Gemma Solanes

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

Ligand-independent Activation Domain in the N Terminus of Peroxisome Proliferator-activated Receptor γ (PPARγ) DIFFERENTIAL ACTIVITY OF PPARγ1 AND -2 ISOFORMS AND INFLUENCE OF INSULIN

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear hormone receptor superfamily, and is an important regulator of adipogenesis and adipocyte gene expression. PPARγ exists as two isoforms, PPARγ1 and PPARγ2, that differ only in their N termini. Both isoforms are activated by ligands that include the antidiabetic thiazoladinedione drugs and 15-deoxy-Δ12, 14-prostaglandin J2, and potential differences in their function have yet to be described. We report that, in addition to a ligand-activated transcriptional activity, when studied under conditions of ligand depletion, intact PPARγ has a ligand-independent activation domain. To identify the basis for this ligand-independent activation, we used GAL4-PPARγ chimeric expression constructs and UAS-TK-LUC in CV1 cells and isolated rat adipocytes. In both cell systems, isolated PPARγ1 and PPARγ2 N termini have activation domains, and the activation function of PPARγ2 is 5–6-fold greater than that of PPARγ1. Insulin enhances the transcriptional effect mediated by both PPARγ1 and PPARγ2 N-terminal domains. These data demonstrate that 1) PPARγ has an N-terminal (ligand-independent) activation domain; 2) PPARγ1 and PPARγ2 N termini have distinct activation capacities; and 3) insulin can potentiate the activity of the N-terminal domain of PPARγ.

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, UCP3is expressed preferentially and at high levels in human skeletal muscle and exists as short and long form transcripts,UCP3 S and UCP3 L.UCP3 S is predicted to encode a protein which lacks the last 37 C-terminal residues of UCP3 L. In the present study, we have defined the intron-exon structure for the human UCP3 gene and determined thatUCP3 S is generated when a cleavage and polyadenylation signal (AATAAA) located in the last intron prematurely terminates message elongation. In addition we have mappedUCP3 to the distal segment of human chromosome 11q13 (between framework markers D11S916 and D11S911), adjacent toUCP2. 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 thatUCP3 is also coincident with these quantitative trait loci raising the possibility that abnormalities in UCP3 are responsible for obesity in these models.

The human uncoupling protein-3 gene promoter requires MyoD and is induced by retinoic acid in muscle cells

The uncoupling protein‐3 (UCP‐3) gene encodes for a mitochondrial protein expressed preferentially in skeletal muscle. UCP‐3 mRNA is expressed in cultured muscle cells (C2C12 or L6E9) only when differentiated, at which stage UCP‐3 is highly induced by all‐trans retinoic acid (RA). Here we report that human UCP‐3 promoter activity is dependent on MyoD and inducible by all trans‐RA. The action of all trans‐RA is increased by co‐transfection with RA receptor (RAR). We have characterized the RA response element that controls the induction by RA in the 5′ noncoding region of the UCP‐3 gene. Deletion and point‐mutation analysis of the hUCP‐3 promoter led us to identify a direct‐repeat element with one base‐pair spacing (DR1) at position −71/−59 responsible for the induction by RA of the activity of the promoter. This DR1 element bound a nuclear protein complex from muscle cells that contain RAR and retinoid X receptor (RXR). In the absence of this element, the promoter became unresponsive to RA, but it was still dependent on MyoD. In conclusion, it has been established that UCP‐3 gene promoter activity is dependent on MyoD, and the first regulatory pathway for UCP‐3 gene promoter regulation has been recognized by identifying RA as a transcriptional activator of the gene.

Thyroid hormones directly activate the expression of the human and mouse uncoupling protein-3 genes through a thyroid response element in the proximal promoter region

The transcription of the human UCP3 (uncoupling protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo. The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo, and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle.

Differential regulation of expression of genes encoding uncoupling proteins 2 and 3 in brown adipose tissue during lactation in mice.

Thermogenic activity in brown adipose tissue (BAT) decreases during lactation; the down-regulation of the gene encoding uncoupling protein 1 (UCP1) is involved in this process. Our studies show that UCP2 mRNA expression does not change during the breeding cycle in mice. In contrast, UCP3 mRNA is down-regulated in lactation but it recovers after weaning, in parallel with UCP1 mRNA. This leads to a decrease in the content of UCP3 in BAT mitochondria during lactation. Lowering the energy-sparing necessities of lactating dams by decreasing litter size or feeding with a high-fat diet prevented the down-regulation of UCP1 mRNA and UCP3 mRNA. In most cases this resulted in a less marked decrease in UCP1 and UCP3 protein in BAT mitochondria owing to lactation. Fasting for 24 h caused a different response in UCP1 and UCP3 mRNA expression: it decreased UCP1 mRNA levels but had no effect on UCP3 mRNA abundance in virgin mice; it even increased UCP3 mRNA expression in lactating dams. These changes did not lead to modifications in UCP1 or UCP3 protein abundance. Whereas acute treatment with peroxisome-proliferator-activated receptor (PPAR)alpha and PPARgamma agonists increased UCP1 mRNA levels only in lactating dams, UCP3 mRNA expression was induced by both kinds of PPAR activator in lactating dams and by PPARalpha agonists in virgin mice. It is concluded that modifications of UCP2 mRNA levels are not part of the physiological adaptations taking place in BAT during lactation. In contrast, the down-regulation of UCP3 mRNA expression and mitochondrial UCP3 content is consistent with a role for the gene encoding UCP3 in the decrease in metabolic fuel oxidation and thermogenesis in BAT during lactation.

Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis†

The mitochondrial uncoupling protein‐3 is a member of the mitochondrial carrier protein family. As a homologue of the thermogenic brown fat uncoupling protein‐1, it possesses a mitochondrial uncoupling activity and thus can influence cell energy metabolism but its exact biological function remains unclear. In the present study, uncoupling protein‐3 was expressed in 293 cells using the tetracycline‐inducible system and its impact on cell bioenergetics and responsiveness to the apoptotic stimulus was determined. The induction of uncoupling protein‐3 expression in mitochondria did not lead to uncontrolled respiratory uncoupling in intact cells. However, it caused a GDP‐inhibition of state 4 respiration and a GDP‐induced re‐polarization of the inner mitochondrial membrane in the presence of fatty acids, in agreement with its expected physiological behavior as an uncoupling protein (UCP). Uncoupling protein‐3 expression did not cause apoptosis per se but increased the responsiveness of the cells to a mitochondrial apoptotic stimulus (i.e., addition of staurosporine in the culture medium). It enhanced caspase 3 and caspase 9 activation and favored cytochrome c release. Moreover, cells in which uncoupling protein‐3 expression had been induced showed a higher mitochondrial Bax/Bcl‐2 ratio essentially due to enhanced translocation of Bax from cytosol to mitochondria. Finally, the induction of uncoupling protein‐3 also increased the sensitivity of mitochondria to open the permeability transition pore in response to calcium. It is concluded that the presence of uncoupling protein‐3 in mitochondria sensitizes cells to apoptotic stimuli involving mitochondrial pathways.