CB1 blockade potentiates down-regulation of lipogenic gene expression in perirenal adipose tissue in high carbohydrate diet-induced obesity.

De novo lipogenesis and hypercaloric diets are thought to contribute to increased fat mass, particularly in abdominal fat depots. CB1 is highly expressed in adipose tissue, and CB1-mediated signalling is associated with stimulation of lipogenesis and diet-induced obesity, though its contribution to increasing fat deposition in adipose tissue is controversial. Lipogenesis is regulated by transcription factors such as liver X receptor (LXR), sterol-response element binding protein (SREBP) and carbohydrate-responsive-element-binding protein (ChREBP). We evaluated the role of CB1 in the gene expression of these factors and their target genes in relation to lipogenesis in the perirenal adipose tissue (PrAT) of rats fed a high-carbohydrate diet (HCHD) or a high-fat diet (HFD). Both obesity models showed an up-regulated gene expression of CB1 and Lxrα in this adipose pad. The Srebf-1 and ChREBP gene expressions were down-regulated in HFD but not in HCHD. The expression of their target genes encoding for lipogenic enzymes showed a decrease in diet-induced obesity and was particularly dramatic in HFD. In HCHD, CB1 blockade by AM251 reduced the Srebf-1 and ChREBP expression and totally abrogated the remnant gene expression of their target lipogenic enzymes. The phosphorylated form of the extracellular signal-regulated kinase (ERK-p), which participates in the CB1-mediated signalling pathway, was markedly present in the PrAT of obese rats. ERK-p was drastically repressed by AM251 indicating that CB1 is actually functional in PrAT of obese animals, though its activation loses the ability to stimulate lipogenesis in PrAT of obese rats. Even so, the remnant expression levels of lipogenic transcription factors found in HCHD-fed rats are still dependent on CB1 activity. Hence, in HCHD-induced obesity, CB1 blockade may help to further potentiate the reduction of lipogenesis in PrAT by means of inducing down-regulation of the ChREBP and Srebf-1 gene expression, and consequently in the expression of lipogenic enzymes.

The biochemistry of drug metabolism-an introduction: part 7. Intra-individual factors affecting drug metabolism.

This review on intra-individual factors affecting drug metabolism completes our series on the biochemistry of drug metabolism. The article presents the molecular mechanisms causing intra-individual differences in enzyme expression and activity. They include enzyme induction by transcriptional activation and enzyme inhibition on the protein level. The influencing factors are of physiological, pathological, or external origin. Tissue characteristics and developmental age strongly influence enzyme-expression patterns. Further influencing factors are pregnancy, disease, or biological rhythms. Xenobiotics, drugs, constituents of herbal remedies, food constituents, ethanol, and tobacco can all influence enzyme expression or activity and, hence, affect drug metabolism.

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

A-kinase anchoring proteins: scaffolding proteins in the heart.

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called "A-kinase anchoring proteins" (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.

Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors.

Three novel members of the Xenopus nuclear hormone receptor superfamily have been cloned. They are related to each other and similar to the group of receptors that includes those for thyroid hormones, retinoids, and vitamin D3. Their transcriptional activity is regulated by agents causing peroxisome proliferation and carcinogenesis in rodent liver. All three Xenopus receptors activate the promoter of the acyl coenzyme A oxidase gene, which encodes the key enzyme of peroxisomal fatty acid beta-oxidation, via a cognate response element that has been identified. Therefore, peroxisome proliferators may exert their hypolipidemic effects through these receptors, which stimulate the peroxisomal degradation of fatty acids. Finally, the multiplicity of these receptors suggests the existence of hitherto unknown cellular signaling pathways for xenobiotics and putative endogenous ligands.

Evidence for tuning adipocytes ICER levels for obesity care.

Abnormal adipokine production, along with defective uptake and metabolism of glucose within adipocytes, contributes to insulin resistance and altered glucose homeostasis. Recent research has highlighted one of the mechanisms that accounts for impaired production of adiponectin (ADIPOQ) and adipocyte glucose uptake in obesity. In adipocytes of human obese subjects and mice fed with a high fat diet, the level of the inducible cAMP early repressor (ICER) is diminished. Reduction of ICER elevates the cAMP response element binding protein (CREB) activity, which in turn increases the repressor activating transcription factor 3. In fine, the cascade triggers reduction in the ADIPOQ and GLUT4 levels, which ultimately hampers insulin-mediated glucose uptake. The c-Jun N-terminal kinase (JNK) interacting-protein 1, also called islet brain 1 (IB1), is a target of CREB/ICER that promotes JNK-mediated insulin resistance in adipocytes. A rise in IB1 and c-Jun levels accompanies the drop of ICER in white adipose tissues of obese mice when compared with mice fed with a chow diet. Other than the expression of ADIPOQ and glucose transport, decline in ICER expression might impact insulin signaling. Impairment of ICER is a critical issue that will need major consideration in future therapeutic purposes.

Transcriptional regulation of MDR1, a gene involved in antifungal drug resistance in Candida albicans

ABSTRACT Upregulation of the Major Facilitator transporter gene MDR1 (Multi_drug Resistance 1) is one of the mechanisms observed in Candida albicans clinical isolates developing resistance to azole antifungal agents. To better understand this phenomenon, the cis-acting regulatory elements present in a modulatable reporter system under the control of the MDR1 promoter were characterized. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H2O2, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements, that are necessary and sufficient to convey the same transcriptional responses to a heterologous promoter (CDR2), were identified within the MDR1promoter. The first element, called BRE (for Benomyl Response Element, -296 to -260 with respect to the ATG start codon), is required for benomyl-dependent MDR1 upregulation and for constitutive high expression of MDR1 in FR2. The second element, termed HRE (for H2O2 Response Element, -561 to -520), is required for H2O2-dependent MDR1 upregulation, but is dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the blip transcription factor Cap1p lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H2O2 and diminished significantly the transient response to benomyl. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a transacting and positive regulatory role in benomyl- and H2O2-dependent transcription of MDR1. However, it is not the only transcription factor involved in the response of MDR1 to benomyl. A minimal BRE element (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also delimited. Genome-wide transcript profiling analyses undertaken with a matched pair of clinical isolates, one of which being azole-resistant and upregulating MDR1, and with an azole-susceptible strain exposed to benomyl, revealed that genes specifically upregulated by benomyl harbour in their promoters Cap1p binding site(s). This strengthened the idea that Cap1p plays a role in benomyl-dependent upregulation of MDR1. BRE-like sequences were also identified in several genes co-regulated with MDR1 in both conditions, which was consistent with the involvement of the BRE in both processes. A set of 147 mutants lacking a single transcription factor gene was next screened for loss of MDR1response to benomyl. Unfortunately, none of the tested mutants showed a loss of benomyl-dependent MDR1 upregulation. Nevertheless, a significant diminution of the response was observed in the mutants in which the MADS-box transcription factor Mcm1p and the C2H2 zinc finger transcription factor orf19.13374p were inactivated, suggesting that Mcm1p and orf19.13374p are involved in MDR1response to benomyl. Interestingly, the BRE contains a perfect match to the binding consensus of Mcm1p, raising the possibility that MDR1may be a direct target of this transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools we have developed during characterization of the cis-acting elements of the MDR1promoter should now serve to elucidate the nature of the components that modulate its activity. RESUME La surexpression du gène MDR1 (pour Résistance Multidrogue 1), qui code pour un transporteur de la famille des Major Facilitators, est l'un des mécanismes observés dans les isolats cliniques de la levure Candida albicans développant une résistance aux agents antifongiques appelés azoles. Pour mieux comprendre ce phénomène, les éléments de régulation agissant en cis dans un système rapporteur modulable sous le contrôle du promoteur MDR1 ont été caractérisés. Dans une souche sensible aux azoles, la transcription de ce rapporteur est transitoirement surélevée en réponse soit au bénomyl soit à l'agent oxydant H2O2, alors que son expression est constitutivement élevée dans une souche résistante aux azoles (souche FR2). Deux éléments de régulation agissant en cis, nécessaires et suffisants pour transmettre les mêmes réponses transcriptionnelles à un promoteur hétérologue (CDR2), ont été identifiés dans le promoteur MDR1. Le premier élément, appelé BRE (pour Elément de Réponse au Bénomyl, de -296 à -260 par rapport au codon d'initiation ATG) est requis pour la surexpression de MDR1dépendante du bénomyl et pour l'expression constitutive de MDR1 dans FR2. Le deuxième élément, appelé HRE (pour Elément de Réponse à l'H2O2, de -561 à -520), est requis pour la surexpression de MDR1 dépendante de l'H2O2, mais n'est pas impliqué dans l'expression constitutive du gène MDR1. Deux sites de fixation potentiels (TTAG/CTAA) pour le facteur de transcription Cap1p ont été identifiés dans l'élément HRE. De plus, l'inactivation de CAP1 abolit la réponse transitoire à l'H2O2 et diminua significativement la réponse transitoire au bénomyl. Cap1p, qui est impliqué dans les réponses de la cellule au stress oxydatif, doit donc jouer un rôle positif en trans dans la surexpression de MDR1 dépendante du bénomyl et de l'H2O2. Cependant, ce n'est pas le seul facteur de transcription impliqué dans la réponse au bénomyl. Un élément BRE d'une longueur minimale (de -290 à -273) a également été défini et est suffisant pour détecter une interaction spécifique in vitro avec des protéines provenant d'extraits bruts de C. albicans. L'analyse du profil de transcription d'une paire d'isolats cliniques comprenant une souche résistante aux azoles surexprimant MDR1, et d'une souche sensible aux azoles exposée au bénomyl, a révélé que les gènes spécifiquement surexprimés par le bénomyl contiennent dans leurs promoteurs un ou plusieurs sites de fixation pour Cap1p. Ceci renforce l'idée que Cap1p joue un rôle dans la surexpression de MDR1dépendante du bénomyl. Une ou deux séquences ressemblant à l'élément BRE ont également été identifiées dans la plupart des gènes corégulés avec MDR1 dans ces deux conditions, ce qui était attendu compte-tenu du rôle joué par cet élément dans les deux processus. Une collection de 147 mutants dans lesquels un seul facteur de transcription est inactivé a été testée pour la perte de réponse au bénomyl de MDR1. Malheureusement, la surexpression de MDR1 dépendante du bénomyl n'a été perdue dans aucun des mutants testés. Néanmoins, une diminution significative de la réponse a été observée chez des mutants dans lesquels le facteur de transcription à MADS-box Mcm1p et le facteur de transcription à doigts de zinc de type C2H2 orf19.13374p ont été inactivés, suggérant que Mcm1p et orf19.13374p sont impliqués dans la réponse de MDR1au bénomyl. Il est intéressant de noter que la BRE contient une séquence qui s'aligne parfaitement avec la séquence consensus du site de fixation de Mcm1p, ce qui soulève la possibilité que MDR1 pourrait être une cible directe de cet activateur transcriptionnel. En conclusion, alors que l'identité des facteurs agissant en trans en se fixant à la BRE et à la HRE reste à être confirmée, les outils que nous avons développés au cours de la caractérisation des éléments agissant en cis sur le promoteur MDR1 peut maintenant servir à élucider la nature des composants modulant son activité.

Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR).

Peroxisome proliferators regulate the transcription of genes by activating ligand-dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPAR alpha and beta but not xPPAR gamma are expressed in oocytes and embryos. In the adult, expression of xPPAR alpha and beta appears to be ubiquitous, and xPPAR gamma is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic-nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in the peroxisomal beta-oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the promoter of the ACO gene. Besides the known xenobiotic activators of PPARs, such as hypolipidemic drugs, natural activators have been identified. Polyunsaturated fatty acids at physiological concentrations are efficient activators of PPARs, and 5,8,11,14-eicosatetraynoic acid (ETYA), which is the alkyne homolog of arachidonic acid, is the most potent activator of xPPAR alpha described to date. Taken together, our data suggest that PPARs have an important role in lipid metabolism.

Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites.

Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.

Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling.

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.

Peroxisome proliferator-activated receptors: finding the orphan a home.

The peroxisome proliferator-activated receptor (PPAR) is a member of the steroid hormone receptor superfamily and is activated by a variety of fibrate hypolipidaemic drugs and non-genotoxic rodent hepatocarcinogens that are collectively termed peroxisome proliferators. A key marker of peroxisome proliferator action is the peroxisomal enzyme acyl CoA oxidase, which is elevated about ten fold in the livers of treated rodents. Additional peroxisome proliferator responsive genes include other peroxisomal beta-oxidation enzymes and members of the cytochrome P450 IVA family. A peroxisome proliferator response element (PPRE), consisting of an almost perfect direct repeat of the sequence TGACCT spaced by a single base pair, has been identified in the upstream regulatory sequences of each of these genes. The retinoid X receptor (RXR) forms a heterodimer with PPAR and binds to the PPRE. Furthermore, the RXR ligand, 9-cis retinoic acid, enhances PPAR action. Retinoids may therefore modulate the action of peroxisome proliferators and PPAR may interfere with retinoid action, perhaps providing one mechanism to explain the toxicity of peroxisome proliferators. Interestingly, a variety of fatty acids can activate PPAR supporting the suggestion that fatty acids, or their acyl CoA derivatives, may be the natural ligands of PPAR and that the physiological role of PPAR is to regulate fatty acid homeostasis. Taken together, the discovery of PPAR has opened up new opportunities in understanding how lipid homeostasis is regulated, how the fibrate hypolipidaemic drugs may act and should lead to improvements in the assessment of human risk from peroxisome proliferators based upon a better understanding of their mechanism of action.

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models.

Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Glycogen synthase 2 is a novel target gene of peroxisome proliferator-activated receptors.

Glycogen synthase 2 (Gys-2) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue, yet little is known about regulation of Gys-2 transcription. The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well. Here, we show that Gys-2 is a direct target gene of PPARalpha, PPARbeta/delta and PPARgamma. Expression of Gys-2 is significantly reduced in adipose tissue of PPARalpha-/-, PPARbeta/delta-/- and PPARgamma+/- mice. Furthermore, synthetic PPARbeta/delta, and gamma agonists markedly up-regulate Gys-2 mRNA and protein expression in mouse 3T3-L1 adipocytes. In liver, PPARalpha deletion leads to decreased glycogen levels in the refed state, which is paralleled by decreased expression of Gys-2 in fasted and refed state. Two putative PPAR response elements (PPREs) were identified in the mouse Gys-2 gene: one in the upstream promoter (DR-1prom) and one in intron 1 (DR-1int). It is shown that DR-1int is the response element for PPARs, while DR-1prom is the response element for Hepatic Nuclear Factor 4 alpha (HNF4alpha). In adipose tissue, which does not express HNF4alpha, DR-1prom is occupied by PPARbeta/delta and PPARgamma, yet binding does not translate into transcriptional activation of Gys-2. Overall, we conclude that mouse Gys-2 is a novel PPAR target gene and that transactivation by PPARs and HNF4alpha is mediated by two distinct response elements.