Microarray analysis during adipogenesis identifies new genes altered by antiretroviral drugs
Pacenti, Moniaa,1; Barzon, Luisaa,1; Favaretto, Francescaa,b; Fincati, Karinaa,c; Romano, Sarab; Milan, Gabriellab; Vettor, Robertob; Palù, Giorgioa
From the aDepartment of Histology, Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy
bDepartment of Medical and Surgical Sciences, University of Padova, Padova, Italy
cCIVEN (Coordinamento Interuniversitario Veneto per le Nanotecnologie), Mestre, Venezia, Italy.
1Note: Monia Pacenti and Luisa Barzon equally contributed to this work.
Received 22 November, 2005
Revised 6 January, 2006
Accepted 12 January, 2006
Correspondence to L. Barzon and G. Palù, Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Via A. Gabelli 63, I-35121 Padova, Italy. Tel: +39 049 8218946; fax: +39 049 8272355; e-mail: email@example.com; firstname.lastname@example.org
Objective: To elucidate the pathogenesis of HAART-associated lipodystrophy, by investigating the effects of antiretroviral drugs on adipocyte differentiation and gene expression profile.
Design and methods: Analysis of gene expression profile by DNA microarrays and quantitative RT–PCR of 3T3-L1 preadipocytes treated with the nucleoside reverse transcriptase inhibitors (NRTI) lamivudine, zidovudine, stavudine, and zalcitabine, and with the protease inhibitors (PI) indinavir, saquinavir, and lopinavir during maturation into adipocytes.
Results: Under standard adipogenic differentiation protocols, PI significantly inhibited adipocyte differentiation, as demonstrated by cell viability assay and Oil Red O staining and quantification, whereas NRTI had mild effects on adipogenesis. Gene expression profile analysis showed that treatment with NRTI modulated the expression of transcription factors, such as Aebp1, Pou5f1 and Phf6, which could play a key role in the determination of the adipocyte phenotype. PI also modulated gene expression toward inhibition of adipocyte differentiation, with up-regulation of the Wnt signaling gene Wnt10a and down-regulation of the expression of genes encoding master adipogenic transcription factors (e.g., C/EBPα and PPARγ), oestrogen receptor β, and adipocyte-specific markers (e.g., Adiponectin, Leptin, Mrap, Cd36, S100A8).
Conclusions: This study identifies new genes modulated by PI and NRTI in differentiating adipocytes. Abnormal expression of these genes, which include master adipogenic transcription factors and genes involved in lipid metabolism and cell cycle control, could contribute to the understanding of the pathogenesis of HAART-associated lipodystrophy.
HAART, a treatment consisting of a combination of protease inhibitors (PI), nucleoside analogue reverse transcriptase inhibitors (NRTI), and non-NRTI, is associated with severe metabolic side-effects, including lipodystrophy . The mechanism at the basis of HAART-associated lipodystrophy seems to involve dysregulation of fat cell growth and differentiation by both PI and NRTI [1–4]. In this study, we used microarray analysis to identify genes that are modulated by PI and NRTI in differentiating 3T3-L1 preadipocytes, a widely used in vitro model of adipogenesis .
3T3-L1 cells (clone 173, American Type Culture Collection, Rockville, Maryland, USA) were grown and differentiated into mature adipocytes according standard protocols and treated with NRTI and PI during differentiation (see Figs 1 and 2). Adipogenic differentiation and lipid content were measured by lipid-specific Oil-Red O staining. Gene expression profile of treated versus untreated 3T3-L1 cells was evaluated by DNA microarray analysis and quantitative real-time RT–PCR (See Fig. 2). For microarray analysis, polyA+ RNA was isolated from 3T3-L1 cells using Micro-Fast Track™ 2.0 Kit (Invitrogen, Milan, Italy) according to the manufacturer's instructions. For each sample, 3 μg polyA+ RNA was reverse transcribed and labelled using SuperScript II reverse transcriptase (Invitrogen), oligodT primers, and Cy3-dCTP or Cy5-dCTP dyes (Amersham Biosciences, Milan, Italy). Labelled cDNA probes were hybridized to microarray glass slides produced by CRIBI core facility (University of Padova, Italy), which contained 1 344 370 mer oligonucleotides (Operon version 1.1, designed on Mouse Unigene clusters). After hybridization, slides were washed and scanned on a Affymetrix 428™ Array Scanner (MWG-Biotech AG, Ebersberg, Germany) and the images analyzed by ImaGene 5.6 and GeneSight 4.1.6 softwares (BioDiscovery, El Segundo, California, USA). Data analysis included hierarchical clustering to group experimental conditions according to expression profile, differential regulation analysis to determine the significance of average change in expression (ratio of drug-treatment/control), and t test to determine the significance of differences between different experimental conditions. P < 0.005 was taken as significant. Microarray experiments were performed in duplicate and repeated two times. Each sample was analyzed in duplicate, including reciprocal labeling of fluorochromes.
Results and discussion
Differentiation and proliferation analysis
Treatment of 3T3-L1 cells during differentiation with the NRTI zidovudine (ZDV) 10 μM and lamivudine (3TC) 20 μM, either alone or in combination, did not significantly alter cell viability and adipogenesis, as evaluated by counting the number of cells and the number adipocytes upon lipid staining (Fig. 1). At variance, treatment with the PI indinavir (IDV) 20 μM and saquinavir (SQV) 20 μM, either alone or in combination with NRTI, significantly reduced the number of differentiated cells, without any evident toxic effect, assayed by total cell counting. This effect was more evident with SQV than with IDV and in late phases of differentiation. In fact, on day 6 and 10, treatment with SQV and IDV reduced by 40% and 30%, respectively, the number of differentiated cells, which were characterized by the presence of smaller lipid droplets than control cells (Fig. 1a). A significant reduction in lipid accumulation of PI-treated 3T3-L1 cells was confirmed by spectrophotometric measurement upon Oil Red O staining (Fig. 1b). The stronger effect of PI than NRTI on adipocyte differentiation could in part be explained by the strong protein binding of PI in vivo, at variance with NRTI, which are not highly protein bound , and therefore the higher concentration of active PI drug in vitro.
Analysis of gene expression profile during adipocyte differentiation
To identify genes that were modulated in the process of adipocyte differentiation, the gene expression profile of 3T3-L1 cells grown in the presence of the differentiation medium was compared with 3T3-L1 preadipocytes grown in medium without supplementation of adipogenic factors. On day 3 of differentiation (early adipogenesis), a total of 1091 genes (8.1% of the expressed genes) were significantly altered as compared to 3T3-L1 preadipocytes. Up-regulated genes encoded adipocyte-specific transcription factors, enzymes involved in lipid and carbohydrate metabolism and adipocytokines, whereas down-regulated genes encoded mainly proteins of the extracellular matrix and cytoskeleton. On day 10 of differentiation (late adipogenesis), a total of 1084 genes were up- or down-regulated with respect to preadipocytes. Over-expressed genes included those encoding adipose tissue-specific marker and adipocytokines, such as aP2, angiotensinogen, haptoglobin, adipsin, adiponectin, CD36 antigen, and fat-specific gene 27. Under-expressed genes included those encoding Wnt signaling molecules, extracellular matrix proteins, and proteins expressed in fibroblasts (e.g., Wnt1 inducible signaling pathway proteins 1 and 2, high mobility group proteins, alpha actin, tissue inhibitor of metalloproteinase, stromal cell derived factor 1). A list of genes modulated during adipogenesis is given in Table 1.
Gene expression profile of 3T3-L1 cells treated with antiretroviral drugs during adipogenic differentiation
Microarray analysis was subsequently performed to investigate the effect of antiretroviral drug combinations on gene expression profile of differentiating 3T3-L1 cells. Since the in vitro effect of antiretroviral drugs on gene expression of 3T3-L1 cells has been shown to occur mainly in the early phases of differentiation [2,7], the time point corresponding to day 3 of differentiation was chosen for microarray experiments. To analyse the effect of NRTI, 3T3-L1 cells were induced to differentiate in the presence or absence of ZDV 10 μM and 3TC 20 μM, whereas to analyse the effect of the combination of two NRTI with a PI, 3T3-L1 cells were grown in the presence of ZDV 10 μM and 3TC 20 μM, associated with either IDV 20 μM or SQV 20 μM. For each treatment protocol, the gene expression profile of drug-treated 3T3-L1 cells was compared with that of untreated (i.e., cultured in the same differentiation medium without antiretroviral drugs) 3T3-L1 cells at day 3 of differentiation. To analyse the global cellular genetic responses after the various treatments, microarray data were subjected to hierarchical clustering analysis, which showed that gene expression profiles of cells treated with the two combinations of PI–NRTI were quite similar, but markedly distinct from that of treatment with only NRTI. Moreover, drug combinations including a PI had a greater impact on cellular gene expression profile than treatment with NRTI, as suggested by the higher number of up- and down-regulated genes. In fact, when 3T3-L1 cells were treated with the combination of NRTI, a total of 58 genes were differentially regulated at 99% confidence level, whereas when cells were treated with the NRTI and IDV or SQV, the number of differentially regulated genes increased to 98 and 132, respectively. Of these genes, 44 were differently regulated and had the same expression pattern in all three treatment conditions and 26 were differently regulated in the two PI-including treatment conditions, but not in the treatment condition including only NRTI.
Genes modulated by NRTI during 3T3-L1 early adipogenic differentiation
Of the 44 genes significantly modulated by NRTI, both alone and in combination with a PI, 32 were up-regulated by treatment, whereas 12 were inhibited (Table 2). Induced genes included transcription regulators and genes involved in signal transduction. Some of these genes showed increased expression during adipogenesis and were further enhanced by antiretroviral drugs, but others, such as Aebp-1 (adipocyte enhancer-binding protein 1) and Timp-2 (tissue inhibitor of metalloproteinase 2), were found to be repressed upon adipocyte differentiation, but markedly induced by treatment with NRTI. Aebp1 was originally characterized as a repressor of transcription of the adipocyte fatty acid binding protein gene aP2 , an important marker of adipocyte late differentiation. Aebp1 seems to correspond to aortic carboxypeptidase-like protein, i.e, a secreted protein associated with the extracellular matrix whose expression is induced during smooth muscle differentiation . Over-expression of this protein in preadipocytes has been reported to inhibit adipogenesis [10,11] and to promote preadipocyte transdifferentiation into smooth muscle-like cells , even though this finding has not been confirmed by other authors . Tissue inhibitors of metalloproteinase are a family of four secreted proteins (TIMP-1 to TIMP-4) that selectively inhibit matrix metalloproteinases. The proteolytic activity of matrix metalloproteinases has been hypothesized to play a critical role in the early step of adipocyte differentiation, as suggested by their differential regulation in adipose tissue and by the demonstration that inhibitors of matrix metalloproteinases decrease C/EBPβ expression and adipocyte differentiation .
Down-regulated genes included Faim (fas apoptotic inhibitory molecule), a negative regulator of apoptosis ; genes encoding transcription factors, such as Pou5f1 and Phf6; and genes encoding lipid metabolizing enzymes up-regulated in mature adipocytes, such as Hadh and Elovl3. Pou5f1 encodes a POU transcription factor expressed by early embryo cells and germ cells, whose activity is essential for maintaining pluripotency in embryonic stem cells . Phf6 encodes a novel zinc finger gene of unknown function. Mutations of the corresponding human gene are responsible of the Borjeson–Forssman–Lehmann syndrome (BFLS; OMIM 301900), a X-linked disorder of intellectual disability, characteristic craniofacial features, hypogonadism, hypometabolism, obesity with marked gynecomastia, swelling of subcutaneous tissue of the face, short stature and epilepsy . Hadh, which encodes hydroxylacyl–coenzyme A dehydrogenase, is a mitochondrial enzyme that catalyses a step of fatty acid β-oxidation. Over-expression of this enzyme allows the mitochondria to maintain a high rate of oxidative phosphorylation and production of ATP, whereas its down-regulation may determine cell death . The Elovl3 gene belongs to the Elovl gene family coding for microsomal enzymes involved in elongation of very long chain fatty acids. Its expression is dramatically increased in mouse brown adipose tissue upon cold stimulation and this induction is under the control of PPARα, whereas it is inhibited by LXR agonists and SREBP-1 activation .
Some genes were modulated only by NRTI treatment, but not by treatment conditions including PI. Up-regulated genes included Bid3, an important inductor of apoptosis through the regulation of mitochondrial function and caspase-3 activation , whereas inhibited genes included the transcription factor Tcfcp2l3. An inactivating mutation of the corresponding human gene TFCP2L3 was identified in a large family with an autosomal dominant form of progressive non-syndromic sensorineural hearing loss .
Genes modulated by PI during 3T3-L1 early adipogenic differentiation
Of the 26 genes significantly modulated by both treatment conditions including NRTI and a PI, but not by NRTI alone, 14 were up-regulated by treatment, whereas 12 were inhibited (Table 2). In particular, treatment with PI markedly induced the transcription factor Prdm1/Blimp1. Prdm1/Blimp1 is a transcriptional repressor with Krüppel-type zinc fingers which has been demonstrated to be essential for B-cell development  and, recently, also to play a critical role in the specification of mouse primordial germ cells and in the regulation of cell fate specification and morphogenetic processes [22,23]. Other genes induced by PI included those encoding extracellular matrix proteins (i.e., laminin gamma 2 and decorin), proteases (i.e., complement component 1r), and proliferins 1–3, which were found to be repressed in differentiated adipocytes. PI also induced expression of Δ-6 fatty acid desaturase (Fad2), a key enzyme in the biosynthesis of polyunsaturated fatty acids, such as arachidonic acid and decosahexaenoic acid, which is expressed in nearly all human tissues . Transcription of Fad2 is induced by SREBP-1c and PPARα ligand activators [25,26], but suppressed by polyunsaturated fatty acids via inhibition of SREBP-1c [25,26] and by PPARγ agonists . Increased Δ-6 desaturase activity has been associated with insulin resistance . Of note is that PI dramatically induced expression of Wnt10a (wingless related MMTV integration site 10a) and repressed Lef1 (lymphoid enhancer factor 1), which are both involved in Wnt signaling pathway. Activation of the Wnt signaling pathway has been shown to inhibit the differentiation of 3T3-L1 preadipocytes by preventing the induction of C/EBPα and PPARγ , to block the development of white and brown adipose tissue and, in differentiated brown adipocytes, to promote their conversion to white adipocytes . Wnt10a encodes a secreted signaling protein and has been demonstrated to be over-expressed in preadipocytes with decreased ability to differentiate into mature brown adipocytes . The effect of Wnt signaling on adipogenesis has been shown to be mediated by both β-catenin-dependent and β-catenin-independent mechanisms . LEF1 and the other members of this family of nuclear transcription factors, in response to Wnt signals, associate with β-catenin and activate Wnt-responsive target genes . Analysis of Lef1-deficient mice indicated that LEF1 may have a function in epithelium-to-mesenchyme signaling networks. In fact, targeted inactivation of Lef1 resulted in a complete block of development of multiple ectodermal appendages, such as teeth, vibrissae, hair, and mammary glands [33,34]. Our results show that Lef1 expression is induced during adipogenesis and inhibited by PI, thus suggesting a role in adipocyte differentiation. In the Lef1-deficient mouse model, Wnt10a is expressed independently of Lef1 in the dental epithelium , in agreement with the discordant expression pattern observed in our study in adipocytes. In addition to Lef1, PI down-regulated expression of several other genes typically expressed in differentiated adipocytes and mainly involved in lipid metabolism, such as Fsp27 (fat specific gene 27), Lep (leptin), Adn (adipsin), Adipoq (adiponectin), and Pfkfb3 (inducible 6-phosphofructo-2-kinase), and already known to be inhibited by PI [2,7], as well as other genes induced during adipogenesis, but less well characterized, such as Mrap, Cd36/FAT, Hist1h4i, G0s2, and S100a8. Mrap encodes melanocortin 2 receptor accessory protein, which has been recently identified as an interacting partner of the ACTH receptor MC2R and has been supposed to have a role in the trafficking MC2R from the endoplasmic reticulum to the cell surface . MRAP was first identified as a protein that is up-regulated upon differentiation of 3T3-L1 cells into adipocytes . Interestingly, Mc2r is also up-regulated in 3T3-L1 cells during differentiation via PPARγ and mediates the lipolytic effects of ACTH . The G0/G1 switch gene (G0s2) is involved in cell cycle regulation and has a temporal pattern of expression similar to that of Mrap, being induced during adipogenesis and further increased by PPARγ agonists . CD36/FAT expression is also induced by PPARγ during adipocyte differentiation . CD36/FAT mediates the uptake and accumulation of lipids in macrophages, adipose tissue and skeletal muscle  and its deficiency has been associated with dyslipidaemia and insulin resistance [41,42]. The role of CD36 in the pathogenesis of HAART-associated dyslipidaemia has already been investigated but with opposing findings. In fact, CD36 expression in circulating monocytes of HIV-infected patients treated with antiretroviral therapy including a PI has been reported to be reduced in a study , but increased in a more recent investigation in a larger population of patients . Similarly, PI have been demonstrated to both inhibit  and induce  CD36 expression in different human cell lines in vitro. S100A8 belongs to the S100 family of calcium-binding proteins and, together with S100A9, is expressed in cells of the myeloid lineage where its is predominantly localized to the cytoplasm . The secreted S100A8/S100A9 complex specifically binds polyunsaturated fatty acids (such as arachidonic acid) in a calcium-dependent manner  and interacts with CD36/FAT to facilitate cellular uptake of fatty acids . Thus, PI could inhibit fatty acid accumulation and adipogenesis by down-regulation of both CD36/FAT and S100A8 expression. Indeed, polyunsaturated fatty acids, and in particular arachidonic acid, have been shown to stimulate adipogenesis probably by acting as PPARγ agonists . In this context, up-regulation of Fads2 (Δ6-fatty acid desaturase) by PI could be the consequence of reduced intracellular polyunsaturated fatty acids. Finally, treatment with PI significantly inhibited expression of Esr2, which encodes oestrogen receptor β (ERβ). This result is in agreement with our findings in HIV-positive patients receiving antiretroviral therapy . Our study demonstrated reduced ERβ mRNA levels in the subcutaneous adipose tissue of lipodystrophy patients, the down-regulation of ERβ expression in the adipose tissue of HIV-positive patients receiving antiretroviral therapy containing PI, and the restoration of ERβ mRNA levels after switching from PI . Thus, ERβ could represent another nuclear transcription factor involved in the cascade of events triggered by PI that lead to impairment of adipocyte differentiation and metabolism. Overall, our observations with microarray experiments are consistent with previous reports which demonstrated that PI down-regulate the expression of lipogenesis genes, such as Fsp27, Lep, Adn, Pfkfb3, and Adipoq, which were reported to be repressed both in in vitro studies [2,7,51–53] and in vivo in the subcutaneous adipose tissue from lipodystrophic HIV-infected patients [7,50,54–56]. Moreover, our microarray study identifies new genes, such as Mrap, Cd36/FAT, and S100a8, that are inhibited in vitro by PI and that are also involved in adipogenesis and adipocyte function. Our results are thus in agreement with the presence of peripheral lipoatrophy in HIV-infected patients treated with PI . Peripheral lipoatrophy, central fat accumulation, and lipomatosis are common problems in adult patients with HIV-1 infection on antiretroviral drugs. Many of the adverse metabolic effects associated with PI therapy, including hypertriglyceridaemia and insulin resistance, resemble those seen in patients with the rare congenital and acquired lipodystrophy syndromes . Thus it has been proposed that peripheral lipoatrophy may be the primary effect caused by PI therapy, which subsequently leads to other adverse effects such as insulin resistance and many other endocrine disturbances ultimately leading to fat redistribution with an increase of the visceral fat and of lipids stored also inside the muscle fibres . Therefore, the generation of the ‘hypertrophic phase’ in fat distribution must be seen as a secondary event linked to the alteration of the endocrine-metabolic milieu and the loss of the capacity by subcutaneous fat cells to store adequately the flux of free fatty acids within the cell and thus preferentially channelling these substrates towards other targets, which are the fat cells of the visceral area and other cells which get ‘fatty’, as is the case of muscle cells and hepatocytes . Although what happens in vivo should be distinguished from what may be seen in in vitro models, our findings, together with the observations by others , support the general hypothesis of a primary damage of the subcutaneous adipose cell.
Quantitative RT–PCR analysis of genes modulated by antiretroviral drugs
To confirm microarray results, the expression of a subset of genes modulated during differentiation and in response to treatment with NRTI and PI was further investigated using quantitative RT–PCR in a time-course experiment (Fig. 2). Overall, quantitative RT–PCR evaluation confirmed results obtained with microarray analysis. Expression of Srebp-1c, C/EBPα, C/EBPβ, and Pparγ mRNA was evaluated as early marker of adipogenesis, whereas expression of Leptin represented a marker of fully differentiated adipocytes. Among these genes, only Leptin expression was shown to be significantly modulated by antiretroviral drugs at microarray analysis and Srebp-1c probes were not represented in our microarray slides. As expected, in 3T3-L1 cells, Srebp-1c, C/EBPα, C/EBPβ, and Pparγ were rapidly and transiently induced during the early phase of adipocyte differentiation, whereas Leptin mRNA levels increased in the late phase of adipogenesis. Treatment with the NRTI ZDV and 3TC did not significantly modify expression of these genes at all time points of adipocyte differentiation, even though a slight decrease of Srebp-1c and Leptin mRNA as compared with untreated control cells could be observed during early and late adipocyte differentiation, respectively. These results are in agreement with Oil Red O staining which did not show a significant inhibition of adipocyte differentiation. At variance with NRTI, the PI SQV and IDV determined a significant inhibition of Srebp-1c, C/EBPα, Pparγ, and Leptin expression, and this inhibition was more evident with SQV than with IDV (Fig. 2). PI also determined a decrease, although not significant, of C/EBPβ transcript levels. The combination of a PI with two NRTI had the same effect on gene expression as treatment with only a PI.
Among the novel genes that microarray analysis demonstrated to be modulated by antiretroviral drugs, Mrap, Fads2, Aebp1, G0s2, Cd36 and Wnt10a were selected for further investigation using quantitative RT–PCR. These genes had different expression profiles during adipogenesis, since G0s2 was transiently induced during the early phase of adipogenesis, Mrap and Cd36 were induced during the late phase of adipogenesis, Aebp1 and Wnt10a were rapidly and markedly reduced during adipogenesis, whereas Fads2 expression remained unchanged during adipocyte differentiation. RT–PCR analysis showed that treatment with NRTI significantly increased Aebp1 mRNA levels as compared to untreated control cells, but had no effect on transcript levels of the other marker genes, thus confirming microarray results. Treatment with PI determined a marked reduction of Mrap, G0s2, and Cd36 mRNA levels and an increase of Wnt10a and Fads2 expression, both at early and late phases of differentiation. Even in this case, the association of a PI with two NRTI did not seem to modify the effect observed with the single drug treatment. The milder effect of NRTI than PI on adipocyte differentiation could in part be accounted to the relatively lower concentration of NRTI than PI used in our study. Although NRTI altered the gene expression profile in differentiating adipocytes, these effects were probably not so marked to be detectable at morphological examination and Oil Red O quantification.
Effect of other drug combinations on adipogenesis
The time-course experiments were replicated with different combinations of anti-retroviral drugs, including the NRTI zalcitabine (ddC) 0.2 μM and stavudine (d4T) 10 μM, which have been more strongly linked to lipoatrophy , and the PI lopinavir (LPV) 10 μM. Moreover, since the effects of NRTI on mitochondria and thus their presumed toxicity is time dependent, the time-course experiment was prolonged to 16 days (Fig. 3). Also with these combinations of NRTI, no significant alterations of cell proliferation or adipogenesis were observed, even after prolonged (16 days’) treatment, whereas the number of differentiated adipocytes was reduced by LPV treatment. This effect of LPV on adipocyte differentiation was paralleled by abnormal expression of genes involved in adipogenesis (Fig. 3).
In this study, by microarray gene expression analysis, we identified genes modulated by NRTI and PI during early adipogenesis. We hypothesize that up-regulation of master transcription factors and modulation of the Wnt signaling pathway by PI could represent a key event leading to inhibition of adipocyte differentiation and down-regulation of expression of adipocyte-specific markers, such as adiponectin, leptin, MRAP, Cd36/FAT, and S100A8. With respect to PI, the effect of NRTI on adipocyte differentiation and gene expression profile was milder, even though NRTI modulated the expression of tissue inhibitors of metalloproteinases and transcription factors, such as Aebp1, which could play an important role in the determination of the adipocyte phenotype. As already demonstrated for ERβ , abnormal expression of these genes could be at the basis of HAART-associated lipodystrophy and could represent a potential target for the treatment of this syndrome.
Monia Pacenti is a recipient of a fellowship from IOV (Istituto Oncologico Veneto).
This work was supported by grants no. 40F.57 from ISS (Istituto Superiore di Sanità) and no. RSF 168/04 from Regione Veneto to Giorgio Palù, and by grant no. 2003061834_006 MIUR-PRIN to Roberto Vettor.
1. Koutkia P, Grinspoon S. HIV-associated lipodystrophy: pathogenesis, prognosis, treatment, and controversies. Annu Rev Med 2004; 55:303–317.
2. Caron M, Auclair M, Vigouroux C, Glorian M, Forest C, Capeau J. The HIV protease inhibitor indinavir impairs sterol regulatory element-binding protein-1 intranuclear localization, inhibits preadipocyte differentiation, and induces insulin resistance. Diabetes 2001; 50:1378–1388.
3. Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI antiviral drugs: an integrated cellular perspective. Nat Rev Drug Discov 2003; 2:812–822.
4. Caron M, Auclair M, Lagathu C, Lombes A, Walker UA, Kornprobst M, et al. The HIV-1 nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro. AIDS 2004; 18:2127–2136.
5. Novakofski J. Adipogenesis: usefulness of in vitro and in vivo experimental models. J Anim Sci 2004; 82:905–915.
6. Boffito M, Back D, Blaschke TF, Rowland M, Bertz RJ, Gerber JG, et al. Protein binding in antiretroviral therapies. AIDS Res Hum Retrov 2003; 19:825–835.
7. Gougeon ML, Penicaud L, Fromenty B, Leclercq P, Viard JP, Capeau J. Adipocytes targets and actors in the pathogenesis of HIV-associated lipodystrophy and metabolic alterations. Antivir Ther 2004; 9:161–177.
8. He GP, Muise A, Li AW, Ro HS. A eukaryotic transcriptional repressor with carboxypeptidase activity. Nature 1995; 378:92–96.
9. Layne MD, Endege WO, Jain MK, Yet SF, Hsieh CM, Chin MT, et al. Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation. J Biol Chem 1998; 273:15654–15660.
10. Abderrahim-Ferkoune A, Bezy O, Astri-Roques S, Elabd C, Ailhaud G, Amri EZ. Transdifferentiation of preadipose cells into smooth muscle-like cells: role of aortic carboxypeptidase-like protein. Exp Cell Res 2004; 293:219–228.
11. Gagnon A, Abaiian KJ, Crapper T, Layne MD, Sorisky A. Down-regulation of aortic carboxypeptidase-like protein during the early phase of 3T3-L1 adipogenesis. Endocrinology 2002; 143:2478–2485.
12. Gagnon A, Landry A, Proulx J, Layne MD, Sorisky A. Aortic carboxypeptidase-like protein is regulated by transforming growth factor beta in 3T3-L1 preadipocytes. Exp Cell Res 2005; 308:265–272.
13. Chavey C, Mari B, Monthouel MN, Bonnafous S, Anglard P, Van Obberghen E, et al. Matrix metalloproteinases are differentially expressed in adipose tissue during obesity and modulate adipocyte differentiation. J Biol Chem 2003; 278:11888–11896.
14. Schneider TJ, Fischer GM, Donohoe TJ, Colarusso TP, Rothstein TL. A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes. J Exp Med 1999; 189:949–956.
15. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998; 95:379–391.
16. Lower KM, Turner G, Kerr BA, Mathews KD, Shaw MA, Gedeon AK, et al. Mutations in PHF6 are associated with Borjeson–Forssman–Lehmann syndrome. Nat Genet 2002; 32:661–665.
17. Tieu K, Perier C, Vila M, Caspersen C, Zhang HP, Teismann P, et al. L-3-hydroxyacyl-CoA dehydrogenase II protects in a model of Parkinson's disease. Ann Neurol 2004; 56:51–60.
18. Jakobsson A, Jorgensen J, Jacobsson A. Differential regulation of fatty acid elongation enzymes in brown adipocytes imply an unique role for Elovl3 during increased fatty acid oxidation. Am J Physiol Endocrinol Metab 2005; 289:E517–E526.
19. Bouillet P, Strasser A. BH3-only proteins — evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death. J Cell Sci 2002; 115:1567–1574.
20. Peters LM, Anderson DW, Griffith AJ, Grundfast KM, San Agustin TB, Madeo AC, et al. Mutation of a transcription factor, TFCP2L3, causes progressive autosomal dominant hearing loss, DFNA28. Hum Mol Genet 2002; 11:2877–2885.
21. Turner CA Jr, Mack DH, Davis MM. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 1994; 77:297–306.
22. Ohinata Y, Payer B, O'Carroll D, Ancelin K, Ono Y, Sano M, et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 2005; 436:207–213.
23. Wilm TP, Solnica-Krezel L. Essential roles of a zebrafish prdm1/blimp1 homolog in embryo patterning and organogenesis. Development 2005; 132:393–404.
24. Cho HP, Nakamura MT, Clarke SD. Cloning, expression, and nutritional regulation of the mammalian Delta-6 desaturase. J Biol Chem 1999; 274:471–477.
25. Matsuzaka T, Shimano H, Yahagi N, Amemiya-Kudo M, Yoshikawa T, Hasty AH, et al. Dual regulation of mouse Delta(5)- and Delta(6)-desaturase gene expression by SREBP-1 and PPARalpha. J Lipid Res 2002; 43:107–114.
26. Tang C, Cho HP, Nakamura MT, Clarke SD. Regulation of human delta-6 desaturase gene transcription: identification of a functional direct repeat-1 element. J Lipid Res 2003; 44:686–695.
27. Riserus U, Tan GD, Fielding BA, Neville MJ, Currie J, Savage DB, et al. Rosiglitazone increases indexes of stearoyl-CoA desaturase activity in humans: link to insulin sensitization and the role of dominant-negative mutation in peroxisome proliferator-activated receptor-gamma. Diabetes 2005; 54:1379–1384.
28. Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, Erickson RL, et al. Inhibition of adipogenesis by Wnt signaling. Science 2000; 289:950–953.
29. Kang S, Bajnok L, Longo KA, Petersen RK, Hansen JB, Kristiansen K, et al. Effects of Wnt signaling on brown adipocyte differentiation and metabolism mediated by PGC-1alpha. Mol Cell Biol 2005; 25:1272–1282.
30. Tseng YH, Butte AJ, Kokkotou E, Yechoor VK, Taniguchi CM, Kriauciunas KM, et al. Prediction of preadipocyte differentiation by gene expression reveals role of insulin receptor substrates and necdin. Nat Cell Biol 2005; 7:601–611.
31. Kennell JA, MacDougald OA. Wnt signaling inhibits adipogenesis through beta-catenin-dependent and -independent mechanisms. J Biol Chem 2005; 280:24004–24010.
32. van Noort M, Clevers H. TCF transcription factors, mediators of Wnt-signaling in development and cancer. Dev Biol 2002; 244:1–8.
33. van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, et al. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev 1994; 8:2691–2703.
34. Kratochwil K, Galceran J, Tontsch S, Roth W, Grosschedl R. FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest of tooth organogenesis in Lef1(−/−) mice. Genes Dev 2002; 16:3173–3185.
35. Metherell LA, Chapple JP, Cooray S, David A, Becker C, Ruschendorf F, et al. Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nat Genet 2005; 37:166–170.
36. Xu A, Choi KL, Wang Y, Permana PA, Xu LY, Bogardus C, et al. Identification of novel putative membrane proteins selectively expressed during adipose conversion of 3T3-L1 cells. Biochem Biophys Res Commun 2002; 293:1161–1167.
37. Noon LA, Clark AJ, King PJ. A peroxisome proliferator-response element in the murine mc2-r promoter regulates its transcriptional activation during differentiation of 3T3-L1 adipocytes. J Biol Chem 2004; 279:22803–22808.
38. Gerhold DL, Liu F, Jiang G, Li Z, Xu J, Lu M, et al. Gene expression profile of adipocyte differentiation and its regulation by peroxisome proliferator-activated receptor-gamma agonists. Endocrinology 2002; 143:2106–2118.
39. Sato O, Kuriki C, Fukui Y, Motojima K. Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor α and γ ligands. J Biol Chem 2002; 277:15703–15711.
40. Abumrad NA, el-Maghrabi MR, Amri EZ, Lopez E, Grimaldi P. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation: homology with human CD36. J Biol Chem 1993; 268:17665–17668.
41. Miyaoka K, Kuwasako T, Hirano K, Nozaki S, Yamashita S, Matsuzawa Y. CD36 deficiency associated with insulin resistance. Lancet 2001; 357:686–687.
42. Aitman TJ. CD36, insulin resistance, and coronary heart disease. Lancet 2001; 357:651–652.
43. Serghides L, Nathoo S, Walmsley S, Kain KC. CD36 deficiency induced by antiretroviral therapy. AIDS 2002; 16:353–358.
44. Meroni L, Riva A, Morelli P, Galazzi M, Mologni D, Adorni F, et al. Increased CD36 expression on circulating monocytes during HIV infection. J Acquir Immune Defic Syndr 2005; 38:310–313.
45. Dressman J, Kincer J, Matveev SV, Guo L, Greenberg RN, Guerin T, et al. HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages. J Clin Invest 2003; 111:389–397.
46. Kerkhoff C, Klempt M, Sorg C. Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9). Biochim Biophys Acta 1998; 1448:200–211.
47. Siegenthaler G, Roulin K, Chatellard-Gruaz D, Hotz R, Saurat JH, Hellman U, et al. A heterocomplex formed by the calcium-binding proteins MRP8 (S100A8) and MRP14 (S100A9) binds unsaturated fatty acids with high affinity. J Biol Chem 1997; 272:9371–9377.
48. Kerkhoff C, Sorg C, Tandon NN, Nacken W. Interaction of S100A8/S100A9-arachidonic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells. Biochemistry 2001; 40:241–248.
49. Gaillard D, Negrel R, Lagarde M, Ailhaud G. Requirement and role of arachidonic acid in the differentiation of pre-adipose cells. Biochem J 1989; 257:389–397.
50. Barzon L, Zamboni M, Pacenti M, Milan G, Bosello O, Federspil G, et al. Do estrogen receptors have a role in the pathogenesis of HIV-associated lipodystrophy? AIDS 2005; 19:531–533.
51. Parker RA, Flint OP, Mulvey R, Elosua C, Wang F, Federson W, et al. Endoplasmic reticulum stress links dyslipidemia to inhibition of proteasome activity and glucose transport by HIV protease inhibitors. Mol Pharmacol 2005; 67:1909–1919.
52. Dowell P, Flexner C, Kwiterovich PO, Lane MD. Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors. J Biol Chem 2000; 275:41325–41332.
53. Caron M, Auclair M, Sterlingot H, Kornprobst M, Capeau J. Some HIV protease inhibitors alter lamin A/C maturation and stability, SREBP-1 nuclear localization and adipocyte differentiation. AIDS 2003; 17:2437–2444.
54. Bastard JP, Caron M, Vidal H, Jan V, Auclair M, Vigouroux C, et al. Association between altered expression of adipogenic factor SREBP1 in lipoatrophic adipose tissue from HIV-1-infected patients and abnormal adipocyte differentiation and insulin resistance. Lancet 2002; 359:1026–1031.
55. Kannisto K, Sutinen J, Korsheninnikova E, Fisher RM, Ehrenborg E, Gertow K, et al. Expression of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma co-activator 1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapy. AIDS 2003; 17:1753–1762.
56. Jan V, Cervera P, Maachi M, Baudrimont M, Kim M, Vidal H, et al. Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients. Antivir Ther 2004; 9:555–564.
57. Grinspoon S, Carr A. Cardiovascular risk and body-fat abnormalities in HIV-infected adults. N Engl J Med 2005; 352:48–62.
58. Gan SK, Samaras K, Thompson CH, Kraegen EW, Carr A, Cooper DA, et al. Altered myocellular and abdominal fat partitioning predict disturbance in insulin action in HIV protease inhibitor-related lipodystrophy. Diabetes 2002; 51:3163–3169.
59. Lemoine M, Barbu V, Girard PM, Kim M, Bastard JP, Wendum D, et al. Altered hepatic expression of SREBP-1 and PPARγ is associated with liver injury in insulin-resistant lipodystrophic HIV-infected patients. AIDS 2006; 20:387–395.
60. Jones SP, Qazi N, Morelese J, Lebrecht D, Sutinen J, Yki-Jarvinen H, et al. Assessment of adipokine expression and mitochondrial toxicity in HIV patients with lipoatrophy on stavudine and zidovudine-containing regimens. J Acquir Immune Defic Syndr 2005; 40:565–572.
HAART; lipodystrophy; adipocytes; Wnt pathway; microarray analysis; nucleoside reverse transcriptase inhibitors; protease inhibitors
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