrophoblasts, cytotrophoblast cells, and HEK-293 cells. This was further confirmed by RT-PCR analysis followed by sequencing. In addition, anti-CaBP28k labeling was associated with cytotrophoblast and syncytiotrophoblast tissues in placental tissue sections and in vitro cultured cells. The presence of CaBP28k protein in these cells was confirmed by Western blotting. Cytotrophoblast cells isolated from human term placenta showed differentiation into syncytiotrophoblasts in culture according to the increase in hCG secretion. Both Ca2+ uptake and hCG secretion by trophoblasts increased gradually and were high at Day 4. Taken together, these data suggest that CaBP28k may play a role in Ca2+ transport or cell development in human trophoblast possibly trough Ca2+ buffering.

Human apolipoprotein D overexpression in transgenic mice induces insulin resistance and alters lipid metabolism

Apolipoprotein D (apoD), a widely expressed lipocalin, has the capacity to transport small hydrophobic molecules. Although it has been proposed that apoD may have multiple tissue-specific, physiological ligands and functions, these have yet to be identified. To gain insight in some of its functions, we generated transgenic mice overexpressing human apoD (H-apoD) under the control of neuron-specific promoters. In Thy-1/apoD and NSE/apoD mice, expression of H-apoD was strong in the nervous system although weakly detected in peripheral organs such as the liver and blood cells. These mice displayed not entirely anticipated metabolic defects. Although they are not obese and have normal lipid concentration in circulation, Thy-1/apoD and NSE/apoD mice are glucose intolerant, insulin resistant, and develop hepatic steatosis. The steatosis and its associated insulin resistance are correlated with impairments in hepatic lipogenesis. However, they are not strongly related with inflammation. This impaired insulin response is not caused by a decrease in circulating leptin or a modulation of adiponectin and resistin levels. These results suggest that variations in the levels and/or sites of apoD expression influence the lipid and glucose metabolism, consolidating apoD as a target for insulin-resistance-related disorders.

Calbindin-D9k (CaBP9k) localization and levels of expression in trophoblast cells from human term placenta.

During pregnancy, the calcium (Ca2+) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca2+ transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca2+ in placental transport in vivo, we questioned whether calbindin-D9k (CaBP9k), which is mainly expressed in duodenum, uterus, and placenta of several mammals, is present in cytotrophoblast cells and syncytiotrophoblasts of human term placenta. We were interested in this protein because of its potential importance in serving as an indicator of Ca2+ availability and utilization in the placenta. Here, we demonstrated that CaBP9k transcript is present in both cell types, with a lower expression in cytotrophoblast cells as compared to syncytiotrophoblasts. Moreover, we showed by immunochemistry that CaBP9k protein was present in cytotrophoblast and syncytiotrophoblast placental tissue sections as well as in cultured cells. The occurrence of CaBP9k protein in trophoblast cells was further confirmed by Western blot analysis. Thus, these results indicate for the first time that CaBP9k is unequivocally expressed by trophoblast cells from human term placenta.